Numbers Of Leukocyte Subsets Research Articles

Targeted DNA Methylation Analysis Facilitates Leukocyte Counts in Dried Blood Samples.

Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application. DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices. In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements. The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing.

Repeated social defeat stress induces neutrophil mobilization in mice: maintenance after cessation of stress and strain-dependent difference in response.

Inflammation has been associated with stress-related mental disturbances. Rodent studies have reported that blood-borne cytokines are crucial for stress-induced changes in emotional behaviours. However, the roles and regulation of leukocytes in chronic stress remain unclear. Adult male C57BL/6N mice were subjected to repeated social defeat stress (R-SDS) with two protocols which differed in stress durations, stress cycles, and housing conditions, followed by the social interaction test. The numbers of leukocyte subsets in the bone marrow, spleen, and blood were determined by flow cytometry shortly after or several days after R-SDS. These leukocyte changes were studied in two strains of mice with different stress susceptibility, C57BL/6N and BALB/c mice. R-SDS with both protocols similarly induced social avoidance in C57BL/6N mice. In the bone marrow, neutrophils and monocytes were increased, and T cells, B cells, NK cells, and dendritic cells were decreased with both protocols. In the blood, neutrophils and monocytes were increased with both protocols, whereas T cells, B cells, NK cells, and dendritic cells were decreased with one of these. Neutrophils and monocytes were also increased in the spleen. Changes in the bone marrow and increased levels of circulating neutrophils were maintained for 6 days after R-SDS. BALB/c mice showed greater social avoidance and increase in circulating neutrophils than C57BL/6N mice. In two strains of mice, chronic stress induced neutrophil mobilization and its maintenance. These effects were strain-related and may contribute to the pathology of mental illness. This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.4/issuetoc.

Intrastriatal injection of preformed alpha-synuclein fibrils alters central and peripheral immune cell profiles in non-transgenic mice

Parkinson’s disease (PD) is characterized by the accumulation of alpha-synuclein (α-syn) inclusions, the major component of Lewy bodies. Extracellular α-syn aggregates act as a damage-associated molecular pattern (DAMP) and the presence of autoantibodies against α-syn species in the cerebrospinal fluid and the serum of PD patients implicate the involvement of innate and adaptive immune responses. In non-transgenic (Tg) mice, intrastriatal injection of preformed fibril (PFF) α-syn results in widespread pathologic α-syn inclusions in the CNS. While the PFF model has been broadly utilized to study the mechanistic relationship between α-syn transmission and other neuropathological phenotypes, the immune phenotypes in this model are not clearly demonstrated. This study aimed to characterize the immune phenotypes during pathologic α-syn propagation by utilizing PFF α-syn–injected non-tg mice. Here, we showed that pathologic α-syn inclusions are prevalent in various brain regions and the gut at 5 months post injection (p.i.), preceding the degeneration of dopaminergic neurons in substantia nigra (SN). We discovered a distinct inflammatory response involving both activation of microglia and astrocytes and infiltration of B, CD4+ T, CD8+ T, and natural killer cells in the brain at 5 months p.i. Moreover, PFF α-syn–injected mice display significant alterations in the frequency and number of leukocyte subsets in the spleen and lymph nodes with minimum alterations in the blood. Our data provide primary evidence that intracerebral-initiated synucleinopathies in non-tg mice alter immune cell profiles both in the CNS and peripheral lymphoid organs. Furthermore, our data provides support for utilizing this mouse model to assess the mechanistic connection between immune responses and synuclein pathology.

Frailty is associated with elevated CRP trajectories and higher numbers of neutrophils and monocytes.

With aging, the human immune system undergoes several changes. The clinical relevance of these changes, however, is relatively unknown. We investigated immunological aspects of human aging in relation to frailty in the Doetinchem Cohort Study (DCS). We calculated a frailty index score based on 36 health parameters for each individual in the DCS with data obtained in the period 2008-2016. The frailty index was used to define three health groups ('healthy', 'intermediate', and 'frail'), stratified by age and sex. In a subcohort (n = 289, 60-85 years, selected by balanced random sampling per frailty group), we collected blood samples between October 2016 and March 2017 to determine absolute numbers of leukocyte subsets. In addition, cytomegalovirus serostatus was assessed. C-reactive protein (CRP) levels were longitudinally assessed in four consecutive plasma samples per individual. These samples had been previously collected (1993-2013) as part of the DCS at regular time intervals and spanning a period of >15 years. We observed higher numbers of myeloid derived neutrophils and monocytes in the frail group compared to the healthy group in both men and women, and, retrospectively, consistently higher CRP concentrations over a period of >15 years. An increase in CRP concentration with age was found in women, but not in men. Frailty was not associated with cytomegalovirus serostatus or with changes in lymphoid derived T-, B-, or NK-cell numbers. Frail elderly, compared to their age- and sex-matched peers, endure a chronic and stable low-grade inflammation, which is associated with a myeloid cell lineage expansion. These findings could help to monitor clinically significant immunological decline in the elderly.

Effect of ex vivo extracorporeal membrane oxygenation flow dynamics on immune response.

Extracorporeal membrane oxygenation is a life-saving support for heart and/or lung failure patients. Despite technological advancement, abnormal physiology persists and has been associated with subsequent adverse events. These include thrombosis, bleeding, systemic inflammatory response syndrome and infection. However, the underlying mechanisms are yet to be elucidated. We aimed to investigate whether the different flow dynamics of extracorporeal membrane oxygenation would alter immune responses, specifically the overall inflammatory response, leukocyte numbers and activation/adhesion surface antigen expression. An ex vivo model was used with human whole blood circulating at 37°C for 6 hours at high (4 L/minute) or low (1.5 L/minute) flow dynamics, with serial blood samples taken for analysis. During high flow, production of interleukin-1β (p < 0.0001), interleukin-6 (p = 0.0075), tumour necrosis factor-α (p = 0.0013), myeloperoxidase (p < 0.0001) and neutrophil elastase (p < 0.0001) were significantly elevated over time compared to low flow, in particular at 6 hours. While the remaining assessments exhibited minute changes between flow dynamics, a consistent trend of modulation in leukocyte subset numbers and phenotype was observed at 6 hours. We conclude that prolonged circulation at high flow triggers a prominent pro-inflammatory cytokine response and activates neutrophil granule release, but further research is needed to better characterize the effect of flow during extracorporeal membrane oxygenation.

Gender Dictates the Relationship between Serum Lipids and Leukocyte Counts in the National Health and Nutrition Examination Survey 1999⁻2004.

Dyslipidemias and leukocytosis are associated with cardiovascular disease and immune disorders. Mechanistic studies have shown lipoprotein metabolism to play a significant role in the regulation of atherosclerosis development and leukocyte activation, whereas lipid-lowering treatments have been shown to exert beneficial anti-inflammatory and immunomodulatory effects in clinical trials. However, the relationship between clinical markers of lipid metabolism and leukocyte counts has not been extensively evaluated at the population level. We aimed to determine whether clinical blood lipid measures are associated with leukocyte counts in the general U.S. population represented in the National Health and Nutrition Examination Survey (NHANES) 1999–2004, and whether differences exist between men and women (n = 5647). We observed a strong positive linear trend between serum triglycerides vs. blood lymphocyte and basophil counts in both men and women, whereas a positive trend between monocytes vs. triglycerides and lymphocytes vs. total cholesterol and LDL-cholesterol (LDL-C) was only detected in women. Conversely, HDL-C was inversely associated with a greater number of leukocyte subsets in men, whereas inverse trends between HDL-C vs. lymphocytes were observed in both men and women. In multiple regression models, a 10% increase in total cholesterol, LDL-C, and triglycerides was associated with a predicted 1.6%, 0.6%, and 1.4% increase in blood lymphocyte counts in women, respectively, whereas no relationship was observed in men. In both men and women, a 10% increase in triglycerides was additionally associated with higher lymphocyte, neutrophil, and basophil counts, whereas 10% increases in HDL-cholesterol were associated with significantly lower lymphocyte, neutrophil, eosinophil, and basophil counts in men, in addition to lower lymphocyte and monocyte counts in women. These findings suggest that clinical lipid markers may be used to predict blood leukocyte distributions, and that a gender-specific relationship exists between distinct classes of serum lipids and immune cell subsets.

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children

BackgroundChildhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children.Methods13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2) and leptin receptor expression on peripheral blood mononuclear cell subsets was performed.ResultsObese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI). The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also higher numbers of immature transition B-cells and intermediate CD14++CD16+ monocytes combined with lower total monocyte numbers, compared to controls. Furthermore, adiponectin receptor 1 expression on nonclassical CD14+CD16++ monocytes was consistently upregulated in obese children pre-intervention, compared to controls. However, none of the differences in leukocyte subset numbers and adipokine receptor expression profiles between obese children and controls remained significant after multiple testing correction.ConclusionsFirst, the distinct adipokine receptor profiles of circulating leukocyte subsets may partly explain the differential impact of adipokines on leukocyte subsets. Second, the similarities in adipokine receptor expression profiles between obese children and normal weight controls suggest that adipokine signaling in childhood obesity is primarily modulated by circulating adipokine levels, instead of adipokine receptor expression.

Effects of nongenetic factors on immune cell dynamics in early childhood: The Generation R Study

Numbers of blood leukocyte subsets are highly dynamic in childhood and differ greatly between subjects. Interindividual variation is only partly accounted for by genetic factors. We sought to determine which nongenetic factors affect the dynamics of innate leukocytes and naive and memory lymphocyte subsets. We performed 6-color flow cytometry and linear mixed-effects modeling to define the dynamics of 62 leukocytesubsets from birth to 6years of age in 1182 children, with 1 to 5 measurements per subject. Subsequently, we defined the effect of prenatal maternal lifestyle-related or immune-mediated determinants, birth characteristics, and bacterial/viral exposure-related determinants on leukocyte subset dynamics. Functionally similar leukocyte populations were grouped by using unbiased hierarchical clustering of patterns of age-related leukocyte dynamics. Innate leukocyte numbers were high at birth and predominantly affected by maternal low education level. Naive lymphocyte counts peaked around 1year, whereas most memory lymphocyte subsets more gradually increased during the first 4years of life. Dynamics of CD4+ T cells were predominantly associated with sex, birth characteristics, and persistent infections with cytomegalovirus (CMV) or EBV. CD8+ T cells were predominantly associated with CMV and EBV infections, and T-cell receptor γδ+ T cells were predominantly associated with premature rupture of membranes and CMV infection. B-cell subsets were predominantly associated with sex, breast-feeding, and Helicobacter pylori carriership. Our study identifies specific dynamic patterns of leukocyte subset numbers, as well as nongenetic determinants that affect these patterns, thereby providing new insights into the shaping of the childhood immune system.

Ablation of CD8α(+) dendritic cell mediated cross-presentation does not impact atherosclerosis in hyperlipidemic mice.

Clinical complications of atherosclerosis are almost exclusively linked to destabilization of the atherosclerotic plaque. Batf3-dependent dendritic cells specialize in cross-presentation of necrotic tissue-derived epitopes to directly activate cytolytic CD8 Tcells. The mature plaque (necrotic, containing dendritic cells and CD8 Tcells) could offer the ideal environment for cross-presentation, resulting in cytotoxic immunity and plaque destabilization. Ldlr−/− mice were transplanted with batf3−/− or wt bone marrow and put on a western type diet. Hematopoietic batf3 deficiency sharply decreased CD8α+ DC numbers in spleen and lymph nodes (>80%; P < 0,001). Concordantly, batf3−/− chimeras had a 75% reduction in OT-I cross-priming capacity in vivo. Batf3−/− chimeric mice did not show lower Tcell or other leukocyte subset numbers. Despite dampened cross-presentation capacity, batf3−/− chimeras had equal atherosclerosis burden in aortic arch and root. Likewise, batf3−/− chimeras and wt mice revealed no differences in parameters of plaque stability: plaque Tcell infiltration, cell death, collagen composition, and macrophage and vascular smooth muscle cell content were unchanged. These results show that CD8α+ DC loss in hyperlipidemic mice profoundly reduces cross-priming ability, nevertheless it does not influence lesion development. Taken together, we clearly demonstrate that CD8α+ DC-mediated cross-presentation does not significantly contribute to atherosclerotic plaque formation and stability.

Porcine circovirus type 2 DNA detection in the uterine tissue of gilts in relation to endometritis and the number of leukocyte subsets in the endometrium

The aim of the present study was to determine the prevalence of porcine circovirus type 2 (PCV2) DNA-positive uterine tissues in gilts in relation to endometritis and the number of leukocyte subsets in the endometrium. Uterine tissues from 32 Landrace × Yorkshire gilts were investigated. The gilts were classified according to both macroscopic and microscopic examination of the endometrium into three groups: normal (n = 13), acute endometritis (n = 11) and chronic endometritis (n = 8). Reproductive cycle was defined according to the ovarian appearance as follicular, luteal and inactive phases. PCV2 DNA was determined using polymerase chain reaction. The number of leukocyte subsets, including lymphocytes, neutrophils, eosinophils, macrophages and plasma cells, in the epithelium, subepithelium and glandular layer of the endometrium was carefully evaluated. It was found that PCV2 DNA was detected in 34.4 % (11/32) of the uteri. According to the lesion of endometritis, PCV2 DNA was detected in 53.9 % (7/13) of normal uteri, 36.4 % (4/11) of acute endometritis, and 0.0 % (0/8) of chronic endometritis (P = 0.038). The PCV2 DNA was detected in 55.0 % (5/9), 20.0 % (4/20) and 66.7 % (2/3) of tissues in follicular, luteal and inactive phases, respectively (P = 0.081). Leukocyte subsets in all tissue layers of the endometrium did not differ significantly between the uterine tissues with and without PCV2 DNA detection (P > 0.05). These findings indicate that PCV2 exists in the reproductive organs of the gilts. The PCV2 persistence can be a risk factor for transmission of the virus to negative sows and potentially cause reproductive failures. Nevertheless, the influence of PCV2 natural infection on the reproductive function of gilt still requires further investigation.

Quantitative &amp;amp; Qualitative Regulation of Peripheral Leukocyte Subsets plus Emotional Hormones by Floor Heating

A floor heating system is a traditionary famous in Asian countries, especially in North East of China and Korea. In this report, we intended to clarify the effect of floor hating system for accessing a peripheral leukocyte regulation in winter season by evidence-based manner more than VAS. We set up two different office rooms heat-controlled by floor heating and air-conditioned by ceiling electric heater. Both qualitative and quantitative approaches determine which are better therapeutic effective recipes for this trial. The elements were evaluated by the total number of peripheral leukocyte, granulocyte and lymphocyte ratio and lymphocyte subsets. For 5 days of working in each conditioned room, the volunteers were changed the room after one-week interval for cooling down. Floor heating system regulated total number of peripheral leukocyte. The mode of the action for the high number of the volunteer was down-regulated. On the contrary, in lower numbers it was up-regulated after the trial. The number of leukocyte subsets, granulocyte and lymphocyte were also regulated by dose dependent manner. Emotional hormone, adrenalin, cortisone and nor-adrenaline were down regulated by floor heating but dopamine was up-regulated by this heating system. The reversal results were obtained by the group who worked in the conditioned room by the system heating from ceiling.

Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study

BackgroundImmune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry.MethodsSix leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons.ResultsOptimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies.ConclusionsLocal performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.

Are Periodontal Bacterial Profiles and Placental Inflammatory Infiltrate in Pregnancy Related to Birth Outcomes?

The aim of this study is to determine whether periodontal clinical parameters, periodontal bacterial profiles, and inflammatory infiltrate in placental chorionic villi are associated with adverse pregnancy results. The authors designed an observational case-control study in 244 postpartum females: mothers with preterm/low-birth weight newborns (n = 91 cases) and mothers with full-term, normal-weight infants (n = 153 controls). Sociodemographic, gynecologic, and periodontal variables were gathered for all participants. Data on placental inflammatory infiltrate in biopsies from 68 cases and 65 controls and the gingival bacterial profile in mothers with periodontitis were gathered, detecting associations with bivariate analyses and constructing a multiple logistic regression model with the number of positive inflammatory cells as the dependent variable. Periodontal values were significantly worse in cases versus controls. Numbers of leukocyte subsets per square millimeters in maternal and fetal vascular spaces were similar between cases and controls. CD45 in maternal placental space was related to the presence of periodontitis (P = 0.029) but not to case or control group (P = 0.264). The anaerobic and commensal bacterial profile in mothers with periodontitis was similar between the groups. Periodontal disease was more severe and a periodontitis diagnosis more frequent in mothers with preterm or low-birth weight versus normal delivery. No differences in anaerobic or commensal bacterial profile were found between mothers with periodontitis in the two groups. Local placental factors, such as the nature of the inflammatory infiltrate and slightly higher expression of cyclooxygenase-2 in the females with these adverse pregnancy outcomes, may be related to a subclinical proinflammatory status that could contribute to triggering premature labor.

Stress-induced effects, which inhibit host defenses, alter leukocyte trafficking

Acute cold restraint stress (ACRS) has been reported to suppress host defenses against Listeria monocytogenes, and this suppression was mediated by beta1-adrenoceptors (β1-ARs). Although ACRS appears to inhibit mainly early innate immune defenses, interference with leukocyte chemotaxis and the involvement of β1-AR (or β2-AR) signaling had not been assessed. Thus, the link between sympathetic nerve stimulation, release of neurotransmitters, and changes in blood leukocyte profiles, including oxidative changes, following ACRS was evaluated. The numbers of leukocyte subsets in the blood were differentially affected by β1-ARs and β2-ARs following ACRS; CD3(+) (CD4 and CD8) T-cells were shown to be decreased following ACRS, and the T cell lymphopenia was mediated mainly through a β2-AR mechanism, while the decrease in CD19(+) B-cells was influenced through both β1- and β2-ARs, as assessed by pharmacological and genetic manipulations. In contrast to the ACRS-induced loss of circulating lymphocytes, the number of circulating neutrophils was increased (i.e., neutrophilia), and this neutrophilia was mediated through β1-ARs. The increase in circulating neutrophils was not due to an increase in serum chemokines promoting neutrophil emigration from the bone marrow; rather it was due to neutrophil release from the bone marrow through activation of a β1-AR pathway. There was no loss of glutathione in any of the leukocyte subsets suggesting that there was minimal oxidative stress; however, there was early production of nitric oxide and generation of some protein radicals. Premature egress of neutrophils from bone marrow is suggested to be due to norepinephrine induction of nitric oxide, which affects the early release of neutrophils from bone marrow and lessens host defenses.

Kinetics of Regulatory T Cells in the Ovalbumin Asthma Model in the Rat

Background: The kinetics of regulatory T cells (T_Reg) in allergic diseases such as asthma are only partly known. Methods: The asthma model in the Fischer rat with ovalbumin (OVA) sensitization and aerosol challenge was used. The relative and absolute numbers of leukocytes, lymphocytes and T_Reg subsets were determined by flow cytometry in the lung interstitium and draining bronchial lymph nodes at different time points after two challenges, and lung function was tested in parallel. Results: The progressive number of challenges resulted in increased relative and absolute numbers of lymphocytes and in particular of T_Reg. The T_Reg number was augmented with each aerosol challenge and was already significantly increased 6 h after the second challenge. The relative (%) and absolute numbers of CD4+ and CD8+ T_Reg and dendritic cells showed different kinetics after two challenges. The leukocyte numbers in the lung did not correlate with lung function. Conclusion: T_Reg increased surprisingly early after challenge in the lung tissue. Relative and absolute numbers of leukocyte subsets should always be calculated. The kinetics of different leukocyte subsets can only be determined when several time points are studied.

Determinants of the NF-κB response to acute psychosocial stress in humans

Previous research has shown that psychosocial stress is associated with an increased activity of the transcription factor nuclear factor-kappaB (NF-κB), a major inducer of inflammatory genes. While considerable individual variation has been noted, factors contributing to this variation have not been described so far.Therefore, 29 healthy participants (35.8±12yrs) were exposed to the Trier Social Stress Test. Blood was collected before and repeatedly afterward for determination of NF-κB activity, leukocyte subset numbers, cortisol, norepinephrine, and in vitro-stimulated IL-6 production. Additionally, age, sex, and ratings of perceived chronic and acute stress were assessed.Regression analyses revealed that older participants showed a lower NF-κB stress response compared to younger adults (β=−.42, p=.026). Higher NF-κB stress responses were associated with lower cortisol stress responses (β=−.37, p=.05), higher pre-stress IL-6 production (β=.38, p=.043), and high chronic in combination with low acute stress, or vice versa (β=−.61, p=.06). Norepinephrine and sex were not associated with NF-κB stress responses (all p⩾.13).In summary, the present study shows for the first time in human psychosocial stress the negative association of cortisol and NF-κB. This parallels results from in vitro studies. Our finding of lower NF-κB stress responses in older age and in people with high chronic and acute stress might be interpreted as an adaptive dampening of NF-κB activity. In the absence of longitudinal data, however, this interpretation remains speculative.

Selection for lean growth and food intake leads to correlated changes in innate immune traits in Large White pigs

Genetic selection is well established as a means of improving productivity in pigs, but the effects of continued selection for increased performance on immunity are not well understood, nor are genetic relationships between performance and immunity. This study compared differences in the levels of a range of immune traits between lines of Large White pigs divergently selected for a number of productivity traits. Selection lines compared were highv. low lean growth under restricted feeding (31 high linev. 10 controlv. 38 low line pigs), high v. low lean growth underad libitumfeeding (18 high line v. 10 controlv. 19 low line pigs), and highv. low food intake (24 high linev. 26 low line pigs). Immune traits measured were total white blood cell numbers (WBC), and the numbers of leukocyte subsets: neutrophils, monocytes, eosinophils, lymphocytes, CD4+cells, CD8α+cells, B cells, γδ T cells and CD11R1+Natural killer (NK) cells. CD4+, γδ T cells and CD11R1+cells were subdivided into subpopulations that were positive or negative for the CD8α marker, and conventional CD8αhigh+cytotoxic T cells were also determined. Pigs were tested underad libitumfeeding conditions from 14 to 24 weeks, and immune traits were assessed at ages 18 and 24 weeks. Line differences were estimated using residual maximum likelihood techniques. Consistent differences in immune trait levels were evident between pigs previously selected for high and low lean growth under restricted feeding: at age 24 weeks, high line pigs had higher basal levels of WBC (39·6v. 27·8×106cells per ml, s.e.d. 2·09, for highv. low line pigs) mainly explained by higher levels of lymphocytes (25·5v. 17·3×106cells per ml, s.e.d. 1·54, for highv. low line pigs) with increased numbers of CD8α+cells (8·19v. 5·15×106cells per ml, s.e.d. 0·14) and CD11R1+cells (5·23v. 2·46×106cells per ml, s.e.d. 0·43), predominantly the CD11R1+CD8α?subpopulation ((3·20v. 1·64×106cells per ml, s.e.d. 0·11). High line pigs also had increased numbers of monocytes (2·64v. 1·83×106cells per ml, s.e.d. 0·35). Similar results were obtained at age 18 weeks. There were no consistent differences between divergent lines in pigs selected for lean growth underad libitumfeeding or food intake. This is the first report to demonstrate that selection for some aspects of performance can influence WBC and leukocyte subset numbers in pigs.

Attenuation of T-lymphocyte demargination and adhesion molecule expression in response to moderate exercise in physically fit individuals

The effects of physical fitness on leukocyte demargination and cellular adhesion molecule (CAM) responses to moderate exercise were examined. We assessed leukocyte subsets and CAM expression before, immediately after, and 10 min after a 20-min treadmill exercise at 65-70% peak oxygen consumption in fit vs. nonfit individuals. Physical fitness was determined by peak oxygen consumption during a treadmill test. Catecholamine levels were determined by radioenzymatic assay, and enumeration of cells and detection of CAM expression were assessed by flow cytometry. As expected, exercise led to significant increases in numbers of leukocyte subsets, regardless of fitness level (P < 0.01). Values returned to near resting levels 10 min after exercise. More importantly, physically fit individuals showed attenuated responses to the moderate-exercise challenge in numbers of CD3(+), CD4(+), CD8(+), memory (CD45RO(+)) CD4, and naive (CD45RA(+)62L(+)) CD4 and CD8 lymphocytes. Postexercise human leukocyte antigen-DR absent memory CD4(+) cell numbers were also lower in fit subjects. Increases in CD62L-expressing CD4(+) and CD8(+) lymphocytes and CD11a- expressing lymphocytes after exercise were also attenuated in fit individuals compared with nonfit individuals (P < 0.05). Catecholamine levels increased to a similar extent (P < 0.01) in both fitness groups. The findings suggest that physical fitness attenuates demargination of selected lymphocyte subsets in response to moderate exercise. Although the differences in plasma catecholamine responses were not significant between the groups, a possible mediating role of the sympathetic system remains to be further investigated. Being physically fit may offset exaggerated immune cell responses to stress.

Twenty-five years of clinical flow cytometry: AIDS accelerated global instrument distribution.

As a cell biologist, one experiences some ambivalencewith an enigmatic sensation on the occasion of the silverjubilee of Clinical Cytometry. This significant milestone iscelebrated with ominous feelings because of the remark-able symbiosis that was forged between the birth of a newclinical diagnostic tool and the emergence of one of themost devastating infectious diseases of the 20th century. Itis less then 25 years ago that the first reports surfacedabout clusters of aggressive opportunistic infections ap-pearing mysteriously in young gay men in some metropol-itan areas in the United States (1,2). The causative agent ofthe epidemic was unknown, yet the disease was wellcharacterized within a very short time span as the ac-quired immunodeficiency syndrome (AIDS). The initialCDC definition of AIDS incorporated leukocyte subsetnumbers from peripheral blood. AIDS’ defining conditionincluded CD4 T-cell counts of 200 or less (3,4). Initially, inmost immunology laboratories, immunophenotyping wasperformed with fluorescent microcopy. But quickly, thechoice of assay method shifted to flow cytometry. As theworld-wide apocalyptic devastation began, cellular immu-nologists engaged in research had at their disposal anarsenal of reagents and instruments just waiting for anopportunity for qualitative assessment of immunodefi-ciency. Therefore, in a very short time, instruments thatcould count T-helper cells were in great demand. Theepidemic had profound impact throughout the Westernworld, but those initial human losses paled in comparisonwith the carnage AIDS delivered and still is delivering inresource-poor regions of the globe. It is estimated that in2003, over 40 million individuals live with AIDS; about10% of this population lives in South Africa.Most of the time, significant scientific progress occursin dull obscurity, but occasionally human curiosity pre-vails and the odd science story proceeds with significantfanfare. The discoverers of the structure of DNA did re-ceive immediate accolades. However, in most instances oftechnical breakthroughs in diagnostics, the assessment ofthe day falls short of appropriate recognition. Invariablythe press selects trivia to report as newsworthy science.For example, the postmortem analysis of the space shuttledisaster received phenomenal coverage. Polemical reportswere bombarding us about how scientists were able todemonstrate that a piece of the foam debris at a certainvelocity could have or could not have damaged criticalceramic tiles attached to the wing on that doomed andmost unfortunate space vehicle. At the same time, thispast July, a subcommittee of the International Union ofPure and Applied Chemists identified the 110th element.The new chart recognizes darmstadtium (Ds) as a uniqueelement. It was formally added to the Periodic Chart. Thislatter event received virtually no publicity, while the sig-nificance of the finding is relatively easy to convey to thepublic, as we all learned about the elements while takinghigh school chemistry. The low profile coverage that isoften associated with our discipline was altered forever 25years ago. Events in cytology during the early 1980s weregoing to unfold in a most uneventful fashion. However,with a newly discovered incredibly devastating lethal dis-ease on the horizon, the role of the underrated flowcytometer changed forever. Because reliable diagnosing ofimmunodeficiency was an essential requirement, clinicalcytometry was born. With flow, rapid diagnosis of AIDSwas possible as early as 1981 (1). Clinical immunopheno-typing was possible just a few years after the discovery ofthe hybridoma technology. It opened the possibility forthe production of mAbs with high affinity and avidity.Between 1978 and 1981, Milstein, Reinhertz, Schlossman,Goldstein, Janossy, and many others were diligently crank-ing out antibody-producing murine clones and were de-fining and assigning function-specific roles to markers onhuman T- and B-cells. Flow cytometry, a laser-based tech-nology, was photogenic and there were no serologicaltests available to feature. Therefore, flow cytometry prof-ited from seemingly spontaneous and persistent presscoverage. The virtuosity and veracity of the investigators’productivity a quarter-century ago expedited research ca-pacity in immunology beyond any expectations. Unfortu-nately, to date the regulatory role of various T-cell subsetshas not been fully resolved. The scholarly struggle to gainmore knowledge in this area, including oncology, hasadvanced, but it is far from over.Thereisalwaysasilverliningtoanyhumancatastrophe.The story of the AIDS pandemic is no exception. In thiscase, 25 years ago we had instrumentation to analyzeperipheral blood cells, but there was no apparent wide-scale clinical application beyond differentiating various

Stimulation of Bronchus-Associated Lymphoid Tissue in Rats by Repeated Inhalation of Aerosolized Lipopeptide MALP-2

Objective: Bronchus-associated lymphoid tissue (BALT) is a part of the integrated mucosal immune system. It may play an important functional role for antigen uptake and induction of specific immune reactions. The aim of this study was to investigate whether it is possible to induce or modulate BALT by the repetitive inhalation of the synthetic lipopeptide MALP-2. Methods: Female Lewis rats (245 ± 19 g) inhaled 25 µg of MALP-2 six times at intervals of 1 week. One week after the last inhalation, they were sacrificed. Cells of the bronchoalveolar lavage and the left lung were investigated by flow cytometry. The middle lobe of the right lung was embedded in paraffin. BALT was semiquantitatively measured in 15 serial cross sections per animal. Results: After repetitive inhalation of the diluent as well as MALP-2, BALT was found. The total area was increased after repetitive treatment with MALP-2. In addition, the preferential incidence of BALT was higher after MALP-2 application, in association with a bronchial diameter of 0.6–1 mm. The cellular analysis revealed no differences in the number of leukocyte subsets between the control and MALP-2 group. Conclusion: MALP-2 is a potent local stimulator and can be used to modulate BALT by repetitive inhalant treatment. The functional significance of enlarged or activated BALT has to be elucidated in future studies.