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Intracellular Production (intracellular + production)
Selected AbstractsSuppression of anti- Candida activity of macrophages by a quorum-sensing molecule, farnesol, through induction of oxidative stressMICROBIOLOGY AND IMMUNOLOGY, Issue 6 2009Shigeru Abe ABSTRACT Farnesol is well known as a quorum-sensing molecule of Candida albicans. To assess the pathological function of farnesol, its effects on macrophage viability and functions including growth inhibitory activities against C. albicans were examined in vitro. Murine macrophages, when cultured in the presence of 56,112 ,M of farnesol for 1,2 hr, decreased their activity inhibiting the mycelial growth of C. albicans and lost their viability. This suppression of macrophage function by farnesol was neutralized by the coexistence of the anti-oxidants probucol and trolox. Macrophages cultured in the presence of farnesol for 2 hr displayed morphological change of nuclei and DNA fragmentation, which suggested apoptosis of the cells. Intracellular production of ROS in the farnesol-treated macrophages was shown by fluorescence of DCFH-DA and increase of peroxidized materials. These effects of farnesol were blocked by probucol or trolox. These results indicate that farnesol lowered viability of the murine macrophages and suppressed their anti- Candida activity, perhaps through induction of ROS. [source] Lipid peroxidation caused by oxygen radicals from Fusobacterium -stimulated neutrophils as a possible model for the emergence of periodontitisORAL DISEASES, Issue 1 2001M Sheikhi OBJECTIVE: The possible contribution of bacteria and polymorphonuclear neutrophils (PMN) to the disease process of periodontitis was evaluated. DESIGN: Fusobacterium nucleatum has been associated with chronic adult periodontitis. Intracellular production and extracellular release of reactive oxygen species (ROS) by PMN stimulated by fusobacteria were evaluated. To estimate the potential extracellular damage that might be caused by the ROS, the lipid peroxidation (LPO) of an exogenous phospholipid, Intralipid, was assayed. METHODS: The ROS production of PMN was studied by the nitroblue tetrazolium and chemiluminescence tests. The levels of malonaldehyde (MDA) and 4-hydroxyalkenals were used to indicate LPO. RESULTS: Fusobacterium nucleatum strains stimulated neutrophils to produce a large amount of ROS, independently of plasma complement factors. The two strains tested induced considerable intracellular, but no extracellular chemiluminescence responses during the first hour, indicating that ROS were released into phagosomes. However an incubation period of 4 h, in the presence of the extracellular lipid resulted in a high degree of LPO, presumably caused by ROS release from the Fusobacterium -stimulated PMN. ROS production and lipid peroxidation could be counteracted by vitamin E. CONCLUSION: In periodontitis local bacteria might stimulate PMN to release ROS, which cause inflammation and destruction. [source] ORIGINAL ARTICLE: TH1 , TH2 Response and the Atopy Risk in Patients with Reproduction FailureAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2009Jana Hanzlikova Problem, Enhanced TH2 activity is characteristic for atopic diseases and is observed also in physiological pregnancy. The immune causes of repeated pregnancy losses and/or repeated in vitro fertilization failure may be associated with TH2 hypoactivity. The association with frequency of atopic diseases is unclear. Method of Study, Intracellular production of IL-4 and IFN-, by peripheral CD4+ T lymphocytes was studied, as well as serum levels of total and allergen specific IgE. Simultaneously skin prick tests with inhalant allergens were performed, and clinical features of atopy were registered by means of a questionnaire. Results, Lower intracellular production of IL-4 by peripheral CD4+ T cells and lower frequency of elevated total and allergen specific IgE were found in women with reproduction failure compared to controls, as well as lower frequency of some symptoms possibly associated with atopy. Conclusion, Our study showed the presence of TH2 hypoactivity in women with reproduction failure, which may be associated with lower occurrence of atopic diseases. [source] Monitoring of monocyte functional state after extracorporeal circulation: A flow cytometry studyCYTOMETRY, Issue 1 2004Silverio Sbrana Abstract Background Cardiovascular surgery with cardiopulmonary bypass (CPB) induces systemic inflammation and postoperative complications depending on pro- and anti-inflammatory mechanisms. Activated polymorphonuclear cells and monocytes may be responsible for morbidity associated with CPB. Knowledge of the monocyte functional state in particular may help to develop protective interventions. Methods Samples were drawn from venous peripheral blood (basal condition, at 4 and 24 h after CPB) and coronary blood (before and after cardioplegic arrest) of 14 patients undergoing cardiac surgery. The following phenotypic and functional parameters of the monocyte population were studied by flow cytometry: surface molecules expression (CD18, CD11a, CD11b, CD14, CD15, CD45, HLA-DR, and Toll-like receptor [TLR]-4), myeloperoxidase (MPO) content, and intracellular cytokine production (tumor necrosis factor [TNF]-,, interleukin [IL]-1,, IL-6, and IL-8). Results Cardiac surgery with CPB induced down-modulation of surface molecules expression on peripheral monocytes, especially at 24 h after CPB, for CD18, CD11a, and CD11b (P < 0.003) and for the CD15 adhesive cluster (P = 0.0028) and HLA-DR (P < 0.001). At 4 h after CPB, downregulation was observed for CD14 (P = 0.004), CD45 (P = 0.014), and CD15 (P = 0.0056). A loss of MPO was detected in venous peripheral (at 24 h after CPB, P = 0.01) or coronary (at reperfusion, P < 0.02) blood. The CD15 cluster complex exhibited a down-modulation in coronary blood (at reperfusion, P = 0.0003). Spontaneous intracellular production of IL-1,, IL-6, and IL-8 decreased at 24 h after CPB (P < 0.05). Conclusions The down-modulation of integrins and adhesive receptor expression and the loss of MPO suggest a strong activation and shedding reaction of circulating monocyte after CPB, further exacerbated by contact with coronary ischemic vessels. The changes of differentiation antigens may reflect the appearance of a partially immature population immediately after CPB. The reduced proinflammatory cytokine production, observed at 24 h after CPB, suggests a functional polarization of circulating monocytes. © 2003 Wiley-Liss, Inc. [source] Microcystin-LR modulates selected immune parameters and induces necrosis/apoptosis of carp leucocytes,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010Anna Rymuszka Abstract Microcystins (MCs) are potent hepatotoxins acting by the inhibition of protein phosphatase 1 and 2A, and may promote liver tumors. Moreover, studies also suggest they are nephrotoxic. The aim of the present study was to assess possible in vitro effects of microcystin-LR (which contains the amino acids leucine and arginine, the most widely studied and distributed variant of all microcystins) on the selected immune functions of the cells isolated from the head kidney of carp. In the experiments, pure microcystin-LR (MC-LR), was used at concentrations of 0.01, 0.1, 0.5, and 1,µg/ml RPMI-1640 medium. Leucocytes (lymphocytes and phagocytes) were isolated by centrifugation on a density gradient. Lymphocyte proliferation, intracellular production of reactive oxygen species by phagocytes, and the presence of apoptotic and/or necrotic cells were assessed. The respiratory burst activity of phagocytic cells was increased at the lowest toxin concentration used in the study, but it was decreased at higher concentrations. Using a sensitive luminescent immunoassay, MC-LR was observed to have no influence on the T-cell proliferation but decreased the proliferation of B lymphocytes. Moreover, it was noted that MC-LR induced necrosis to a higher degree than apoptosis in fish leucocytes. The results of the present study suggest the modulatory potency of microcystin-LR on fish leucocytes. Environ. Toxicol. Chem. 2010;29:569,574. © 2009 SETAC [source] Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Dorota Rogacka Glutamine:fructose-6-phosphate amidotransferase (GFAT) and N -acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O -glycosylation, one of key post-translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT-PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3,±,0.2,nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1,±,0.2,nmol/min/mg protein) in a significant part, involved a sodium-dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50,µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6,mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577,584, 2010. © 2010 Wiley-Liss, Inc. [source] Autologous nucleus pulposus primes T cells to develop into interleukin-4-producing effector cells: An experimental study on the autoimmune properties of nucleus pulposusJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2009Andrea Geiss Abstract An autoimmune response to herniated nucleus pulposus has been proposed to constitute a pathophysiologic mechanism for inducing sciatica based on the fact that nucleus pulposus under normal conditions is excluded from the development of immunological tolerance. The manifestation of an autoimmune response comprises different steps starting with antigen capture, continuing with activation of T helper (TH) cells and ending with production of autoantibodies. Activated TH cells differentiate into either TH1 cells, predominately producing proinflammatory cytokines such as interferon , (IFN,) or a TH2 subset mainly producing anti-inflammatory cytokines such as interleukin-4 (IL-4). The aim of the present study was to examine if exposure of autologous nucleus pulposus (NP) to the immune system for 3 weeks is potent enough to prime TH cells to differentiate into TH2 cells. The study was performed in a pig model allowing the exposure of NP to the immune system. To assess the polarization of TH cells the intracellular production of IFN, and IL-4 was measured in T cells by using flow cytometry. The revealed predominant production of IL-4 together with low production of IFN, in T cells after NP exposure to the immune system indicates that nucleus pulposus may prime TH cells to develop into IL-4-producing TH2 cells after being exposed to the immune system, for example, in association with disc herniation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:97,103, 2009 [source] Alterations of Mitochondria in Peripheral Blood Mononuclear Cells of Vitiligo PatientsPIGMENT CELL & MELANOMA RESEARCH, Issue 5 2003Maria Lucia Dell'Anna The possible role for a defective mitochondrial functionality in the pathogenesis of vitiligo was investigated by measuring intracellular levels of reactive oxygen species and of antioxidants, the activity of Krebs cycle enzymes, as well as the effects of inhibitors of the electron transport chain, in peripheral blood mononuclear cells from patients with active or stable disease vs. normal subjects. Plasma glyoxal levels were also determined in the same groups of subjects as an index of systemic oxidative stress. In patients with vitiligo in active phase, we observed an increased intracellular production of reactive oxygen species with a consequent imbalance of the prooxidant/antioxidant equilibrium, whereas plasma did not show apparent alterations in glyoxal levels, ruling out a systemic oxidative stress. In patients with stable disease, the balance between pro-oxidants and anti-oxidants seems to be maintained. Moreover, a marked increase in the expression of mitochondrial malate dehydrogenase activity and a specific sensitivity to electron transport chain complex I inhibitor were observed. Overall, these data provide further evidence for an altered mitochondrial functionality in vitiligo patients. [source] ORIGINAL ARTICLE: Comparative Analysis of Peripheral Natural Killer Cells in the Two Phases of the Ovarian CycleAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2010Ageliki Pantazi Problem Changes in endometrial Natural Killer (NK) cells during the luteal phase of the ovarian cycle are important in initiating/maintaining a subsequent pregnancy. In the present study it was investigated whether during the menstrual cycle changes occur also in peripheral blood (PB) NKs. Method of study Blood samples during the follicular and the luteal phase were collected from 30 women without fertility problems. Samples were analyzed by flow-cytometry for: (1) NK cells (CD3,CD16+CD56+) and (2) intracellular production of interferon-, (IFN-,) by NK cells. For the comparison and correlation of the two populations between the two phases, Wilcoxon signed-rank test and Spearman's Coefficient were used. Results The differences in percentages of CD3,CD16+CD56+ cells and that of CD3,CD16+CD56+/IFN-,+ cells between the follicular and the luteal phase were not statistically significant (10.61 ± 5.11 versus 9.76 ± 4.57 and 6.48 ± 7.90 versus 7.30 ± 6.77, respectively, P > 0.05). The correlation between the two variables (NK% and NK/IFN-,%) was weakly positive (P = 0.07) only in the follicular phase. Conclusion The study did not reveal menstrual cycle-depended changes in PB NK cells. Thus, a suggestion to measure these cells in a specific phase of the cycle in order to predict the outcome of a subsequent pregnancy in women with fertility problems is objected. [source] ORIGINAL ARTICLE: TH1 , TH2 Response and the Atopy Risk in Patients with Reproduction FailureAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2009Jana Hanzlikova Problem, Enhanced TH2 activity is characteristic for atopic diseases and is observed also in physiological pregnancy. The immune causes of repeated pregnancy losses and/or repeated in vitro fertilization failure may be associated with TH2 hypoactivity. The association with frequency of atopic diseases is unclear. Method of Study, Intracellular production of IL-4 and IFN-, by peripheral CD4+ T lymphocytes was studied, as well as serum levels of total and allergen specific IgE. Simultaneously skin prick tests with inhalant allergens were performed, and clinical features of atopy were registered by means of a questionnaire. Results, Lower intracellular production of IL-4 by peripheral CD4+ T cells and lower frequency of elevated total and allergen specific IgE were found in women with reproduction failure compared to controls, as well as lower frequency of some symptoms possibly associated with atopy. Conclusion, Our study showed the presence of TH2 hypoactivity in women with reproduction failure, which may be associated with lower occurrence of atopic diseases. [source] Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Rebekka Biedendieck Abstract A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His6 -tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525,537. © 2006 Wiley Periodicals, Inc. [source] Process Technology for Production and Recovery of Heterologous Proteins with Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 6 2006Mehmedalija Jahic Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion. [source] Dapsone suppresses human neutrophil superoxide production and elastase release in a calcium-dependent mannerBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2005T. Suda Summary Background, Dapsone (4,4,-diaminodiphenyl sulphone) is a powerful therapeutic tool in many skin diseases including neutrophilic dermatoses. The drug has an outstanding therapeutic efficacy against many skin diseases characterized by neutrophil-rich infiltrates; however, mechanisms of its action are poorly understood. Objectives, We investigated the effects of dapsone on respiratory and secretory functions of human neutrophils triggered by the chemotactic peptide N -formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the physiological agonist C5a, and phorbol myristate acetate (PMA). Methods, Human neutrophils were isolated from venous blood obtained from healthy donors. We detected extracellular production of superoxide (O2,) by cytochrome C reduction assay, and intracellular production of O2, by flow cytometry. Neutrophil elastase release was measured by the cleavage of the specific elastase substrate N -methoxysuccinyl-Ala-Ala-Pro-Val- p -nitroanilide. Measurement of cytosolic free calcium concentration was performed using the calcium-reactive fluorescence probe, Fluo-3. Results, Dapsone suppressed intra- and extracellular production of O2, and elastase release triggered by fMLP and C5a, but not by PMA. Both fMLP and C5a signalled the above pathways by inducing calcium influx, but PMA functions bypassed calcium influx. Dapsone was capable of antagonizing the induction of calcium influx. Conclusions, These findings suggest that one mechanism of the anti-inflammatory action of dapsone is inhibition of calcium-dependent functions of neutrophils including release of tissue-damaging oxidants and proteases in the affected skin. [source] | |||||||||||||