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Peripheral Blood Leucocytes (peripheral + blood_leucocyte)
Selected AbstractsInvolvement of 15-lipoxygenase and prostaglandin EP receptors in aspirin-triggered 15-hydroxyeicosatetraenoic acid generation in aspirin-sensitive asthmaticsCLINICAL & EXPERIMENTAL ALLERGY, Issue 7 2008M. Jedrzejczak-Czechowicz Summary Background The mechanism of aspirin (acetylsalicylic acid: ASA) hypersensitivity in asthmatic patients is related to arachidonic acid metabolism abnormalities, and specific triggering by ASA of 15-hydroxyeicosatetraenoic acid (15-HETE) generation was observed in leucocytes from aspirin-sensitive (AS) but not from aspirin-tolerant (AT) asthmatics. Objective The aim of this study was to identify the enzymatic pathway involved in ASA-induced 15-HETE generation in AS asthmatics and to assess the regulatory role of prostaglandin EP receptors. Methods Peripheral blood leucocytes (PBLs) were isolated from AS (n=18) and AT (n=20) asthmatics and challenged with ASA, with and without pre-incubation with caffeic acid (CA) [15-lipoxygenase (15-LO) inhibitor] or prostaglandin receptor non-specific (misoprostol, sulprostone) and specific EP1,4 receptors agonists. Eicosanoids were measured in supernatants using specific immunoassays. Results Aspirin triggered 15-HETE generation in PBLs of AS asthmatics (mean increase 292%) but not in AT asthmatics and inhibited prostaglandin2 (PGE2) generation in both groups of patients to the same degree. Leucocytes from AS patients produced less PGE2, both before and after ASA incubation. Pre-incubation of PBLs with CA decreased basal 15-HETE production in all patients and completely inhibited ASA-induced 15-HETE generation in AS asthmatics. CA did not change basal PGE2 production but enhanced induced by ASA inhibition of PGE2. Non-specific agonists of EP receptors (misoprostol and sulprostone) did not affect basal 15-HETE production but inhibited in a dose-dependent manner the ASA-induced increase of 15-HETE generation in AS asthmatics. On the contrary, in AT asthmatics, pre-incubation of PBLs with misoprostol or sulprostone resulted in a significant increase in 15-HETE generation after addition of ASA (200 ,m). EP1,3 receptor agonists inhibited (range 72,94%) the ASA-induced 15-HETE production significantly. Conclusion Our study demonstrated that ASA-triggered 15-HETE generation involves the activation of 15-LO and is modulated by prostaglandin EP1,3 receptors. The relevance of these observations to the mechanism of in vivo ASA-induced asthmatic attack remains to be established. [source] Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface-only versus intracellular antigens with the Fix & PermÔ reagent,,§CYTOMETRY, Issue 1 2010Elaine Sobral da Costa Abstract Staining for intracellular markers with the Fix & PermÔ reagent is associated with variations in the scatter properties of leucocytes, limiting automated analysis of flow cytometry (FCM) data. Here, we investigated those variables significantly contributing to changes in the light scatter, autofluorescence, and bcl2 staining characteristics of peripheral blood (PB) leucocytes, after fixation with Fix & PermTM. Our major aim was to evaluate a new mathematical approach for automated harmonization of FCM data from datafiles corresponding to aliquots of a sample treated with cell-surface-only versus Fix & Perm intracellular staining techniques. Overall, neither the anticoagulant used nor sample storage for <24 h showed significant impact on the light scatter and fluorescence properties of PB leucocytes; similarly, the duration of the fixation period (once >15 min were used) had a minimum impact on the FCM properties of PB leucocytes. Conversely, changes in cell/protein concentrations and the fixative/sample (vol/vol) ratio had a clear impact on the light scatter features of some populations of leucocytes. Accordingly, lower cell/protein concentrations were associated with lower scatter values, particularly for the neutrophils. Such changes could be partially corrected through the use of higher fixative to sample volume ratios. Despite the variable changes detected between aliquots of the same sample treated with cell surface-only versus intracellular staining procedures, the new mathematical approach here proposed and evaluated for automated harmonization of common parameters in both datafiles, could correct the FCM profiles of leucocytes derived from cells undergoing conventional fixation/permeabilization procedures, and made them indistinguishable from those corresponding to aliquots of the same sample treated with cell-surface-only staining techniques. © 2009 Clinical Cytometry Society [source] Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsivenessIMMUNOLOGY, Issue 4 2007M. Maximina Bertha Moreno-Altamirano Summary Monocytes constitute 5,10% of total human peripheral blood leucocytes and remain in circulation for several days before replenishing the tissue macrophage populations. Monocytes display heterogeneity in size, granularity and nuclear morphology, and in the expression of cell membrane molecules, such as CD14, CD16, CD32, CD64, major histocompatibility complex class II, CCR2, CCR5, among others. This has led to the suggestion that individual monocyte/macrophage populations have specialized functions within their microenvironments. This study provides evidence for the occurrence of two peripheral blood monocyte subpopulations on the basis of their differential expression of GM1, a sphingolipid found mostly in lipid rafts, a CD14+ GM1low population and a CD14+ GM1high population comprising about 97·5% and 2·5% of total CD14+ cells, respectively. GM1 expression correlates with functional differences in terms of endocytic activity, susceptibility to mycobacterial infection, and response to lipopolysaccharide (LPS) (modulation of Toll-like receptor-4 expression). CD14+ GM1low cells proved to be less endocytic and more responsive to LPS, whereas CD14+ GM1high cells are more endocytic and less responsive to LPS. In addition, during monocyte to macrophage differentiation in vitro, the percentage of CD14+ GM1high cells increases from about 2·5% at day 1 to more than 50% at day 7 of culture. These results suggest that GM1low and GM1high monocytes in peripheral blood, represent either different stages of maturation or different subsets with specialized activities. The expression of CD16 on GM1high favours the first possibility and, on the other hand that up-regulation of GM1 expression and probably lipid rafts function is involved in the monocyte to macrophage differentiation process. [source] The preparation of periapical lesions for flow cytometryINTERNATIONAL ENDODONTIC JOURNAL, Issue 2 2000K. Fernando Aim To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry. Methodology PA lesions were mechanically agitated, with and without proteolysis. This was with either 0.2% collagenase alone, or in combination with 0.02% DNA-ase in serial incubations until all tissue was digested. The efficacy of each method was assessed by counting total cell yield and cell viability. Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment. Results Disaggregation of PA lesions was superior if collagenase was present, but cell clumping was problematic unless the DNA-ase was also added, and serial digestion with this combination produced optimal cell yield and viability. Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80%. Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6,10%) CD45RB + cells. Conclusions Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required. [source] Investigation into possible DNA damaging effects of ultrasound in occupationally exposed medical personnel , the alkaline comet assay studyJOURNAL OF APPLIED TOXICOLOGY, Issue 3 2005Verica Garaj-Vrhovac Abstract In the present paper the possible DNA damaging effects of ultrasound in occupationally exposed medical personnel were investigated using the alkaline comet assay. The extent of DNA migration in peripheral blood leucocytes was measured. Parameters of the comet assay were studied in 30 medical workers occupationally exposed to ultrasound and in 30 corresponding unexposed control subjects. It was found that the subjects who were occupationally exposed to ultrasound for various periods of time showed a highly significant increase in levels of DNA damage compared with the control. The results obtained have confirmed the usefulness of the alkaline comet assay as a sensitive biodosimetric method, reflecting the current level of DNA damage and[sol ]or repair in peripheral blood leucocytes of ultrasound-exposed subjects. In spite of their limitations, the results of the present investigation indicate that individuals occupationally exposed to ultrasound may experience an increased genotoxic risk, emphasizing the need for more research into the nature and extent of the biological consequences to medical personnel working with ultrasonic equipment. Copyright © 2005 John Wiley & Sons, Ltd. [source] Alternative splicing of cyclooxygenase-1 gene: altered expression in leucocytes from patients with bronchial asthma and association with aspirin-induced 15-HETE releaseALLERGY, Issue 6 2007M. L. Kowalski Background:, Cyclooxygenase-1 (COX-1) is a key enzyme involved in generation of prostanoids, important mediators and modulators of asthmatic inflammation. In a subpopulation of aspirin-sensitive asthmatics (ASA) inhibition of COX-1 by nonsteroidal anti-inflammatory drugs results in activation of inflammatory cells and development of symptoms. Alternatively spliced variants of COX-1 lacking 111 bp from exon 9 were described previously but have never been identified in human leucocytes peripheral blood leucocytes (PBL) or upper airway epithelial cells. We aimed to assess the expression of spliced variants of COX-1 mRNA in PBLs from patients with asthma and in healthy subjects (HS) referring the expression to patients characteristics (including ASA-sensitivity) and to aspirin-triggered 15-hydroxyeicosatetraenoic acid (15-HETE) generation. Methods:, The study included 30 patients with ASA, 30 patients with aspirin-tolerant asthma (ATA) and 30 HS serving as controls. Nasal polyps for epithelial cell cultures were obtained from 10 patients with chronic rhinosinusitis. Expression of full length and spliced variants of COX-1 enzyme was detected by RT-PCR and presented as the ratio of full-length COX-1 to alternatively spliced COX-1 mRNA [COX-1 alternative splicing index (COX-1 AS index)]. Release of eicosanoids (PGE2 and 15-HETE) by PBLs was measured with enzyme immunoassay. Results:, In both PBLs and airway epithelial cells the expression of full-length product prevailed over spliced variants of COX-1 enzyme. Cyclooxygenase-1 AS index was significantly lower in asthmatics as compared to HS (1.96 ± 0.71 vs 2.41 ± 0.99, P < 0.05) indicating the relatively higher expression of the alternative transcript in asthmatic patients. Cyclooxygenase-1 AS index was not different between ASA and ATA groups (mean 1.90 ± 0.66 vs 2.02 ± 0.76, respectively, P = 0.39). There was no significant association between COX-1 AS index and mean daily dose of inhaled glucocorticosteroids or pulmonary function tests (FEV1, FVC) but in ASA group a weak correlation with daily dose of oral glucocorticosteroids was found (r = 0.39; P = 0.03). In ASA patients there was a significant positive correlation between the COX-1 AS index and the percentage of aspirin-triggered increase in 15-HETE generation (r = 0.51; P < 0.03). Conclusions:, Alternatively spliced variants of COX-1 mRNA are differently expressed in patients with bronchial asthma and may be associated with aspirin-triggered 15-HETE generation. [source] Effects of substitution of dietary fish oil with a blend of vegetable oils on liver and peripheral blood leucocyte fatty acid composition, plasma prostaglandin E2 and immune parameters in three strains of Atlantic salmon (Salmo salar)AQUACULTURE NUTRITION, Issue 6 2009I.K. PETROPOULOS Abstract Duplicate groups of three genetic strains of Atlantic salmon smolts were cultured on diets containing either fish oil (FO) or a blend of vegetable oils (VO). Fatty acid compositions of liver and peripheral blood leucocytes were compared. The effect of different strains and diets on innate immune parameters and plasma prostaglandin E2 were also measured. Two strains were selected as being either ,fat' or ,lean' in terms of muscle adiposity. The third strain was a commercial stock (MH). Total replacement of dietary FO with VO resulted in reduced docosahexaenoic (DHA; 22:6n -3) and eicosapentaenoic acids (EPA; 20:5n -3) in liver, while oleic (18:1n -9), linoleic (18:2n -6) and ,-linolenic (18:3n -3) acids were all increased in VO-fed fish. Fatty acid compositions of blood leucocytes showed similar changes. Evaluation of innate immune function showed that in the fat strain, circulating leucocytes were significantly lower in VO fish. The lean strain also had significantly higher serum lysozyme activity than MH fish. Reduced haematocrit was seen in VO lean fish compared with FO lean fish. This study provides evidence of strain-induced differences in liver and leucocyte fatty acid compositions and innate immunity in Atlantic salmon fed either FO- or VO-based diets. [source] Effect of plant active compounds on immune response and disease resistance in Cirrhina mrigala infected with fungal fish pathogen, Aphanomyces invadansAQUACULTURE RESEARCH, Issue 10 2009Ramasamy Harikrishnan Abstract The antibacterial activity of individual and mixed medicinal plant compounds, azadirachtin (Az), camphor (Ca) and curcumin (Cu), was tested at 100, 200, 300, 400, 500, 600 and 700 ppm (mg L,1) against fungal fish pathogen, Aphanomyces invadans, in vitro. At the lower concentrations between 100 and 300 ppm, the mixture of the tri-herbal (Az+Ca+Cu) compound yielded a higher (P>0.05) zone of inhibition (ZI) of 7 mm than the positive control; the maximum ZI values (8,15 mm) were realized between 400 and 700 ppm (P<0.05). At the lowest minimum inhibitory concentration (MIC) the tri-herbal compound (100 ppm) yielded 13 colony-forming units; hence, this dose can be effectively used at the lowest concentration of 100 mg L,1 to ward off the growth of A. invadans in vitro. In Cirrhina mrigala, intramuscular administration (100 ,L) of the selected doses of 100, 400 and 700 ppm (mg L,1) significantly enhanced (P<0.05) the serum lysozyme activity (Ly), production of reactive oxygen species and reactive nitrogen species (RNS or NO) by peripheral blood leucocytes on the 10th, 20th and 30th day. A priori administration of the compound in the fish (100 ppm on 30th day) decreased the percentage mortality when challenged with the pathogen while in the untreated group the mortality increased (P<0.05). This study indicates that intramuscular administration of the tri-herbal compound Az+Ca+Cu at a concentration of 100 ppm could augment the immune response in C. mrigala against A. invadans. [source] A variant TGFBI corneal dystrophy from G623D mutation with an unusual amyloidogenic phenotypeACTA OPHTHALMOLOGICA, Issue 2009HT AGOSTINI Purpose To present a unique corneal dystrophy never before described in a German family carrying the Gly623Asp mutation of the TGFBI gene with late clinical onset. Methods Clinical documentation and isolation of genomic DNA from peripheral blood leucocytes were obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were sequenced. 5 corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against Keratoepithelin (KE) 2 and 15. Results Specimens showed changes in Bowman's layer and the adjacent stroma. Congo red-positive amyloid deposits were found within the epithelium in one cornea, in Bowman's layer and in the anterior stroma of all specimens, also showing KE2- but not KE15-immunostaining. EM revealed deposits located in the anterior stroma and Bowman's layer and the basal area of some epithelial cells. These areas were strongly Alcian blue-positive but negative in the Masson-Trichrom-stain. Only affected patients had a heterozygous missense mutation in exon 14 of the TGFBI gene (G->A transition at nucleotide 1915) with the change Gly623Asp in the keratoepithelin protein. Conclusion In contrast to the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al., our cases presented with Salzmann nodular degeneration like clinical features with KE2 positive amyloid. The reason for this now "meeting the expectation histological phenotype" is unclear. The histological findings emphasize that this is a unique corneal dystrophy which shares no clinical characteristics with Reis-Bücklers dystrophy and should be treated as a distinct entity. [source] Levels of cysteinyl leukotriene receptor mRNA in human peripheral leucocytes: significantly higher expression of cysteinyl leukotriene receptor 2 mRNA in eosinophilsCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2001H. Mita Background Cysteinyl leukotrienes (CysLTs) have been implicated as important contributors in the pathophysiology of asthma and their biological effects are mediated by at least two distinct G-protein-coupled receptors. cDNA sequences of cysteinyl leukotriene receptor 1 (CysLTR1) and cysteinyl leukotriene receptor 2 (CysLTR2) have recently been elucidated. Objectives Our aim is to explore gene expression and the comparative expression of CysLTR1 mRNA and CysLTR2 mRNA in human peripheral blood leucocytes. Methods Gene expression of CysLTR1 and CysLTR2 mRNAs in human peripheral blood eosinophils, neutrophils, monocytes and T lymphocytes has been measured by competitive reverse transcription-polymerase chain reactions using RNA or DNA competitors. Results(a) When cellular levels of CysLTR1 mRNA were normalized to those of G3PDH mRNA, the relative concentration of CysLTR1 mRNA in eosinophils (43.8 ± 37.2, n = 29) was significantly higher than that in neutrophils (18.7 ± 23.3, n = 11), monocytes (0.93 ± 1.1, n = 10) and T lymphocytes (3.4 ± 2.4, n = 11). (b) When measured using each DNA competitor, mRNAs for both types of CysLTR coexisted in each type of leucocyte. The ratio of CysLTR1 mRNA to CysLTR2 mRNA was significantly lower in eosinophils (0.65 ± 0.42, n = 12) than in neutrophils (6.9 ± 4.9, n = 12), monocytes (1.8 ± 0.9, n = 10) and T lymphocytes (4.5 ± 5.7, n = 10). (c) Human umbilical vein endothelial cells expressed CysLTR2 mRNA, but not CysLTR1 mRNA. Conclusion These studies reveal that CysLTR1 mRNA and, in particular, CysLTR2 mRNA are abundantly expressed at high levels in eosinophils, raising the possibility that CysLTR2 may have an important physiological role in eosinophils and a CysLTR2 antagonist may be a good target for preventing signal transduction by CysLTs in eosinophils. [source] HLA-A and HLA-B transcription decrease with ageing in peripheral blood leucocytesCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2001C. Le Morvan Immunosenescence involves modifications of humoral and cellular immunity. In a previous study, we have shown a locus-dependent reduction of HLA class-I cell surface expression on peripheral lymphocytes and monocytes with advancing age. Here we report the quantitative analysis of HLA-A and -B transcripts from PBL of 54 healthy subjects aged 21,90 years. Using a competitive RT-PCR method, we observed a significant decrease of HLA-A (P < 0·0001) and -B (P = 0·0025) mRNA contents with increasing age. Secondly, to investigate this locus-dependent alteration of HLA class-I transcription, we performed EMSA using nuclear extracts from PBL of five young (24,31-year-old) and 5 elderly (58,69 years old) donors with locus A and B sequences of the Enh-A as probes. No qualitative variation of EMSA profiles appeared between the two groups of donors with 6 and 4 bandshift for the locus A and B, respectively. Quantitatively, we observed a significant increase of B4 intensity in the elderly group compared to the young group (P < 0·05). These results suggest that the variation of DNA binding protein could contribute to the lower transcription of HLA-A and -B with ageing. These alterations of HLA class-I expression at the transcriptional level could lead to the unresponsiveness of CD8 T cells due to default of antigen presentation with ageing. [source] | |||||||||||||||||||||||||