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H Culture (h + culture)
Selected AbstractsNeutrophil apoptosis in preeclampsia, do steroids confound the relationship?JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 5 2004Akiko Fuchisawa Abstract Aim:, To investigate the influence of maternal corticosteroid administration on neutrophil apoptosis in early onset preeclampsia. Methods:, We investigated five groups: early onset preeclampsia (EOPET, <34 weeks, n = 10); late-onset preeclampsia (LOPET, ,34 weeks, n = 7); normotensive intrauterine growth restriction (nIUGR, n = 11); normal pregnancy (NPC, n = 22); and non-pregnancy (n = 10). We examined, by flow cytometry, spontaneous neutrophil apoptosis after 18 h culture (hypodiploid DNA, Annexin V binding, propidium iodide [PI] permeability). Results:, For the 10 women with EOPET exposed to betamethasone in the previous 48 h, we found that neutrophil apoptosis was not inappropriately inhibited, in contrast to our previous findings in women not thus exposed. Neither LOPET nor nIUGR differed from normal pregnancy. Conclusion:, Betamethasone alters the rate of spontaneous neutrophil apoptosis in EOPET. The anti-inflammatory influence of betamethasone may explain some of the differences between our previous and present findings with respect to neutrophil apoptosis in EOPET. Corticosteroids ameliorate the course of antenatal and postnatal preeclampsia. These results may reflect the mechanisms that underlie the transient improvements seen with antenatal dexamethasone use. [source] Upregulation of co-stimulatory molecule expression and dendritic cell marker (CD83) on B cells in periodontal diseaseJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2002Rangsini Mahanonda T cells and their cytokines are well known for their important role in the pathogenesis of periodontitis. To date, the role of antigen presenting cells (APCs), which are known to be critical in the regulation of T cell response, has been poorly investigated in periodontitis. In this study, we analyzed the expression of co-stimulatory molecules (CD80 and CD86) and CD83, which is a marker of mature dendritic cells, on gingival cells that were isolated from severe periodontitis tissues, with the use of flow cytometry. Significant upregulation of CD86 and CD83 expression was detected in periodontitis lesions, and most of this occurred on B cells. In vitro peripheral blood mononuclear cell cultures showed that stimulation with different periodontopathic bacteria, that included Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Actinomyces viscosus, upregulated both CD86 and CD83 expression on B cells. Therefore, the presence of plaque bacteria may be responsible for the enhanced expression seen in vivo on gingival B cells. APC function by bacterial-activated B cells was further investigated using allogeneic mixed leukocyte reactions. After 24 h culture with either A. actinomycetemcomitans or P. gingivalis, these activated B cells performed as potent APCs in mixed leukocyte reactions, and they stimulated T cells to produce high levels of gamma interferon and minimal interleukin-5. In conclusion, periodontopathic bacterial-induced B cell activation with upregulation of CD86 and CD83 may be associated with enhanced APC function. The results of this study suggest, therefore, that infiltrated gingival B cells have a possible role as APCs in the regulation and maintenance of local T cell response in periodontitis. [source] The Effect of Vascular Endothelial Growth Factor on in vitro Embryonic Heart Development in RatsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2004H. Ülger Summary In vitro effects of vascular endothelial growth factor (VEGF) on heart development and total embryonic growth were investigated in 84 rat embryos (obtained from nine pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), in <30 kDa + >50 kDa serum fractions [retenate (R)], and in R + VEGF. After 24-h culture, the embryos from each group were harvested and divided into two groups. One group was analysed morphologically and biochemically to obtain embryo protein content, the second group was serially sectioned and examined by light microscopy. Morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in R had significant embryonic retardation, whereas the addition of VEGF to R increased embryonic growth and development. The morphological scores for WRS, R and R + VEGF were 57.7 ± 0.87, 46.6 ± 1.90 and 52.1 ± 0.97, somite numbers were 26.5 ± 0.47, 20.1 ± 0.63 and 24.4 ± 0.46, crown-rump lengths were 3 ± 0.07, 2.4 ± 0.06 and 2.7 ± 0.06 mm, and embryo protein contents were 160.5 ± 7.41, 98.2 ± 4.81 and 141.1 ± 10.96 ,g per embryo, respectively. The results of histological examination of heart development were similar. The hearts of embryos grown in R were unseptated and tubular. The atrioventricular endocardial cushions were incompletely developed. The addition of VEGF to R improved heart development. There were no gross morphological differences in the cardiac development between embryos grown in WRS and R + VEGF. In both groups, development of the muscular interventricular septum had begun. Development of the atrioventricular cushions was also similar in both groups and had caused narrowing of the atrioventricular canals, but the atrial septation was not observed. [source] Cytocompatibility, interactions, and uptake of polyethyleneimine-coated boron nitride nanotubes by living cells: Confirmation of their potential for biomedical applicationsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2008Gianni Ciofani Abstract Boron nitride nanotubes (BNNTs) have unique physical properties, which can be exploited in the biomedical field. Hence, the surprising lack of reported studies on their biocompatibility and interactions with living cells, addressed by the present paper which deals the results of such an investigation based on 72 h culture of human neuroblastoma cell line (SH-SY5Y) in the presence of an aqueous suspension of polyethyleneimine (PEI)-coated BNNTs. BNNTs conjugated with fluorescent markers (quantum dots) are employed to enable tracking of their uptake by living cells. The results demonstrate good cytocompatibility together with unequivocal BNNT cellular uptake by an energy-dependent endocytic process. Biotechnol. Bioeng. 2008;101: 850,858. © 2008 Wiley Periodicals, Inc. [source] Long,term culture of multibacillary leprosy macrophages isolated from skin lesions: a new model to study Mycobacterium leprae,human cell interactionBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2007D.F. Moura Summary Background, Leprosy is characterized by a disease spectrum having two polar clinical forms dependent on the presence or not of cell-mediated immunity. In the tuberculoid forms, granuloma-activated macrophages kill Mycobacterium leprae in conjunction with a Th1 response while, in multibacillary (MB) lesions, M. leprae nonactivated macrophages infiltrate the nerves and internal organs together with a Th2 response. The functional properties and activation pathways of macrophages isolated from patients with MB leprosy remain only partially understood. Objectives, To establish an ex vivo methodology capable of evaluating the activation pathways, grade and fate of cultured macrophages isolated from MB lesions. Methods, Skin biopsies from patients with borderline tuberculoid, bordeline lepromatous and lepromatous leprosy (LL) were characterized by immunohistochemistry and transcriptional analysis. To isolate inflammatory cells, a portion of the samples was submitted to enzymatic digestion. These same cells, maintained in culture for a minimum 7-day period, were characterized morphologically and via flow cytometry at different culture time points. Cytokine [interferon (IFN)-,, tumour necrosis factor (TNF)-, and interleukin (IL)-10] mRNA levels were quantified by real-time polymerase chain reaction and protein secretion in the culture supernatants was measured by enzyme-linked immunosorbent assay and the nitric oxide levels by Griess reagent. Results, RNA expression in tuberculoid and MB lesions showed the profile expected of characteristic Th1 and Th2 responses, respectively. The inflammatory cells in all biopsies were successfully isolated. Although the number of cells varied between biopsies, it was highest in LL biopsies. The frequency of isolated CD14+ and CD3+ cells measured by flow cytometry correlated with the percentages of macrophages and lymphocytes in the lesions. Throughout the culture period, CD68+ macrophages showed morphological changes. A progressive increase in cell number and reduction of infected cells were perceptible in the cultures. In contrast to the biopsies, TNF-,, IFN-, and IL-10 expression in the tuberculoid and MB leprosy cells in 24-h culture and the cytokine levels in the supernatants did not differ significantly. During the culture period, cytokine expression in the MB cells progressively declined, whereas, from days 1 to 7, nitrite levels progressively increased. After day 40, the remaining macrophages were able to ingest fluorescein isothiocyanate-labelled M. leprae. These data need to be confirmed. Conclusions, This study confirmed the feasibility of obtaining ex vivo macrophages from leprosy lesions and keeping them in long-term culture. This procedure may open new pathways to studying the interaction between M. leprae and human macrophages, which might, in turn, lead to the development of therapeutic tools capable of overcoming the specific anergy found in patients with MB leprosy. [source] T cell inflammatory response, Foxp3 and TNFRS18-L regulation of peripheral blood mononuclear cells from patients with nasal polyps-asthma after staphylococcal superantigen stimulationCLINICAL & EXPERIMENTAL ALLERGY, Issue 9 2010C. A. Pérez Novo Cite this as: C. A. Pérez Novo, M. Jedrzejczak-Czechowicz, A. Lewandowska-Polak, C. Claeys, G. Holtappels, P. Van Cauwenberge, M. L. Kowalski and C. Bachert, Clinical & Experimental Allergy, 2010 (40) 1323,1332. Summary Background Staphylococcal superantigens may modulate airway inflammatory disease. Objective We assessed the effect of Staphylococcus aureus enterotoxin B (SEB) on T cell activation in patients with nasal polyps and asthma, and its possible link to aspirin hypersensitivity. Methods Leucocytes were isolated from five healthy subjects (controls), five asthmatics with nasal polyps without (NP-ATA) and five with aspirin-induced asthma (NP-AIA). Cells were incubated with increasing concentrations of SEB for 4 and 18 h. Release of TH1/TH2 cytokines was assessed by Cytometric Bead-Array. Foxp3 and TNFRS18-L expression were analysed by qPCR and flow cytometry. Results After 4 and 18 h, SEB significantly increased IFN-gamma, IL-4, TNF-alpha, IL-5 and IL-2 concentrations in supernatants of both NP polyp groups compared with controls. Baseline Foxp3 was significantly decreased in both NP-asthma groups. Incubation with SEB for 4 h induced a limited up-regulation of Foxp3 in NP-AIA patients, which was switched off consecutively. Foxp3 was significantly up-regulated in the control group after 18 h, but not in the NP-asthmatic groups. In parallel, TNFRS18-L mRNA significantly increased after 18 h in the NP-asthma groups compared with control subjects. This molecule was highly expressed in CD11c+CD14+ cells and its levels increased after 18 and 24 h culture in the NP-asthma patients. Conclusion SEB induces both TH1 and TH2 pro-inflammatory responses in patients with nasal polyps and asthma regardless of the presence of aspirin hypersensitivity. The nature of this response may be linked to a basal deficiency of Foxp3 observed in the NP-asthmatic patients and/or to the up-regulation of TNFRS18-L on monocytes/dendritic cell precursors. [source] Differential expression and localization of neuronal intermediate filament proteins within newly developing neurites in dissociated cultures of Xenopus laevis embryonic spinal cordCYTOSKELETON, Issue 1 2001Jayanthi Undamatla Abstract The molecular subunit composition of neurofilaments (NFs) progressively changes during axon development. In developing Xenopus laevis spinal cord, peripherin emerges at the earliest stages of neurite outgrowth. NF-M and XNIF (an ,-internexin-like protein) appear later, as axons continue to elongate, and NF-L is expressed after axons contact muscle. Because NFs are the most abundant component of the vertebrate axonal cytoskeleton, we must understand why these changes occur before we can fully comprehend how the cytoskeleton regulates axon growth and morphology. Knowing where these proteins are localized within developing neurites and how their expression changes with cell contact is essential for this understanding. Thus, we examined by immunofluorescence the expression and localization of these NF subunits within dissociated cultures of newly differentiating spinal cord neurons. In young neurites, peripherin was most abundant in distal neuritic segments, especially near branch points and extending into the central domain of the growth cone. In contrast, XNIF and NF-M were usually either absent from very young neurites or exhibited a proximal to distal gradient of decreasing intensity. In older neurites, XNIF and NF-M expression increased, whereas that of peripherin declined. All three of these proteins became more evenly distributed along the neurites, with some branches staining more intensely than others. At 24 h, NF-L appeared, and in 48-h cultures, its expression, along with that of NF-M, was greater in neurites contacting muscle cells, arguing that the upregulation of these two subunits is dependent on contact with target cells. Moreover, this contact had no effect on XNIF or peripherin expression. Our findings are consistent with a model in which peripherin plays an important structural role in growth cones, XNIF and NF-M help consolidate the intermediate filament cytoskeleton beginning in the proximal neurite, and increased levels of NF-L and NF-M help further solidify the cytoskeleton of axons that successfully reach their targets. Cell Motil. Cytoskeleton 49:16,32, 2001. © 2001 Wiley-Liss, Inc. [source] Effect of triclosan on interferon-, production and major histocompatibility complex class II expression in human gingival fibroblastsJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 10 2000Manal Mustafa Abstract Background, aims: The effect of triclosan (2,4,4,-trichloro-2,-hydroxyl-diphenyl ether) on the production of interferon-, (IFN-,) and the expression of major histocompatibility complex (MHC) class II antigen was studied in human gingival fibroblasts isolated from 4 individuals. Methods/Results: AII cell lines demonstrated high IFN-, production in 24-h cultures of human gingival fibroblasts stimulated by phytohemagglutinin (PHA) (5 ,g/ml). Human gingival fibroblasts showed a high expression of MHC class II when stimulated with 500 and 1000 pg/ml rIFN-, in 7-day cultures. Treatment of the cells with triclosan (0.5 ,g/ml) reduced both IFN-, production and MHC class II expression in human gingival fibroblast cultures. Similar inhibitory effects on IFN-, production and MHC class II expression were observed when the anti-inflammatory agent dexamethazone (1 ,M) was used. Conclusion: The present study further supports the view that the agent has an anti-inflammatory effect in addition to its antibacterial capacity. [source] Effect of leptin on activation and cytokine synthesis in peripheral blood lymphocytes of malnourished infected childrenCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2007L. Rodríguez Summary Malnutrition compromises immune function, resulting in reduced resistance to infection. Recent animal and human studies have suggested that leptin is capable of modulating the immune response and that its levels, which are regulated by nutritional status, fall rapidly during starvation. Leptin deficiency is associated with impaired cell-mediated immunity, an increased incidence of infectious disease and an associated increase in mortality. The purpose of this study was to examine the effect of leptin on activation and cytokine production in peripheral blood T cells from malnourished children. The data obtained in the present study demonstrate that leptin produced an increase in the percentage of CD4+ and CD8+ cells producing interleukin (IL)-2 and interferon (IFN)-, in 24-h cultures. Moreover, leptin decreased the percentage of CD4+ and CD8+ cells producing IL-4 and IL-10, and enhanced activation of circulating T cells when co-stimulated by phorbol 12-myristate 13 acetate (PMA),ionomycin. Leptin enhanced the expression of activation markers CD69 and CD25 in both CD4+ and CD8+ cells after 5 h of stimulation. In conclusion, the results obtained show that leptin modulates CD4+ and CD8+ cell activation towards a T helper 1 (Th1) phenotype by stimulating the synthesis of IL-2 and IFN-,. In contrast, leptin decreases IL-4 and IL-10 production. Moreover, leptin enhanced the expression of CD69 and CD25 on CD4+ and CD8+ cells after stimulation with PMA,ionomycin. [source] | |||||||||||||