| 161. |
- Wihlmark, Ulf
(författare)
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The effects of reactive oxygen species and lipofuscin on the function and health of the retinal pigment epithelium
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Age-related macular degeneration (ARJVID) is a common cause of central vision loss in elderly people. Specific treatment is possible only for selected patients. A dysfunction of the retinal pigment epithelial (RPE) cells has been proposed to help explain the pathogenesis of ARMD. In the normal turnover of photoreceptor outer segments (POS), membranes rich in polyunsaturated fatty acids (PUPAs) are shed and phagocytised by the RPE. PUFAs are highly susceptible to free radical damage, causing peroxidation and subsequent formation of products with fluorescence similar to Schiff bases, a component of lipofuscin. With increasing age, lipofuscin accumulates in the RPE cells, and it has been suggested that lipofuscin could be detrimental to RPE function through free radical generation or interference with the autophagocytic capacity of cells having lipofuscin-loaded lysosomes.To study the effect of oxidative stress on lipofuscin accumulation, rabbit RPE cell cultures were kept at an ambient oxygen concentration of either 8 % or 40 %. To simulate the normal phagocytic function of RPE cells, bovine POS were added daily. The lipofuscin-specific autofluorescence was measured after 1, 2 and 3 weeks. RPE cells cultured under normobaric hyperoxic conditions (40 % oxygen) showed significantly higher levels of lipofuscin-like autofluorescence than those kept at nonnobaric and probably normooxic conditions (8 % oxygen) after 1, 2 as well as after 3 weeks. For both oxygen concentrations, the lipofuscin accumulation level was increased after 2 weeks of POS exposure and had increased even further after 3 weeks. The results suggest an involvement of oxidative mechanisms in the formation of lipofuscin from phagocytised POS by RPE cells.In the second study, bovine POS were photo-oxidised, and turned into a lipofuscin-like material, by irradiation with UV light. Transmission electron microscopy of irradiated POS showed loss of the normal stacks of the disk membranes with conversion into an amorphous osmiophilic, electron dense mass. The formation of thiobarbituric acid reactive substances (TEARS), estimated during the irradiation process, indicated lipid peroxidation. The later decline in TEARS indicates fragmentation of the peroxides and conjugation offonned aldehydes with proteins under the formation of more stable Schiff bases and their secondary reaction products, e.g. lipofuscin. Irradiated POS also showed a strong granular yellow auto-fluorescence. RPE cell cultures, kept at 21 % ambient oxygen, were fed daily for 3, 5 or 7 days with either UVperoxidised POS, native POS or culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-specific autofluorescence compared to cells exposed tonativePOS after 3 days, 5 days and 7 days and to the non-exposed control cells. The lipofuscin content of ce11s exposed to irradiated POS increased significantly between days 3 and 7. Ultrastructural studies showed much more numerous and larger lipofuscin-like inclusions in RPE cells fed irradiated POS compared to cells exposed to native POS. In the control cells, lipofuscin-like granules were small and sparse. It appears that exposing RPE cells to previously peroxidised POS, thus artificially converted into lipofuscin-like material and obviously not digestible by the lysosomal enzymes, accelerates the fonnation of severely lipofuscin-loaded cells.A well-known physical property of lipofuscin is its yellowish autofluorescence when irradiated by blue light. Such energy transformation is known to induce photo-oxidative processes since oxygen present in the immediate surroundings would be activated into reactive oxygen metabolites. RPE cells are constantly exposed to visible light during the time the subject is awake. Consequently, in RPE cells exposed to light, the membranes of the lysosomes surrounding enclosed lipofuscin would be subjected to oxidative stress, which may result in damage, with leakage to the cytosol of lysosomal hydrolytic enzymes and ensuing cellular degeneration.To test this hypothesis, cultures of heavily lipofuscin-loaded RPE cells were blue-lightirradiated and compared to relevant controls. Following irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin loaded blue-lightexposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type.,The aim of the last investigation was to study whether heavy loading with lipofuscin of RPE lysosomes would affect the further phagocytic ability of the cells.In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS for 4 weeks, resulting in a pronounced lipofuscin accumulation of the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at 0 and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POSderived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly higher levels in unloaded control cells than in lipofuscin-loaded ones.In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytoplasmic POS emitting reddish fluorescence, while fewer POS were fagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the fagocytic ability of the cells, showed significantly higher levels in control cells than in lipofuscin-loaded ones. Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity.The suggested mechanisms may be of relevance in the pathogenesis of ARMD.
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| 162. |
- Wirén, Michael
(författare)
-
Growth and integrity of the small intestine in malnutrition and trauma
- 1995
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The small intestine is an active metabolic organ constituting a functional and immunologic barrier to toxins and microbes in the intestinal lumen. Injuries are repaired by rapid cell replication, which depends on nutritional and humoral growth factors. Glutamine has been suggested to be the most important nutrient for the enterocytes. In the present studies, the effects of glutamine were evaluated using experiments with cultured cells and postoperative supplementation in animals and humans.Growth of two enterocyte-like epithelial cell lines, CaCo-2 and HT 29, was studied at different glutamine concentrations, and compared to effects of growth factors and energy substrates. Glutamine effects in starved, operated rats were evaluated by weight, DNA, protein analysis, 3H-thymidine incorporation in intestinal mucosa and urinary recovery of orally administered polyethylene glycols. Three different balanced and complete enteral preparations with no glutamine, 2% (normal) and 4% were used postoperatively . Growth parameters and tissue and plasma concentrations of humoral growth factors were studied 3 and 8 days after intestinal resection in the rat. In a clinical study with total parenteral nutrition for five days after major abdominal surgery, nitrogen balance and humoral growth factors in plasma were evaluated in patients receiving glutamine-containing dipeptide (Gly-Gln) amino acid solution, compared to conventional amino acid solution.In the cell cultures, glutamine was shown to be of importance both as a trophic factor and as a metabolic substrate, particularly in cells of intestinal origin. In the animal model of malnutrition and surgery, 3H-thymidine incorporation was higher in the supplemented group compared to glutamine-free and also higher in all operated groups compared to controls. The permeability study showed a higher uptake of small polyethylene glycol molecules in glutamine-supplemented animals, parallel to increases in thymidine incorporation. After major intestinal resection in rats, no major benefit on growth by glutamine supplementation could be found after one week. Rapid PYY increases in plasma and higher IGF-II concentration in ileal mucosa were found. Stimulation of IGF-II concentration suggested an auto- or paracrine action in regulating growth. In the clinical study, no significant differences were seen in the levels of transthyretin, retinol binding protein or nitrogen balance, compared to patients recieving conventional amino acid solution. A positive correlation between insulin and nitrogen balance was found inglutamine treated patients.It is concluded that glutamine has important effects as a nutritional substrate for enterocytes and stimulates their proliferation and absorptive capacity in refeeding after malnutrition and surgical trauma in the rat. After intestinal resection, glutamme has no major effects upon growth one week after surgery, but the production of growth factorsincreases earlier in glutamine supplemented animals. In clinical use, a glutamine supplemented amino acid solution appeared no better than conventional amino acd solution. It could, howeyer, represent a more balanced way of supportmg protem metabolism after trauma, by the interaction with insulin and other growth factors.
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| 163. |
- Wu, Zhengyang
(författare)
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Studies of Vibrio cholerae toxins (zonula occludens toxin and hemagglutinin/protease) and their effects on epithelial cells
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Intestinal epithelium is important for absorption of nutrients and for defense against harmful substances in the gut. The permeability and barrier functions of epithelia are mainly controlled by intercellular tight junctions. Vibrio cholerae, a noninvasive enteropathogen, produces several toxins which affect the intestinal epithelium Besides the hypertoxic cholera toxin (CT), it has also been suggested to produce two other putative enterotoxins, zonula occludens toxin (ZOT) and accessory cholera enterotoxin (Ace). Since the suggested effects ofZOT on the tight junctions have been shown to be reversible, ZOT could be useful for studying the mechanisms of regulation of the tight junctions. The gene encoding ZOT has been cloned and sequenced, but the ZOT protein was not purified or characterized. Therefore, the first task in this Ph. D. study was isolation and characterization of WT. PCR-derived fragments containing zot were cloned into expression vectors. These vectors were then introduced into E. coli or V. cholerae. However, ZOT could only be expressed as insoluble inclusion bodies in E. coli. When extra signal sequences were used to export it, no stable and reproducible ZOT signals could be found. Neither was there any detectable amount of ZOT -protein in culture supematants of V. cholerae. Moreover, no ZOT-specific effects on cultured epithelial cells, i.e. MDCK-I, Caco-2, and HT-29, could be found in the culture supematants of V. cholerae stralu 395, in which the ZOT-activities were initially found. On the other hand, a V. cho/erae stralu that did not contalu any of the known enterotoxins showed severe cytotoxic effects on the epithelial cells. Further investigations revealed that the V. cholerae hemagglutinin/protease (HA/P) was the responsible agent. HA/P did not affect the apical microvilli of the cells but caused distinct reorganization of a tight junction-associated protein Z0-1, as well as reanangement of the F-actin cytoskeleton, as studied with confocal microscopy. These cytotoxic effects of HA/P were reduced by endogenous nitric oxide (NO). HA/P could only cause negligible alterations in the lateral diffusion of ganglioside GMI receptors of er on the cell surface, as assessed with fluorescence recovery after photo bleaching (FRAP). However, extended challenge of MDCK-I cells with HA/P inhibited the uptake of CT by the cells.
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| 164. |
- Yang, Ping
(författare)
-
Perinuclear Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antineutrophil cytoplasmic antibodies (ANCAs) are a group of autoantibodies which are specific for granulocyte antigens. By indirect immunofluorescence (llF) on ethanol-fixed neutrophils, ANCAs can be divided into two types: a cytoplasmic staining pattern (C-ANCA) or a perinuclear staining pattern (P-ANCA). Their pathogenic role and initiating stimuli are unknown.This study analysed P-ANCA in monozygotic twins with inflammatory bowel disease. In ulcerative colitis (UC), P-ANCA occurred in 9 of 14 (64.3%) monozygotic twins and in 13 of 21 (61.9%) non-twin subjects, which was significantly different compared with healthy controls who were positive in three of 52 (5.8%) subjects (pSeventy-six UC patients after proctocolectomy with ileal pouch-anal anastomosis (JP AA) including 28 patients who had had pouchitis and 48 patients who had not, were analysed regarding the correlation between P-ANCA and pouchitis. P-ANCA was found in 49n6 (64.5%) iu UC patients after the operation. Furthermore, we found that patients with recent (512 months) or ongoing pouchitis were all P-ANCA positive and pouchitis patients with higher P-ANCA titres were more prone to have frequent relapses.To screen the occurrence of P-ANCA and detect the target antigen(s), 36 patients with UC, 37 patients with CD, 38 patients with collagenous colitis (CC) and 190 controls were studied. P-ANCA was found in a higher frequency in UC (23/36. 63.9%) than in CD (2/37. 5.4%). CC (4/38, 10.5%) or controls (4/190. 2.1 %). The antigens of P-ANCA were not found to be associated with reactivity to lactoferrin (L:f), !3-glucuronidase (j3- Glc), myeloperoxidase (M:PO) or proteinase 3 (PR3).ANCAs were analysed by IIF on unfixed neutrophils or cells fixed by either ethanol, acetone or parafonnaldehyde in 21 sera from patients with UC, 17 sera from patients with vasculitides including 8 with PR3-positive C-ANCA and 9 with MPO-positive P-ANCA. Established ANCA patterns were most clear with ethanol-fixed neutrophils. The PR3-positive C-ANCA pattern was the same on unfixed or fixed cells irrespective of fixation method. However, the staining was brightest and the serum titres were higher with ethanol-fixed cells. Furthermore, we confirmed that the antigen of UC associated P-ANCA was better exposed on ethanol- than on acetone- or paraformaldehyde-fixed cells. In addition, anti-MPO positive sera tested on the very same day with freshly prepared paraformaldehyde-fixed neutrophils gave a C-ANCA pattern. Two or more days after preparation of the neutrophils anti-MPO-positive sera gave a mixed C- and P-ANCA pattern. Neutrophils kept unfixed for a few days or more and then treated with parafonnaldebyde gave a P-ANCA pattern, indicating diffusion of MPO from the azurophilic granules to the periphery of the nucleus, a process that is strongly enhanced by ethanol fixation.Six anti-MPO-negative P-ANCA-positive sera from patients with UC, six antiMMPO-positive P-ANCA, five anti-PR3-positive C-ANCA and ten antinuclear antibody (ANA)-positive serum samples were tested with 15 different GramHnegative and Gram-positive bacterial strains. We found that soluble material from live E. coli and P. mirabilis has the capacity to decompose the anti. genic substrate of neutrophils responsible for both MPOpositive and MPO-negative P-ANCA. most probably brought about by enzymatic activity. Anti-PR3-positive CANCA was not affected which suggests substrate specificity of the proposed enzymatic activity.
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| 165. |
- Yang, Yanqi
(författare)
-
Hemocompatibility of materials for use in prosthetic heart valves.
- 1997
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Thromboembolism (valve thrombosis and systemic embolism) is the main drawback of mechanical heart valve prostheses. The patients carrying these valves have to be subjected to life-long anticoagulant therapy to reduce thromboembolism. This therapy does not completely prevent these complications and may, if not properly controlled, even lead to life-threatening bleeding problems. Hemocompatibility of a mechanical heart valve is related to its engineering design and the construction material. To improve hemocompatibility of a mechanical heart valve, not only design but also valve material must be improved. Therefore the search for new materials or surface coatings that are more hemocompatible than those currently used must continue. The purpose of the present investigation was to develop an in vivo method, and to evaluate and compare hemocompatibility of some materials currently used, and for potential use, in ·prosthetic heart valves. Pyrolytic carbon (PyC), titanium (Ti), cobalt-chromium (CC), glutaraldehyde-treated bovine pericardium (Pe) and some new materials such as titanium nitride (TiN) and diamond-like carbon (DLC) were evaluated in three series of sheep experiments. The test materials were implanted in the central veins in the first and second series, and in the descending aorta in the third series. Up to four different materials could be tested simultaneously in each animal. No anticoagulant was given. After two hours of exposure to flowing blood, the test surfaces were explanted and prepared for photography and scanning electron microscopy (SEM). Thrombus area (the area covered by thrombus) was measured on close-up photographs of each surface using planimetry. Blood cell adhesion and blood-surface interaction were observed with SEM. The results showed more thrombi on PyC and Pe than on Ti and TiN. Leukocytes were the main type of blood cells adhering to PyC and DLC, and erythrocytes to Ti and TiN. Different materials exhibited different patterns of blood-surface interaction. Thrombus composition was largely related to the pattern of cell adhesion, indicating that the mechanism of early thrombus formation might be different on different surfaces. The results suggested that the method is practical and reliable. Under the present conditions PyC was not as hemocompatible as the metals currently used. TiN was more hemocompatible than PyC. Due to its combination of excellent hemocompatibility and wear resistance, TiN may be a promising new surface coating material for metallic components of mechanical heart valves, blood pumps and other devices in contact with blood.
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| 166. |
- Yin, Dazhong
(författare)
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Age pigments and the biochemical basis of their formation
- 1995
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The autofluorescent, yellow-brown pigments that accumulate over time inbiological organisms are called age pigments. Lipofuscin, known as a hallmark of aging, refers to intracellular age pigments that accumulate mainly in the lysosomes of postrnitotic cells. This dissertation reports studies on the biochemical nature of lipofuscin and other age pigments, including their origin, mechanisms of formation, composition, structure and other properties.Since lipofuscin-extracts and lipid peroxidation products exhibit similar fluorescent characteristics, oxidative stress-induced lipid peroxidation is linked with age pigment formation. The initiation of lipid peroxidation resulting from oxygen free radicals was studied in a Tween 20-emulsified linoleic acid model system. Fenton reagents did not result in hydrogen abstraction-related diene conjugation. Also, different cOmbinations of Fe(II) and Fe(III) did not support the Fe(II):Fe(Ill) (1: 1) optimum ratio hypothesis. It is, therefore, concluded that perferryl ions or chelator-Fe-02 complexes are responsible for the first-chain initiation of lipid peroxidation in this model system.To elucidate a mitochondrion-lysosome hypothesis of lipofuscinogenesis, the oxidative stress-induced formation of lipofuscin-like fluorophores was studied in a testtube model using lysosomal-mitochondrial fractions. The sequential formation of TEARS, protein carbonyls and lipofuscin-like fluorophores was found. These findings add support to the concept that lipofuscin fonns in secondary lysosomes as a result of iron-catalyzed oxidative reactions involving autophagocytosed materials.Further, lipofuscin-like fluorescence was obtained during ascorbic acidautoxidation and from reaction products between ascorbic acid and amino compounds. The reaction between ascorbic acid and glutamine shows that such fluorophore formation is oxidation-dependent. On the basis of a comparison of the fluorophore formation mechanisms of lipid peroxidation, ascorbate oxidation and glycation reactions, we propose that carbonyl-protein crosslinking is a common biochemical reaction in aging processes.Striking discrepancy exists between the orange-yellow fluorescence of lipofuscin in situ and the blue fluorescence of lipofuscin-extracts. A concentration-dependent fluorescence shift was discovered using different lipofuscin-related fluorophores. The methodological difference between microfluorometry, by which lipofuscin is studied in highly condensed form, and spectrofluorometry, by which lipofuscin is studied at a very low concentration, is also clarified.These studies suggest that crosslinking between carbonyls and amino compounds may represent the major biochemical process responsible for the formation of age pigments and lipofuscin-like fluorophores.
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| 167. |
- Yngve, Smedberg
(författare)
-
On the reporting of dental health, time for dental care, and the treatment panorama : Methodological studies of measuring and analysing dental care outcomes within a Swedish Public Dental Service organization
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The thesis included five methodological studies and one caries epidemiological investigation, the general aim being to study how to measure and report dental health, time for dental care, treatment panorama, and dental care outcomes, within a Public Dental Service organization. The specific aims were to monitor dental clinic activities using a time study method, to apply time study results of a dental health-related patient group system for the 3-19 year age groups, and to compare time study results with corresponding results from computerized systems used for reporting dental care. Other specific aims were to compare longitudinal caries index data results between cohort and cross-sectional samples, to analyse caries index for extreme caries groups among adolescents leaving organized dental care, and — using time series methods — to analyse dental health development of the 15-19 year age groups. Results from the time studies portrayed the dental clinic as a working unit, showed that reported values can represent dental care only for intervention procedures, and indicated that clinic patterns were not adapted to the health situation of the patient groups. Longitudinal cohort attempts gave different values from those of the cross-sectional year classes, which should be the primary focus when presenting caries index mean values in dental health reviews. Caries freegroups from 15 to 19 years of age seem to be stable in their caries development in about 60%-80% of cases; while the 20% groups with the highest index values accounted for about 80% of all approximal lesions. In times of major economic adjustment, dental health for adolescents in Göteborg was an example of sustainable dental health development. A model system for monitoring, analysing, and reporting dental health and dental care outcomes within a dental care-giving organization calls for several conditions, for example, a dental healthrelated patient group system, and a rationale for the choice of dental team models. These areas could be gathered into a system where contemporary socio-economic factors and dental research results interact with performed dental care, and also with different methods for reporting and evaluating dental health, dental care costs, and the demand for dental care competence.
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| 168. |
- Yu, Guo
(författare)
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Polyunsaturated fatty acids in relation to childhood and maternal allergic diseases
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this thesis was to study polyunsaturated fatty acid (PUPA) in relation to the appem11nce of anergic diseases in children and mothers in late pregnancy and during the lactation period and to the development of atopy in children, as well as the influence of maternal PUF A on their babies.The levels of docosahexaenoic acid (DHA, C22:6n-3) and total n-3 long-chain polyunsaturated fatty acids (LCP) were lower (p = 0.01 and< 0.05) and the ratio of total n-6 to n-3 LCP was higher (p < 0.01) in serum phospholipids (PL) in 22 allergic school children than in 23 controls. The levels of docosapentaenoic acid (DPA, C22:5n-3) and the ratio of arachidonic acid (AA, C20:4n-6) to its precursor dihomo-y-linolenic acid (DHGLA, C20:3n-6) were also lower in 12 children with positive skin prick test (SPT), as compared to SPT negative children (both p < 0.05). Higher levels of C20:2n-6 and lower EPA were recorded in allergic children with serum IgE above the median level (56kU/L), as compared to those with lower IgE levels,The levels of DHGLA, eicosapentaenoic acid (EPA, C20:5n-3), DPA and DHA were all lower in milk total lipid (TL) obtained from atopic, as compared to non-atopic mothers after one month oflactation (all p < 0.05). Similarly, the lower levels of DHGLA, AA, EPA, DPA and DHA were also observed in serum 1L of the atopic mothers at the time of delivery (all P < 0.05), while the levels of a-linolenic acid and C20:2n-6 were higher. Several ratios of n-6 to n- 3 LCP in mature milk at 1 and 3 months were significantly higher in atopic than non-atopic mothers (all p < 0.05). An n-3 fatty acid C20:4n-3 was present in human milk, but not in the blood samples of the mothers and children.The levels of most of the individual PUFA con·elated well in blood of non-allergic children and in colostrum and mature milk of non-atopic mothers (r > 0.5, p < 0.01 as significant), but the correlations were largely absent in the atopic groups. Furthmore, a correlation between linoleic acid (LA, C18:2n-6) and AA levels was observed in serum samples of non-allergic (r = 0.64, p < 0.001), but not allergic mothers at delivery (r = 0.25, p > 0.05).There were some correlations between AA and its precursor DHGLA and metabolite C22:4n-6 and between several n-3 and n-6 LCP, i.e. DPA and C22:4n-6, DHA and DHGLA and AA in semm phospholipids from cord blood of children without a family history of allergy (FHA) and who did not develop allergic diseases during the first 6 years of life. These relationships between the LCP levels were not seen in children with a FHA and in those who developed allergic diseases in later life. Furthermore, an abnormal PUF A composition in maternal blood and breast milk was related to the appearance of allergy in their children.The LA levels correlated in 22 non-allergic mothers and their babies at the time of delivery (r = 0.53, p = 0.01). Furthermore, the serum levels of maternal DHGLA correlated with some LCP levels in cord serum of their babies, i.e. AA, C22:4 and DHA (all r= 0.65, p = 0.001). None of these relationships were observed in 25 allergic mothers and their babies.The findings confirmed an abnormal PUF A composition in allergic children, in allergic mothers at the time of delivery and early lactation period and in newborn infants who developed allergic diseases during the first 6 years of life. The latter finding indicates that an abnormality of PUF A composition may be primary in allergic diseases. An impaired 0-6-desaturase activity in allergic diseases could not be confirmed, The presence of n-3 series fatty acid C20:4n-3 in human milk but not blood samples may contribute to the anti-inflammatory effect of human milk,
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| 169. |
- Yuan, Xi Ming, 1959-
(författare)
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LDL oxidation, iron, lysosomes, and macrophages in early atherosclerosis
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxidation of low density lipoprotein (LDL) may result in its uptake by macrophages with ensuing foam cell formation. Thus, oxidised LDL (oxLDL) may play an important role in atherogenesis. Extensive in vitro evidence is in favour of the notion that LDL oxidation by cells present in atherosclerotic plaques requires the presence of transition metals. The mechanisms by which LDL becomes oxidised in vivo and the effects of oxLDL on macrophages and foam cells, though, remain unknown.fu the first part of the present study we wanted to learn about the involvment of iron in the process of LDL oxidation by human macrophages; whether iron may be exocytosed following cellular exposure to different iron compounds; and if such exocytosis would affect LDL oxidation, and its uptake by macrophages. Human monocyte-derived macrophages (HMDMs) were exposed initially to different simple iron compounds (100 ~M), or haemoglobin (25 or 50 ~g/ml) for 24 hours. Following rinsing LDL (50 or 150 ~g/ml) was added in fresh culture medium without serum. After another 24 hours the concentrations of iron and thiobarbituric acid-reactive substances (TEARS), as well as the electrophoretic mobility of LDL, were found increased in the medium. Neutral lipids and phospholipids accumulated in a granular, lysosome-like, pattern and the cells acquired a foam cell-like morphology. Lipofuscin-specific autofluorescence was markedly increased in all iron and LDL-exposed cells. A linear correlation was found between lipofuscin formation, and the concentration of iron-complexes to which the HI\IDMs earlier had been pre-exposed.The second part of the study was designed (i) to establish a model of erythrophagocytosis by macrophages, and (ii) to study the iron-sequestration within secondary lysosomes and ironexocytosis by these cells following the degradation of erythrocytes. The binding and uptake of UV-irradiatedredblood cells (UV-RBC) by human macrophages and J-774 cells were greatly stimulated compared to that of native e1ythrocytes. The uptake resulted in lysosomal accumulation of iron in a low-molecular-weight form, as shown by autometallography. Following the exposure to UV -RBC or ferric iron a much enhanced amount of cytosolic ferritin was demonstrated in macrophages by immunocytochemistry. Ensuing exocytosis of iron to the culture medium was demonstrated by atomic absorption spectroscopy.The third part of the study aimed to localise the occurrence of iron in early atherosclerotic lesions from a number of consecutive autopsy cases with evident, general atheromatosis. With the SSM, we found foam cells to contain heavy metals with a mainly lysosomal localization. On the basis of the hypothesis that such a lysosomal accumulation of iron might be due to erythrophagocytosis by migrating tissue-bound macrophages that later develop into foam cells, we designed an in vitro model system in which human monocyte-derived macrophages were exposed to artificially aged, UV -exposed erythrocytes. The capacity of macrophages to oxidise LDL was found to be much enhanced following erythrophagocytosis, and the process was shown to involve secretion of iron. Consequently, LDL oxidation was greatly inhibited by desferrioxamine.The effect of oxLDL on cellular viability and lysosomal membrane stability was examined on cultured murine J -77 4 cells and human monocyte-derived macrophages (HMDMs) in the fourth part of this study. The acridine orange (AO) relocalisation test was applied to study the lysosomal integrity of living cells. UVoxLDL dramatically reduced cell proliferation at a concentration of 25 Jlglml. Incubation with 5 JlM copper alone, normally used to induce LDL oxidation, was also toxic. In contrast to the effects of oxLDL, in concentrations up to 75 J-Lg/ml, native LDL (nLDL) stimulated J-774 cell replication. Incubation with UVoxLDL (25-75 j.ig/nal) altered the cellular AO-uptake, depending on time and dose; the lysosomal accumulation decreased while the cytosolic accumulation increased. This shift indicates damaged lysosomal membranes with decreased intralysosomal and increased cytosolic proton (H+) concentration. Cells initially exposed to UVoxLDL changed into foam cells and subsequently assumed an apoptotic-type morphology.The fifth part of this study aimed to investigate the nature of accumulated iron, and its possible relation to the apoptotic process in human atherosclerotic lesions. Pronounced fenitinaccumulation, occurrence of low-molecular-weight iron, and apoptosis concerned mainlyCD68-positive iron-rich cells (macrophages) within the atherosclerotic lesions. No ferritin- or CD 68-positivity was found in normal coronary arteries from young forensic-autopsy cases, while a moderate number of such cells were observed in the intima of normal-looking vessel areas from the clinical cases with general atherosclerosis. In the atheroma intima, ferritin and iron were found in many CD68-positive macrophages which frequently were surrounded by erythrocytes. A substantial number of apoptotic cells within the intima, media, and adventitia were registered in all atherosclerotic lesions examined, although mainly in the macrophageenriched area of the atheroma shoulder.In conclusion: A. Lysosomal iron may be exocytosed from HMDMs, following a previous uptake of simple iron compounds or Hb, promoting oxidation, uptake of LDL, and foam cell formation. B. Macrophage erythrophagocytosis is a useful model for the study of the lysosomal sequestration of iron in cell-mediated LDL oxidation. Iron is accumulated within the macrophage acidic vacuolar apparatus and subsequently exocytosed. C. Iron promotes lipofuscin formation in the LDL-macrophage system, supporting the concept that lipofuscin accumulates in lysosomes as a result of iron-catalyzed lipid peroxidation. D. Iron, stored as ferritin, may occur in macrophages, and macrophage-derived foam cells as a consequence of erythrophagocytosis or phagocytosis of apoptotic cells. E. OxLDL, but not native LDL, is cytotoxic to macrophages. The cytotoxic effect of oxLDL may result from oxidative damage of lysosomal membranes, with ensuing destabilisation and leakage into the cytosol of lysosomal contents, such as hydrolytic enzymes. F. Dysregulated iron- and ferritin-metabolism within macrophage/foam cells suggest that iron/ferritin may be associated with ongoing apoptosis in vivo, contributing to the instability of atherosclerotic plaques.
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| 170. |
- Zalavary, Stefan
(författare)
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Adenosine Regulation of Neutrophil Function
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The neutrophil granulocyte is an essential component of the human immune system. When rrricrobes invade the body, the highly motile neutrophils quickly leave the vascular compartment and approach, engulf and kill the intruders by releasing a battery of antimicrobial substances. Adenosine is an endogenous purine nucleoside that is supposed to regulate the inflammatory response, for example, by modulating the activity of neutrophils through occupation of A1 and Az receptors. The aim of the present work was to characterize the effects of adenosine on neutrophil functions, particularly phagocytosis and the production of reactive oxygen species (ROS).Adenosine modulated IgG~mediated phagocytosis in neutrophils in a Ca2+dependent way via stimulatory At and inhibitory Az receptors. The Az receptor~mediated inhibition was associated with an elevation of cAMP, which probably activated protein kinase A (PKA) and thereby interfered with actin dynarrrics and expression of BZ integrins. Production of ROS induced by the chemotactic peptide N~formyl-methionylleucyl~ phenylalanine (fMLP) or IgG~opsonized yeast particles was not affected by occupancy of the At receptor, but was, via a cAMP~dependent mechanism, inhibited by engagement of the A2 receptor. Adenosine also reduced L-selectin-induced production of ROS. The inhibition of ROS production by adenosine was more pronounced during fMLP stimulation than during L~selectin~ and IgG~mediated activation, probably because the latter entailed removal of extracellular adenosine through increased activity of adenosine dearrrinase (ADA).Platelets participate in inflammatory reactions, for instance, by affecting neutrophil actiyity. In the present work, platelets inhibited !MLP~stimulated extracellular generation of ROS by inducing the neutrophils to release adenosine and to form a peripheral actin barrier. In contrast, platelets potentiated IgG~mediated phagocytosis and generation of ROS in neutrophils. This involved engagement of neutrophil Pz receptors by plateletderived ATP and enhanced formation of cortical actin filaments. Expanded ADA activity associated with !gO-stimulation counteracted the inhibitory effects of adenosine accumulated during neutrophil-platelet interaction.In conclusion, this work accentuates the importance of adenosine, both exogenously applied and endogenously formed, as an inflammatory agent modulating the activity of the neutrophil granulocyte.
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