The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Redox dysregulation in schizophrenia : implication for oligodendrocyte maturation and structural connectivity

Schizophrenia, which results from an interaction between gene and environmental factors, is a psychiatric disorder characterized by reality distortion. The clinical symptoms, which are generally diagnosed in late adolescence or early adulthood, partly derive from altered brain connectivity especially in prefrontal cortex. Disruption of neuronal networks implies oligodendrocyte and myelin abnormalities in schizophrenia pathophysiology. The mechanisms of these impairments are still unclear. Converging evidences indicate a role of redox dysregulation, generated by an imbalance between pro-oxidants and antioxidant defense mechanisms, in the development of schizophrenia pathophysiology. In particular, genetic and biochemical data indicate impaired synthesis of glutathione, the main cellular antioxidant and redox regulator. As oligodendrocyte maturation is dependent on redox state, we evaluated whether abnormal redox control could contribute to oligodendrocyte and myelin impairments in schizophrenia. We found that glutathione in prefrontal cortex of early psychosis patients and control subjects positively correlated with white matter integrity. We then further explored the interplay between glutathione and myelin using a translational approach. Our data showed that in mice with genetically impaired glutathione synthesis, oligodendrocyte late maturation as well as myelination was delayed in the anterior cingulate cortex. Specifically, oligodendrocyte number and myelin levels were lowered at peripubertal age, coincident in time with the peak of myelin- related gene expression during normal brain development. These data suggest that early adolescence is a vulnerable developmental period during which an adequate redox control is required for oligodendrocyte maturation and active myelination process. Consistently, oxidative stress mediated by psychosocial stress also delayed myelination in peripubertal mice. At cellular levels, impaired glutathione synthesis altered oligodendrocyte development at several levels. Using oligodendrocyte progenitor cells cultures, our data showed that glutathione deficiency was associated with (i) cell cycle arrest and a reduction in oligodendrocyte proliferation, and (ii) an impairment in oligodendrocyte maturation. Abnormal oligodendrocyte proliferation was mediated by upregulation of Fyn kinase activity. Consistently, under oxidative stress conditions, we observed abnormal regulation of Fyn kinase in fibroblasts of patients deficient in glutathione synthesis. Together, our data support that a redox dysregulation due to glutathione deficit could underlie myelination impairment in schizophrenia, possibly mediated by dysregulated Fyn pathway. Better characterization of Fyn mechanisms would pave the way towards new drug targets. -- La schizophrénie est une maladie psychiatrique qui se définit par une distorsion de la perception de la réalité. Les symptômes cliniques sont généralement diagnostiqués durant l'adolescence ou au début de l'âge adulte et proviennent de troubles de la connectivité, principalement au niveau du cortex préfrontal. Les dysfonctionnements des réseaux neuronaux impliquent des anomalies au niveau des oligodendrocytes et de la myéline dans la pathophysiologie de la schizophrénie. Les mécanismes responsables des ces altérations restent encore mal compris. Dans le développement de la schizophrénie, des évidences mettent en avant un rôle de la dérégulation rédox, traduit par un déséquilibre entre facteurs pro-oxydants et défenses antioxydantes. Des données génétiques et biochimiques indiquent notamment un défaut de la synthèse du glutathion, le principal antioxydant et rédox régulateur des cellules. Etant donné que la maturation des oligodendrocytes est dépendante de l'état rédox, nous avons regardé si une dérégulation rédox contribue aux anomalies de la myéline dans le cadre de la schizophrénie. Dans le cortex préfrontal des sujets contrôles et des patients en phase précoce de psychose, nous avons montré que le glutathion était positivement associé à l'intégrité de matière blanche. Afin d'explorer plus en détail la relation entre le glutathion et la myéline, nous avons mené une étude translationnelle. Nos résultats ont montré que des souris ayant un déficit de la synthèse du glutathion présentaient un retard dans les processus de maturation des oligodendrocytes et de la myélinisation dans le cortex cingulaire antérieure. Plus précisément, le nombre d'oligodendrocytes et le taux de myéline étaient uniquement diminués durant la période péripubertaire. Cette même période correspond au pic de l'expression des gènes en lien avec la myéline. Ces données soulignent le fait que l'adolescence est une période du développement particulièrement sensible durant laquelle un contrôle adéquat de l'état rédox est nécessaire aux processus de maturation des oligodendrocytes et de myélinisation. Ceci est en accord avec la diminution de myéline observée suite à un stress oxydatif généré par un stress psychosocial. Au niveau cellulaire, un déficit du glutathion affecte le développement des oligodendrocytes à différents stades. En effet, dans des cultures de progéniteurs d'oligodendrocytes, nos résultats montrent qu'une réduction du taux de glutathion était associée à (i) un arrêt du cycle cellulaire ainsi qu'une diminution de la prolifération des oligodendrocytes, et à (ii) des dysfonctionnements de la maturation des oligodendrocytes. Par ailleurs, au niveau moléculaire, les perturbations de la prolifération étaient générées par une augmentation de l'activité de la kinase Fyn. Ceci est en accord avec la dérégulation de Fyn observée dans les fibroblastes de patients ayant une déficience en synthèse du glutathion en condition de stress oxydatif. Les résultats de cette thèse soulignent qu'une dérégulation rédox induite par un déficit en glutathion peut contribuer aux anomalies des oligodendrocytes et de la myéline via le dysfonctionnement des voies de signalisation Fyn. Une recherche plus avancée de l'implication de Fyn dans la maladie pourrait ouvrir la voie à de nouvelles cibles thérapeutiques.

Using flow cytometry for in situ monitoring of antimicrobial compound production in the biocontrol bacterium Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens strain CHA0 is able to protect plants against a variety of pathogens, notably by producing the two antimicrobial compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the expression of these compounds is affected by many biotic factors, such as fungal pathogens, rhizosphere bacteria as well as plant species. Therefore, the influence of some plant phenolic compounds on the expression of DAPG and PLT biosynthetic genes has been tested using GFP-based reporter, monitored by standard fluometry and flow cytometry. In situ experiments were also performed with cucumber plants. We found that several plant metabolites such as IAA and umbelliferone are able to modify significantly the expression of DAPG and PLT. The use of flow cytometry with autofluorescents proteins seems to be a promising method to study rhizobacteria-plant interactions.

Artificial neural network study of whole-cell bacterial bioreporter response determined using fluorescence flow cytometry.

Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease.

Introduction. Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia. Methods. We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q10 levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX™ and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker™ Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells. Results. We found reduced levels of coenzyme Q10, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy. Conclusions. These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.

Antigenic heterogeneity of clones and subclones from human melanoma cell lines demonstrated by a panel of monoclonal antibodies and flow microfluorometry analysis.

Cells from two melanoma cell lines, Me43 and GLL-19, were cloned in methylcellulose cultures and 20 randomly selected colonies from each line were picked up by micromanipulation, expanded in liquid cultures, and considered as clones of the original cell lines. The antigenic cell surface phenotype of these clones defined by panel of 12 monoclonal antibodies (MAb) was analyzed by flow microfluorometry (FMF) using a fluorescence-activated cell sorter (FACS II) and compared with the known stable phenotype of the parent cell line. The antibody panel consisted of eight MAb against melanoma-associated antigens, two MAb against monomorphic determinants of HLA-DR (la) and HLA-ABC, respectively, one MAb against the common acute lymphoblastic leukemia antigen (CALLA) and one MAb against carcinoembryonic antigen used as control. A remarkable heterogeneity in terms of qualitative and quantitative expression of the cell surface antigens studied was observed among and within the different clones. The single-cell origin of the clones was assessed by comparing the clonogenic cell frequency, determined by limiting dilutions in microculture plates, with the cloning efficiency observed in Petri dishes. Both techniques using methylcellulose medium gave the same percentages of growing colonies. Cells from four Me43 clones were recloned in methylcellulose and the phenotype of five randomly selected subclones from each clone was analysed using the same panel of monoclonal antibodies. Each subclone also displayed heterogeneity with individual phenotypes different from that of the original clone and from the parental Me43 cell line. The antigen expression by individual cells in situ within clones was analyzed on frozen sections from colonies using the same panel of MAb and a biotin-avidin immunoperoxidase method. The results confirmed the marked heterogeneity of antigen expression within and among colonies, as indicated by the FMF analysis.

The return of increased blood pressure after discontinuation of antihypertensive treatment is associated with an impaired post-ischemic skin blood flow response.

OBJECTIVE: To assess the post-ischemic skin blood flow response after withdrawal of antihypertensive therapy in hypertensive patients with normal blood pressure during treatment. DESIGN AND METHODS: Twenty hypertensive patients (group A) with a normal clinic blood pressure (<140/ 90 mmHg) receiving antihypertensive treatment (any monotherapy; one pill per day for at least 6 months) had their treatment discontinued. Before medication withdrawal and 2, 4, 12 and 24 weeks thereafter, the following measurements were made: clinic blood pressure, home blood pressure (three times per week, morning and evening) and skin blood flow response to a 5 min forearm arterial occlusion (using laser Doppler flowmetry). The patients were asked to perform an ambulatory blood pressure recording at any time if home blood pressure was > or =160/95 mmHg on two consecutive days, and treatment was initiated again, after determination of the skin hyperemic response, if daytime ambulatory blood pressure was > or =140/90 mmHg. The same studies were performed in 20 additional hypertensive individuals in whom antihypertensive treatment was not withdrawn (group B). The allocation of patients to groups A and B was random. RESULTS: The data fom 18 patients in group A who adhered strictly to the procedure were available for analysis. Seven of them had to start treatment again within the first 4 weeks of follow-up; four additional patients started treatment again during the next 8 weeks (group A1). The seven other patients remained untreated (group A2). The skin hyperemic response decreased significantly in patients in group A1 and returned to baseline values at the end of the study, when there were again receiving antihypertensive treatment. In patients in group A2 a significant attenuation of the hyperemic response was also observed. This impaired response was present even at the end of the 6 month follow-up, at which time the patients were still untreated but exhibited a significantly greater blood pressure than before drug discontinuation. The hyperemic response of patients who did not stop treatment (group B) did not change during the course of the study. CONCLUSIONS: Our findings show a decrease in the postischemic skin blood flow response after withdrawal of antihypertensive treatment in hypertensive patients. This impaired response may be due to the development of endothelial dysfunction, vascular remodeling, or both, and might contribute to the return of blood pressure to hypertensive values after withdrawal of antihypertensive therapy.

Coronary vasomotor control in obesity and morbid obesity: contrasting flow responses with endocannabinoids, leptin, and inflammation.

OBJECTIVES: This study sought to investigate abnormalities in coronary circulatory function in 2 different disease entities of obese (OB) and morbidly obese (MOB) individuals and to evaluate whether these would differ in severity with different profiles of endocannabinoids, leptin, and C-reactive protein (CRP) plasma levels. BACKGROUND: There is increasing evidence that altered plasma levels of endocannabinoids, leptin, and CRP may affect coronary circulatory function in OB and MOB. METHODS: Myocardial blood flow (MBF) responses to cold pressor test from rest and during pharmacologically induced hyperemia were measured with N-13 ammonia positron emission tomography/computed tomography. Study participants (n = 111) were divided into 4 groups based on their body mass index (BMI) (kg/m(2)): 1) control group (BMI: 20 to 24.9, n = 30); 2) overweight group (BMI: 25 to 29.9, n = 31), 3) OB group (BMI: 30 to 39.9, n = 25); and 4) MOB group (BMI ≥40, n = 25). RESULTS: The cold pressor test-induced change in endothelium-related MBF response (ΔMBF) progressively declined in overweight and OB groups when compared with the control group [median: 0.19 (interquartile range [IQR] 0.08, 0.27) and 0.11 (0.03, 0.17) vs. 0.27 (0.23, 0.38) ml/g/min; p ≤ 0.01, respectively], whereas it did not differ significantly between OB and MOB groups [median: 0.11 (IQR: 0.03, 0.17) and 0.09 (-0.01, 0.19) ml/g/min; p = 0.93]. Compared with control subjects, hyperemic MBF subjects comparably declined in the overweight, OB, and MOB groups [median: 2.40 (IQR 1.92, 2.63) vs. 1.94 (1.65, 2.30), 2.05 (1.67, 2.38), and 2.14 (1.78, 2.76) ml/g/min; p ≤ 0.05, respectively]. In OB individuals, ΔMBF was inversely correlated with increase in endocannabinoid anandamide (r = -0.45, p = 0.044), but not with leptin (r = -0.02, p = 0.946) or with CRP (r = -0.33, p = 0.168). Conversely, there was a significant and positive correlation among ΔMBF and elevated leptin (r = 0.43, p = 0.031) and CRP (r = 0.55, p = 0.006), respectively, in MOB individuals that was not observed for endocannabinoid anandamide (r = 0.07, p = 0.740). CONCLUSIONS: Contrasting associations of altered coronary endothelial function with increases in endocannabinoid anandamide, leptin, and CRP plasma levels identify and characterize OB and MOB as different disease entities affecting coronary circulatory function.

Validation d'une nouvelle grille de codage de la régulation émotionnelle avec le Laboratory Temperament Assessment Battery (Lab-TAB)

L'objectif de cette étude est de valider une nouvelle grille de codage pour le Lab-TAB (Goldsmith et Rothbart, 1999). Cette grille mesure l'intensité émotionnelle et les comportements de régulation chez l'enfant âgé de six mois. L'accord interjuge se révèle satisfaisant. Les analyses factorielles ont mis en évidence trois facteurs - attention, niveau d'activité et détournement actif - retrouvés dans la littérature comme stratégies de régulation émotionnelle. Les analyses de consistance interne démontrent une bonne homogénéité de ces facteurs. La validité externe se révèle satisfaisante aux niveaux concourant et prédictif. Enfin, cet outil de codage permet de distinguer les prématurés et les nés à terme. L'ensemble de ces résultats indiquent que l'outil élaboré présente de bonnes qualités psychométriques.

Genetic clusters and sex-biased gene flow in a unicolonial Formica ant.

BACKGROUND: Animal societies are diverse, ranging from small family-based groups to extraordinarily large social networks in which many unrelated individuals interact. At the extreme of this continuum, some ant species form unicolonial populations in which workers and queens can move among multiple interconnected nests without eliciting aggression. Although unicoloniality has been mostly studied in invasive ants, it also occurs in some native non-invasive species. Unicoloniality is commonly associated with very high queen number, which may result in levels of relatedness among nestmates being so low as to raise the question of the maintenance of altruism by kin selection in such systems. However, the actual relatedness among cooperating individuals critically depends on effective dispersal and the ensuing pattern of genetic structuring. In order to better understand the evolution of unicoloniality in native non-invasive ants, we investigated the fine-scale population genetic structure and gene flow in three unicolonial populations of the wood ant F. paralugubris. RESULTS: The analysis of geo-referenced microsatellite genotypes and mitochondrial haplotypes revealed the presence of cryptic clusters of genetically-differentiated nests in the three populations of F. paralugubris. Because of this spatial genetic heterogeneity, members of the same clusters were moderately but significantly related. The comparison of nuclear (microsatellite) and mitochondrial differentiation indicated that effective gene flow was male-biased in all populations. CONCLUSION: The three unicolonial populations exhibited male-biased and mostly local gene flow. The high number of queens per nest, exchanges among neighbouring nests and restricted long-distance gene flow resulted in large clusters of genetically similar nests. The positive relatedness among clustermates suggests that kin selection may still contribute to the maintenance of altruism in unicolonial populations if competition occurs among clusters.

Chromosomal rearrangements and gene flow over time in an inter-specific hybrid zone of the Sorex araneus group.

Most hybrid zones have existed for hundreds or thousands of years but have generally been observed for only a short time period. Studies extending over periods long enough to track evolutionary changes in the zones or assess the ultimate outcome of hybridization are scarce. Here, we describe the evolution over time of the level of genetic isolation between two karyotypically different species of shrews (Sorex araneus and Sorex antinorii) at a hybrid zone located in the Swiss Alps. We first evaluated hybrid zone movement by contrasting patterns of gene flow and changes in cline parameters (centre and width) using 24 microsatellite loci, between two periods separated by 10 years apart. Additionally, we tested the role of chromosomal rearrangements on gene flow by analysing microsatellite loci located on both rearranged and common chromosomes to both species. We did not detect any movement of the hybrid zone during the period analysed, suggesting that the zone is a typical tension zone. However, the gene flow was significantly lower among the rearranged than the common chromosomes for the second period, whereas the difference was only marginally significant for the first period. This further supports the role of chromosomal rearrangements on gene flow between these taxa.