Circulating Leukocyte Counts Research Articles

Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease.

Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.

Cardiometabolic and immune response to exercise training in patients with metabolic syndrome: retrospective analysis of two randomized clinical trials.

Metabolic syndrome (MetS) is defined by the presence of central obesity plus ≥two metabolic/cardiovascular risk factors (RF), with inflammation being a major disease-driving mechanism. Structured endurance exercise training (ET) may positively affect these traits, as well as cardiorespiratory fitness (V̇O2peak). We explore individual ET-mediated improvements of MetS-associated RF in relation to improvements in V̇O2peak and inflammatory profile. MetS patients from two randomized controlled trials, ExMET (n = 24) and OptimEx (n = 34), had performed 4- or 3-months supervised ET programs according to the respective trial protocol. V̇O2peak, MetS-defining RFs (both RCTs), broad blood leukocyte profile, cytokines and plasma proteins (ExMET only) were assessed at baseline and follow-up. Intra-individual changes in RFs were analysed for both trials separately using non-parametric approaches. Associations between changes in each RF over the exercise period (n-fold of baseline values) were correlated using a non-parametrical approach (Spearman). RF clustering was explored by uniform manifold approximation and projection (UMAP) and changes in RF depending on other RF or exercise parameters were explored by recursive partitioning. Four months of ET reduced circulating leukocyte counts (63.5% of baseline, P = 8.0e-6), especially effector subtypes. ET response of MetS-associated RFs differed depending on patients' individual RF constellation, but was not associated with individual change in V̇O2peak. Blood pressure lowering depended on cumulative exercise duration (ExMET: ≥102 min per week; OptimEx-MetS: ≥38 min per session) and baseline triglyceride levels (ExMET: <150 mg/dl; OptimEx-MetS: <174.8 mg/dl). Neuropilin-1 plasma levels were inversely associated with fasting plasma triglycerides (R: -0.4, P = 0.004) and changes of both parameters during the ET phase were inversely correlated (R: -0.7, P = 0.0001). ET significantly lowered effector leukocyte blood counts. The improvement of MetS-associated cardiovascular RFs depended on individual basal RF profile and exercise duration but was not associated with exercise-mediated increase in V̇O2peak. Neuropilin-1 may be linked to exercise-mediated triglyceride lowering.

Gram-negative Bacteria are Associated With Sensorineural Hearing Loss in Chronic Otitis Media.

Chronic otitis media (COM) is an inflammatory disease that commonly presents with otorrhea and hearing loss. Bacteria-induced inflammation can cause inner ear damage, leading to sensorineural hearing loss (SNHL). This study aimed to compare the prevalence and severity of SNHL in patients with gram-negative versus gram-positive cultures and examine associations between the concentrations of circulating monocytes and neutrophils with bacteria species and SNHL. This was a retrospective study. Cholesteatoma or chronic suppurative otitis media patients with otorrhea were enrolled. Middle ear secretions were collected using sterile swabs under an otoscope, and sent for bacterial detection within 30 min. Pure tone audiometry and circulating leukocyte counts were recorded and analyzed in patients infected with different pathogens. Logistic regression analysis was used to identify the risk factors associated with SNHL. A total of 137 patients were enrolled, including 45 patients infected with gram-negative bacteria, 41 with gram-positive bacteria, 20 with polymicrobial infection, and 31 with no bacterial growth. Logistic regression analysis showed that bacterial culture positive infections (OR = 7.265, 95% CI 2.219-23.786, p = 0.001) were an independent risk factor for SNHL. Patients with gram-negative bacteria had higher risks of SNHL (p < 0.0001) and more severe hearing loss (p = 0.005) than those with gram-positive bacteria. COM patients infected with gram-negative bacteria showed an increase in circulating monocytes, which correlated with the occurrence of SNHL (p = 0.0343). Gram-negative bacteria are associated with elevated circulating monocyte counts and have a higher risk of severe SNHL. 4 Laryngoscope, 134:3335-3341, 2024.

Abstract 19053: Peripheral Eosinophil Count is Associated With Left Ventricular Systolic Dysfunction During Spontaneous Coronary Artery Dissection

Background: Spontaneous coronary artery dissection (SCAD) is an underdiagnosed condition disproportionally affecting young women, particularly during the peripartum period and/or in the presence of systemic inflammatory and autoimmune diseases. Tissue-level studies have demonstrated inflammatory infiltrates with adventitial or peri-adventitial eosinophilia at the site of SCAD lesions. Less is known regarding how systemic markers of immune activation associate with cardiac dysfunction in SCAD. Hypotheses: Higher eosinophil count and Neutrophil-to-Lymphocyte Ratio (NLR) associate with LV systolic dysfunction in SCAD patients. Methods: In individuals diagnosed with SCAD who underwent CBC with differential and echocardiography within the same presenting hospital encounter, we performed age- and sex-adjusted logistic regressions with eosinophil count (% of differential) and NLR (% of differential) as primary exposures and LVEF&lt;55% as the dichotomous endpoint. A p&lt;0.05 was considered statistically significant. We explored associations of neutrophil, lymphocyte, monocyte, and basophil counts with LVEF in secondary analyses. Results: In SCAD patients with complete CBC and LVEF data (N=36; mean age 49.9 years at diagnosis, 28 female), peripheral eosinophil count was associated with significantly higher odds of systolic dysfunction [OR of LVEF&lt;55% per every 1% higher eosinophil count = 1.87; 95% CI 1.04, 3.36; p=0.04]. NLR ratio was not associated with LV systolic dysfunction (OR 1.05; 95% CI 0.94, 1.18; p=0.35) nor were other circulating leukocyte fractions. Conclusion: Peripheral eosinophil count, but not other circulating leukocyte counts, was associated with LV systolic dysfunction as measured by LVEF &lt;55%. These findings provide complementary support to pathology-based evidence of eosinophilia observed in SCAD lesions and require confirmation in future longitudinal studies of changes in LV function following SCAD events.

Association between circulating leukocytes and arrhythmias: Mendelian randomization analysis in immuno-cardiac electrophysiology.

Cardiac arrhythmia is a common disease associated with high mortality and morbidity. Circulating leukocyte counts, which serve as a biomarker for assessing systemic immune status, have been linked to arrhythmias in observational studies. However, observational studies are plagued by confounding factors and reverse causality, whether alterations in circulating leukocyte components are causally associated with arrhythmias remains uncertain. The present study explored this question based on genetic evidence. We performed Mendelian randomization (MR) analysis to evaluate whether alterations in leukocyte counts affect aggregated risk of all types of arrhythmia or risk of five specific types of arrhythmia. Single-nucleotide polymorphisms serving as proxies for leukocyte differential counts were retrieved from the Blood Cell Consortium, and statistical data on arrhythmias were obtained from the UK Biobank), FinnGenand a meta-analysis of genome-wide association studies for atrial fibrillation. We applied inverse variance-weighted method as the primary analysis, complemented by a series of sensitivity analyses. Bidirectional analyses were conducted to assess reverse causality. Finally, multivariable MR was performed to study the joint effects of multiple risk factors. We found that genetically predicted differential leukocyte counts were not significantly associated with aggregated occurrence of all types of arrhythmia. In contrast, each 1-standard deviation increase in lymphocyte count was associated with 46% higher risk of atrioventricular block (OR 1.46, 95% CI 1.11-1.93, p=0.0065). A similar effect size was observed across all MR sensitivity analyses, with no evidence of horizontal pleiotropy. Reverse MR analysis suggested that atrioventricular block was unlikely to cause changes in lymphocyte count. Primary MR analysis based on the inverse-variance weighted method suggested that changes in neutrophil count alter risk of right bundle branch block, and changes in basophil count alter risk of atrial fibrillation. However, these causal relationships were not robust in sensitivity analyses. We found no compelling evidence that neutrophil or lymphocyte counts cause atrial fibrillation. Our data support higher lymphocyte count as a causal risk factor for atrioventricular block. These results highlight the importance of immune cells in the pathogenesis of specific cardiac conduction disorders.

One night of sleep fragmentation does not affect exercise-induced leukocyte trafficking or mitogen-stimulated leukocyte oxidative burst in healthy men

PurposeThis study examined whether one night of sleep fragmentation alters circulating leukocyte counts and mitogen-stimulated oxidative burst by leukocytes at rest and in response to an acute bout of vigorous exercise. MethodsIn a randomised crossover design, nine healthy men (mean ± SD: age 22 ± 2 years; BMI 24.9 ± 1.9 kg/m2) were exposed to one night of fragmented or uninterrupted sleep before cycling for 45 min at 71% ± 4% V̇O2peak. Finger-tip blood samples were collected at rest, immediately post-exercise and one-hour post-exercise. Total leukocytes, lymphocytes, monocytes and neutrophils were counted. Leukocyte oxidative burst was assessed in whole blood by measuring Reactive Oxygen Species (ROS) production with luminol-amplified chemiluminescence after stimulation with phorbol 12-myristate 13-acetate (PMA). ResultsExercise elicited the expected trafficking pattern of leukocytes, lymphocytes, monocytes and neutrophils. Compared to rest, PMA-stimulated ROS production was increased one-hour post-exercise (+73% ± 65%; p = 0.019; data combined for fragmented and uninterrupted sleep). There were no statistically significant effects of fragmented sleep on leukocyte, lymphocyte, monocyte, and neutrophil counts or on ROS production at rest, immediately post-exercise or one-hour post-exercise (p > 0.05). However, with fragmented sleep, there was a +10% greater lymphocytosis immediately post-exercise (fragmented +40% ± 37%; uninterrupted +30% ± 35%; p = 0.51) and a −19% smaller neutrophilia by one-hour post-exercise (fragmented +103% ± 88%; uninterrupted +122% ± 131%; p = 0.72). ConclusionFragmented sleep did not substantially alter the magnitude or pattern of exercise-induced leukocyte trafficking or mitogen-stimulated oxidative burst by leukocytes.

Plasma Trimethylamine-N-oxide following Cessation of L-carnitine Supplementation in Healthy Aged Women.

L-carnitine supplementation elevates plasma trimethylamine-N-oxide (TMAO), which may participate in atherosclerosis development by affecting cholesterol metabolism. The aim of the current study was to determine the effect of increased plasma TMAO on biochemical markers in the blood following cessation of L-carnitine supplementation. The follow-up measurements were performed on subjects who completed 24 weeks of L-carnitine or placebo supplementation protocol. Blood samples were taken after finishing the supplementation and then 4 and 12 months following the supplementation withdrawal. Four months after cessation of L-carnitine supplementation, plasma TMAO concentration reached a normal level which was stable for the following eight months. During this period, no modifications in serum lipid profile and circulating leukocyte count were noted. TMAO implications in health and disease is widely discussed. The results of this study demonstrate no adverse effects of elevated plasma TMAO, induced by L-carnitine, on the measured parameters at 4 and 12 months after withdrawal of supplementation.

Immunomodulatory effects of whole yeast cells and capsicum in weanling pigs challenged with pathogenic Escherichia coli1.

An experiment was conducted to evaluate growth performance, fecal bacterial counts, frequency of diarrhea, and clinical blood parameters in weanling pigs inoculated with enterotoxigenic Escherichia coli (ETEC) who were fed a whole yeast cell (WYC) product and capsicum, a plant essential oil. Weanling pigs (34 barrows and 30 gilts, 21 d of age, 5.90 ± 1.03 kg BW) were allotted to experimental treatments in a randomized complete block design based on litter, sex, and initial BW. Four pigs were individually housed within each containment chamber and assigned to 1 of 4 dietary treatments, which included a control diet without or with 0.2% WYC (CitriStim; ADM, Decatur, IL) or 10 ppm of capsicum (XTract 6933; Pancosma, Geneva, Switzerland), provided either alone or in combination. After receiving diets for 13 d, pigs were orally inoculated with F18+ ETEC and maintained on their assigned diets for an additional 10 d; a separate cohort of 12 pigs receiving the control diet was sham-inoculated using PBS. Body and feeder weights were recorded, and fecal swabs collected, on 0, 5, and 10 d postinoculation (DPI), with blood sampled at 0, 2, 7, and 10 DPI for isolation of peripheral blood mononuclear cells. Pigs challenged with ETEC and fed diets containing WYC or capsicum alone had a higher frequency of diarrhea when compared with pigs receiving diets without those compounds (P < 0.05). Total fecal bacterial counts in pigs fed the combination of additives were highest when compared with either additive alone (interaction, P = 0.03) at 10 DPI. Blood leukocyte counts were increased in challenged pigs receiving the combination of additives compared with all other challenged treatment groups (interaction, P = 0.04). The addition of WYC increased (main effect, P = 0.01) lymphocyte counts at 7 DPI. Proportions of CD8+ and CD4+CD8+ cells were lower in pigs fed the combination of additives compared with pigs fed either additive alone at 0 and 7 DPI. In conclusion, these data indicate that the combination of the 2 additives elicited higher ETEC shedding and circulating leukocyte counts, and reduced the proportions of cytotoxic and memory T-cells than either additive alone.

Analysis of Early Stroke-induced Changes in Circulating Leukocyte Counts using Transcriptomic Deconvolution.

Growing evidence suggests that stroke alters the phenotype of the peripheral immune system; better characterization of this response could provide new insights into stroke pathophysiology. In this investigation, we employed a deconvolution approach to informatically infer the cellular composition of the circulating leukocyte pool at multiple timepoints following stroke onset based on whole blood mRNA expression. Microarray data generated from the peripheral blood of 23 cardiovascular disease controls and 23 ischemic stroke patients at 3, 5, and 24 hours post-symptom onset were obtained from a public repository. Transcriptomic deconvolution was used to estimate the relative counts of nine leukocyte populations based on the expression of cell-specific transcripts, and cell counts were compared between groups across timepoints. Inferred counts of lymphoid cell populations including B-cells, CD4+ T-cells, CD8+ T-cells, γδ T-cells, and NK-cells were significantly lower in stroke samples relative to control samples. With respect to myeloid cell populations, inferred counts of neutrophils and monocytes were significantly higher in stroke samples compared to control samples, however inferred counts of eosinophils and dendritic cells were significantly lower. These collective differences were most dramatic in samples collected at 5 and 24 hours post-symptom onset. Findings were subsequently confirmed in a second dataset generated from an independent population of 24 controls and 39 ischemic stroke patients. Collectively, these results offer a comprehensive picture of the early stroke-induced changes to the complexion of the circulating leukocyte pool, and provide some of the first evidence that stroke triggers an acute decrease in eosinophil counts.

Associations between CT-determined visceral fat burden, hepatic steatosis, circulating white blood cell counts and neutrophil-to-lymphocyte ratio.

Visceral adiposity is associated with cardiovascular disease, an association that may be mediated in part by inflammation. We hypothesized that regional measures of visceral adiposity would associate with commonly obtained clinical measures of immune status. We consecutively studied 3,291 subjects (mean age, 49.8±9.8 years) who underwent an annual cardiovascular risk survey. Peri-cardial (PCF) and thoracic peri-aortic adipose tissue (TAT) volumes were determined by dedicated computed tomography (CT) software (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA). Hepatic steatosis was assessed by abdominal ultrasonography. We explored cross-sectional associations between visceral fat measures and high-sensitivity C-reactive protein (hs-CRP), leukocyte counts, and the neutrophil-to-lymphocyte ration (NLR). Among 3,291 study participants, we observed positive linear associations between PCF and TAT, higher degree of hepatic steatosis and hs-CRP, various leukocyte counts, either total and its differential counts, and NLR (all trend p<0.001). Multi-variate linear and logistic regression models showed independent associations between PCF/TAT (ß-Coef: 0.14/0.16, both p<0.05) and total WBC counts, with only TAT further demonstrated significant relations with neutrophil counts and NLR (both p<0.05) and independently identified abnormally high WBC and NLR (Odds ratio: 1.18 & 1.21, both p<0.05). C-statistics showed significant incremental model prediction for abnormally high WBC and NLR (both ΔAUROC<0.05) when TAT was superimposed on traditional cardiovascular risks and biochemical information. Greater visceral adiposity burden and hepatic steatosis may be associated with higher circulating leukocyte counts and markers for atherosclerosis, with more pronounced influences for peri-aortic adiposity. Our data suggested the differential biological impacts for region-specific visceral adiposity.

Abstract 639: Deletion of Alternatively Spliced Extra Domain A of Cellular Fibronectin Stabilizes Advanced Rupture-Prone Plaques in Hyperlipidemic Mice

Background: The most important clinical manifestation of atherosclerosis is rupture of advanced plaques. The fibronectin containing alternatively spliced extra domain A (Fn-EDA) is abundant in extracellular matrix around macrophages and endothelial cells in advanced human plaques. In vitro studies suggest that Fn-EDA is a ligand for TLR4 and upregulates MMP9, which has been shown to enhance elastin degradation and promote plaque rupture in advanced plaques of hyperlipidemic apolipoprotein E-deficient (Apoe -/- ) mice. The mechanistic role of Fn-EDA in advanced plaques remains unknown. Hypothesis: Fn-EDA/TLR4 signaling in activated macrophages contributes to plaque vulnerability by upregulating MMP9 during advanced atherosclerosis. Methods and Results: We compared atherosclerotic plaques in the brachiocephalic artery (a model artery for study of plaque vulnerability) of female Fn- EDA -/- Apoe -/- and Apoe -/- mice at 50 weeks of age (44 weeks on high-fat Western diet). Fn- EDA -/- Apoe -/- mice exhibited decreased plaque size and characteristics of stable plaques, with decreased necrosis, reduced influx of activated macrophages, increased SMC staining and greater collagen content compared to Apoe -/- mice (P&lt;0.05, n=11-13 mice/group), although cholesterol and triglyceride levels, and circulating leukocyte counts were similar. Purified cellular FN, which contains EDA, potentiated the NFκB-mediated inflammatory pathway (increased phospho-NFκB p65/ NFκB p65, TNFα and IL1β) and MMP9 protein expression (gelatin zymography) in a dose-dependent manner in purified macrophages from Fn-EDA -/- Apoe -/- mice but not from Fn-EDA -/- TLR4 -/- Apoe -/- mice. Using immunohistochemistry, we demonstrated colocalization of macrophage TLR4 and Fn-EDA within advanced atherosclerotic plaques in murine brachiocephalic arteries and human coronary arteries. Genetic deletion of TLR4 in Apoe -/- mice stabilized advanced plaques, and parameters of plaque stability were comparable between TLR4 -/- Apoe -/- and Fn-EDA -/- TLR4 -/- Apoe -/- mice. Conclusions: These findings suggest that Fn-EDA/TLR4 signaling in macrophages is a key mechanism that upregulates MMP9, and thereby promotes plaque vulnerability in advanced atherosclerosis.

Effect of elastic-band exercise on muscle damage and inflammatory responses in Taekwondo athletes

INTRODUCTION: Elastic bands offer variable elastic resistance (ER) throughout a range of motion and their incorporation with exercise movements has been used for variable strength training and rehabilitation purposes. Objective: Investigate the effect of acute bout of progressive elastic-band exercise on muscle damage and inflammatory response in Taekwondo athletes (TKD) compared with untrained ones.METHODS: Fourteen (TKD, n = 7 and untrained, n = 7) men performed 3 sets of progressive resistance elastic exercise. Blood samples were taken pre-exercise and also immediately and 24h post exercise. Delayed onset muscle soreness (DOMS), creatine kinase (CK) and lactate dehydrogenase (LDH) activity, total leukocyte counts, interleukin-6 and C-reactive protein (CRP) were analyzed.RESULTS: Only DOMS increased in untrained group, but elevation of DOMS was observed in both groups (TKD and untrained) at 24h after exercise (p&lt;0.05). CK and LDH activity increased in both groups significantly. Also TKD group only showed CK increasing 24h post exercise (p&lt;0.05). Total circulating leukocyte counts increased immediately in post exercise experiments and decreased in 24h ones in both groups (p&lt;0.05). Serum IL-6 immediately increased in both groups and 24h post exercises but there was no significant difference between immediate and 24h post exercise experiments in TKD group. Furthermore, CRP just increased 24h after exercise in both groups (p&lt;0.05).CONCLUSION: Progressive resistance elastic exercise induced muscle damage and inflammation in TKD athletes, but also had smaller changes in comparison with untrained group and other forms of exercise.

Depletion of polymorphonuclear leukocytes has no effect on preterm delivery in a mouse model of Escherichia coli-induced labor

The objective of the study was to investigate the role of polymorphonuclear leukocytes (PMNs) in a mouse model of Escherichia coli-induced labor. Intraperitoneal injection of rabbit antimouse PMN antiserum or control was performed in CD-1 mice 29 hours and 5 hours prior to laparotomy and intrauterine injection of either killed E coli or phosphate-buffered saline on day 14.5 of pregnancy. Preterm delivery was defined as delivery of at least 1 pup within 48 hours. Circulating leukocyte counts were determined manually or by flow cytometry at the time of surgery and 8, 24, and 48 hours afterward. Maternal and fetal tissues were analyzed in a separate group of animals 8 hours after surgery. Pretreatment with anti-PMN antiserum significantly decreased the numbers of circulating leukocytes and the proportion of neutrophils among all leukocytes by 70-80% at surgery and at least 8 hours thereafter. Neutrophil depletion significantly reduced 2 markers of neutrophil activation in the uterus and placenta (neutrophil elastase and myeloperoxidase activity) and neutrophil infiltration into gestational tissues in bacterially treated animals to baseline (control) levels but did not affect preterm birth rates. The large E coli-induced increases in uterine inflammatory markers (interleukin-1β, tumor necrosis factor, chemokine ligand-5, cyclooxygenase-2) were not affected or were only minimally affected by neutrophil depletion. Although PMN antiserum reduces both neutrophil number and activity, it does not diminish sensitivity to bacterially induced delivery or meaningfully alter the expression of inflammatory markers in the mouse model. Preterm birth and inflammation in this model are not likely to depend on neutrophil function.

Reproductive and immune effects of chronic corticosterone treatment in male White's treefrogs, Litoria caerulea.

Amphibian populations are declining globally. The potential contribution of glucocorticoid hormones to these declines has received little attention, but chronic elevation of glucocorticoids has been linked to a suite of negative outcomes across vertebrate taxa. Recently, chronic environmental stress has been associated with precipitous declines in sperm count and sperm viability in White's treefrogs (Litoria caerulea), but the mechanism remains unknown. In order to determine whether corticosterone is responsible for suppressing reproductive and immune function in this species, we elevated circulating concentrations of corticosterone in 10 male captive-bred frogs via transdermal application for 7 days. We compared sperm count, sperm viability, splenic cell count and circulating leucocyte counts in corticosterone-treated frogs with those in untreated control frogs. Chronic application of exogenous corticosterone led to supraphysiological circulating concentrations of corticosterone, but had no effect on sperm count or viability. However, corticosterone-treated frogs demonstrated a significant decrease in circulating eosinophils, which are immune cells implicated in fighting a variety of pathogens, including extracellular parasites. These findings suggest that although chronic elevation of circulating corticosterone is not necessarily associated with reproductive suppression in this species, it may cause immunosuppression. Thus, chronic glucocorticoid elevations in amphibians might enhance susceptibility to infection with pathogens and parasites, and their potential contributions to global population declines warrant further study.

Bone marrow cell transplantation time-dependently abolishes efficacy of granulocyte colony-stimulating factor after stroke in hypertensive rats.

We aimed to determine a possible synergistic effect of granulocyte colony-stimulating factor (G-CSF) and bone marrow-derived mononuclear cells (BM MNC) after stroke in spontaneously hypertensive rats. Male spontaneously hypertensive rats were subjected to middle cerebral artery occlusion and randomly assigned to daily injection of 50 μg/kg G-CSF for 5 days starting 1 hour after stroke (groups 1, 2, and 3) with additional intravenous transplantation of 1.5×10E7 BM MNC per kilogram at 6 hours (group 2) or 48 hours (group 3) after stroke, or control treatment (group 4). Circulating leukocyte counts and functional deficits, infarct volume, and brain edema were repeatedly assessed in the first week and first month. G-CSF treatment led to a significant neutrophilia, to a reversal of postischemic depression of circulating leukocytes, and to a significantly improved functional recovery without affecting the infarct volume or brain edema. BM MNC cotransplantation was neutral after 6 hours, but reversed the functional effect of G-CSF after 48 hours. Short-term investigation of combined G-CSF and BM MNC treatment at 48 hours indicated splenic accumulation of granulocytes and transplanted cells, accompanied by a significant rise of granulocytes in the circulation and the ischemic brain. G-CSF improved functional recovery in spontaneously hypertensive rats, but this effect was abolished by cotransplantation of BM MNC after 48 hours. In the spleen, transplanted cells may hinder the clearance of granulocytes that were massively increased by G-CSF. Increased circulation and infiltration of granulocytes into the ischemic brain may be detrimental for stroke outcome.

No Effects of Two Anesthetic Agents on Circulating Leukocyte Counts or Resistance to Trematode Infections in Larval Amphibians

Abstract Pharmacological anesthetics are used frequently in aquatic animal husbandry and research. Although several studies have investigated the effects of these anesthetic agents on immune responses in fish, none have assessed such effects in amphibians. To address this disparity, we exposed Osteopilus septentrionalis tadpoles to 0.005% benzocaine, 0.1% tricaine methanesulfonate (MS-222), or artificial spring water, and quantified circulating white blood cells and susceptibility to infection by larval trematodes. Exposure to neither MS-222 nor benzocaine elevated circulating white blood cells relative to water-exposed tadpoles. Furthermore, anesthetic treatment did not affect resistance to larval trematode infection. These results indicate that the anesthetics tested here probably do not affect tadpole immune function and should aid researchers in determining anesthetic usage.

Oxidative Burst of Circulating Neutrophils Following Traumatic Brain Injury in Human

Besides secondary injury at the lesional site, Traumatic brain injury (TBI) can cause a systemic inflammatory response, which may cause damage to initially unaffected organs and potentially further exacerbate the original injury. Here we investigated plasma levels of important inflammatory mediators, oxidative activity of circulating leukocytes, particularly focusing on neutrophils, from TBI subjects and control subjects with general trauma from 6 hours to 2 weeks following injury, comparing with values from uninjured subjects. We observed increased plasma level of inflammatory cytokines/molecules TNF-α, IL-6 and CRP, dramatically increased circulating leukocyte counts and elevated expression of TNF-α and iNOS in circulating leukocytes from TBI patients, which suggests a systemic inflammatory response following TBI. Our data further showed increased free radical production in leukocyte homogenates and elevated expression of key oxidative enzymes iNOS, COX-2 and NADPH oxidase (gp91phox) in circulating leukocytes, indicating an intense induction of oxidative burst following TBI, which is significantly greater than that in control subjects with general trauma. Furthermore, flow cytometry assay proved neutrophils as the largest population in circulation after TBI and showed significantly up-regulated oxidative activity and suppressed phagocytosis rate for circulating neutrophils following brain trauma. It suggests that the highly activated neutrophils might play an important role in the secondary damage, even outside the injured brain. Taken together, the potent systemic inflammatory response induced by TBI, especially the intensively increase oxidative activity of circulating leukocytes, mainly neutrophils, may lead to a systemic damage, dysfunction/damage of bystander tissues/organs and even further exacerbate secondary local damage. Controlling these pathophysiological processes may be a promising therapeutic strategy and will protect unaffected organs and the injured brain from the secondary damage.

The Normal Limits, Subclinical Significance, Related Metabolic Derangements and Distinct Biological Effects of Body Site-Specific Adiposity in Relatively Healthy Population

BackgroundThe accumulation of visceral adipose tissue that occurs with normal aging is associated with increased cardiovascular risks. However, the clinical significance, biological effects, and related cardiometabolic derangements of body-site specific adiposity in a relatively healthy population have not been well characterized.Materials and MethodsIn this cross-sectional study, we consecutively enrolled 608 asymptomatic subjects (mean age: 47.3 years, 27% female) from 2050 subjects undergoing an annual health survey in Taiwan. We measured pericardial (PCF) and thoracic peri-aortic (TAT) adipose tissue volumes by 16-slice multi-detector computed tomography (MDCT) (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA) and related these to clinical characteristics, body fat composition (Tanita 305 Corporation, Tokyo, Japan), coronary calcium score (CCS), serum insulin, high-sensitivity C-reactive protein (Hs-CRP) level and circulating leukocytes count. Metabolic risk was scored by Adult Treatment Panel III guidelines.ResultsTAT, PCF, and total body fat composition all increased with aging and higher metabolic scores (all p<0.05). Only TAT, however, was associated with higher circulating leukocyte counts (ß-coef.:0.24, p<0.05), serum insulin (ß-coef.:0.17, p<0.05) and high sensitivity C-reactive protein (ß-coef.:0.24, p<0.05). These relationships persisted after adjustment in multivariable models (all p<0.05). A TAT volume of 8.29 ml yielded the largest area under the receiver operating characteristic curve (AUROC: 0.79, 95%CI: 0.74–0.83) to identify metabolic syndrome. TAT but not PCF correlated with higher coronary calcium score after adjustment for clinical variables (all p<0.05).ConclusionIn our study, we observe that age-related body-site specific accumulation of adipose tissue may have distinct biological effects. Compared to other adiposity measures, peri-aortic adiposity is more tightly associated with cardiometabolic risk profiles and subclinical atherosclerosis in a relatively healthy population.

Interpreting Stress Responses during Routine Toxicity Studies

Stress often occurs during toxicity studies. The perception of sensory stimuli as stressful primarily results in catecholamine release and activation of the hypothalamic-pituitary-adrenal (HPA) axis to increase serum glucocorticoid concentrations. Downstream effects of these neuroendocrine signals may include decreased total body weights or body weight gain; food consumption and activity; altered organ weights (e.g., thymus, spleen, adrenal); lymphocyte depletion in thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophils with decreased lymphocytes and eosinophils); and altered reproductive functions. Typically, only some of these findings occur in a given study. Stress responses should be interpreted as secondary (indirect) rather than primary (direct) test article-related findings. Determining whether effects are the result of stress requires a weight-of-evidence approach. The evaluation and interpretation of routinely collected data (standard in-life, clinical pathology, and anatomic pathology endpoints) are appropriate and generally sufficient to assess whether or not changes are secondary to stress. The impact of possible stress-induced effects on data interpretation can partially be mitigated by toxicity study designs that use appropriate control groups (e.g., cohorts treated with vehicle and subjected to the same procedures as those dosed with test article), housing that minimizes isolation and offers environmental enrichment, and experimental procedures that minimize stress and sampling and analytical bias. This article is a comprehensive overview of the biological aspects of the stress response, beginning with a Summary (Section 1) and an Introduction (Section 2) that describes the historical and conventional methods used to characterize acute and chronic stress responses. These sections are followed by reviews of the primary systems and parameters that regulate and/or are influenced by stress, with an emphasis on parameters evaluated in toxicity studies: In-life Procedures (Section 3), Nervous System (Section 4), Endocrine System (Section 5), Reproductive System (Section 6), Clinical Pathology (Section 7), and Immune System (Section 8). The paper concludes (Section 9) with a brief discussion on Minimizing Stress-Related Effects (9.1.), and a final section explaining why Parameters routinely measured are appropriate for assessing the role of stress in toxicology studies (9.2.).

Effect of daily mixed nutritional supplementation on immune indices in soldiers undertaking an 8-week arduous training programme

The aim was to investigate the influence of a daily mixed nutritional supplement during an 8-week arduous training programme on immune indices and mediators including circulating leucocyte counts; bacterially stimulated neutrophil degranulation; interleukin-6 (IL-6), cortisol and saliva secretory immunoglobulin-A (SIgA). Thirty men (mean (SD): age 25 (3)years; body mass, 80.9 (7.7)kg) received a habitual diet (CON, n=15) or received a habitual diet plus an additional food supplement (SUP, n=15). From weeks 0-6, CON received 14.0MJday(-1) and SUP received 19.7MJday(-1), and during a final 2-week field exercise in weeks 7 and 8, CON received 17.7MJday(-1) and SUP received 21.3MJday(-1). Blood and saliva were taken at rest after an overnight fast at weeks 0, 6 and 8. Body mass loss over the 8 weeks was greater in CON (CON, 5.0 (2.3); SUP, 1.6 (1.5)kg: P<0.001). Training-induced decreases in circulating total leucocytes (CON: weeks 0, 8.0 (2.1); weeks 8, 6.5 (1.6) 10(9)l(-1), P<0.01), lymphocytes (21%, P<0.01) and monocytes (20%, P<0.01) were prevented by the nutritional supplement. Saliva SIgA secretion rate increased approximately twofold by week 8 in SUP (P<0.01) and was greater at week 8 compared with CON (P<0.01). Circulating neutrophils, bacterially stimulated neutrophil degranulation, IL-6 and cortisol were similar in CON and SUP at week 8. In conclusion, a daily mixed nutritional supplement prevented the decrease in circulating total leucocytes, lymphocytes and monocytes and increased saliva SIgA output during an 8-week arduous training programme. The increase in saliva SIgA with nutritional supplementation during training may reduce susceptibility to upper respiratory infection.