Antioxidants may contribute in the fight against ageing: an in vitro model.

Elderly humans have altered cellular redox levels and dysregulated immune responses, both of which are key events underlying the progression of chronic degenerative diseases of ageing, such as atherosclerosis and Alzeimer's disease. Poorly maintained cellular redox levels lead to elevated activation of nuclear transcription factors such as NFkB and AP-1. These factors are co-ordinately responsible for a huge range of extracellular signalling molecules responsible for inflammation, tissue remodelling, oncogenesis and apoptosis, progessess that orchestrate many of the degenerative processess associated with ageing. It is now clear that levels of endogenous anti-oxidants such as GSH decrease with age. This study aimed to investigate the potential of exogenous anti-oxidants to influence inflammatory responses and the ageing process itself. We investigated the potential of the dietary antioxidant, quercetin, to reverse the age related influences of GSH depletion and oxidative stress using in vitro human umbilical vein endothelial cells (HUVEC) and human skin fibroblast (HSF) cell models. Oxidative stress-induced inflammatory responses were investigated in a GSH depletion and a Phorbol 12-myristate 13-acetate (PMA)-induced stress model. As measured with a sensitive HPLC fluorescence method, GSH in HUVEC was depleted by the addition of L-buthionine-[S,R]-sulfoxiniine (BSO), a gamma-glutamylcysteine synthetase inhibitor, to the culture medium at a concentration of 0.25 mM. Time course studies revealed that the GSH half-life was 4.6 h in HUVEC. GSH depletion by BSO for 24 h led to a slight increase in intracellular adhesion molecule - 1 (ICAM1) expression and prostaglandin E2 (PGE2) secretion in both types of cells. However, GSH depletion markedly enhanced PMA-induced ICAM and PGE2 production in HUVEC. Responses were progressively elevated following prolonged BSO treatment. Inhibition studies showed that 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a protein kinase C (PKC) inhibitor, not only abolished most of PMA-induced ICAM-1 expression and PGE2, production, but also eliminated GSH depletion-enhanced PMA stimulation. This enhancement was also inhibited by supplementation with quercetin. The results clearly demonstrate that GSH depletion increased the susceptibility of vascular endothelial cells and fibroblasts to oxidative stress associated inflammatory stimuli. This increased in vitro susceptibility may be extrapolated to the in vivo situation of ageing, providing a useful model to study the influence of micronutrients on the ageing process. In conclusion, these data suggest that dietary antioxidants could play a significant role in the reduction of inflammatory responses.

Modelling coccidial infection in chickens: emphasis on vaccination by in-feed delivery of oocysts.

Trickle immunization of poultry by incorporating wildtype strains of coccidia in the feed has been shown to be an effective and practical means of controlling coccidiosis. Here we develop a mathematical model of the life cycle of Eimeria tenella and estimate the model parameters from extant experimental studies. Numerical solutions of the model compare well with experiment. The model provides quantitative estimates of the required trickle immunization hitherto only available after extensive time-consuming trials.

Inter-relationship between mucosal and systemic immunity determining the balance between damage and defense in the bovine gut in response to environmental antigens.

At weaning, the complex interactions of macromolecular components embracing maternally derived antibodies and antigens of microbial and dietary origin, grossly influence the immune response, directing it towards protective or damaging reactions in the intestinal mucosa. In young calves, passively acquired maternal antibody surprisingly enhances the systemic IgG1 mediated type III hypersensitivity reactions to dietary antigen. Furthermore, unresponsiveness to orally administered protein antigen in the young calf fails to develop. Additionally, antigens from gram negative bacteria can influence the development of IgE mediated hypersensitivity reactions. In the normal deleterious gut reactions to protein antigens from the lumen, IgE responses infrequently occur, and are transient. However, these can be reinforced by simultaneous challenge with protein and microbial antigen. These observations bring some new perspectives on health and nutrition of young farm animals at weaning.

Hypersensitivity to dietary components in young farm animals: immunisation of calves for IgE antibody responses.

An immunisation schedule is described that reproducibly induces long latent period skin sensitising antibody (immunoglobulin) in calves by a combined oral/parenteral route. This response is only directed towards the protein component of the antigen and is boosted upon challenge injection.

Hypersensitivity to dietary components in young farm animals: isolation and partial purification of bovine immunoglobulin E.

An antibody of known specificity and active in long (72 hours) latent period passive cutaneous anaphylactic reactions, was isolated and partially purified from bovine serum. This antibody was not associated with immunoglobulins IgG, IgM or IgA. A rabbit antiserum raised against this antibody and used as an immunoabsorbent, successfully recovered skin sensitising antibody from bovine reaginic serum.

Soya protein antibodies in man: their occurrence and possible relevance in coeliac disease.

Circulating antibodies to soya-derived protein antigens have been measured in patients with duodenitis, Crohn's disease, ulcerative colitis and coeliac disease. Significantly raised antibody titres were found frequently in the coeliac group, particularly those patients showing a suboptimal response to a gluten-free diet, but rarely in subjects with other gastrointestinal diseases. Antisoya activity was not necessarily accompanied by antibodies to other common dietary antigens. We suggest that some coeliacs may have an associated dietary soya sensitivity which could adversely influence their response to gluten withdrawal.

Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets.

Serum antibody responses to ingested aqueous alcohol-extracted soya proteins were studied in thirty-six pre-ruminant calves. Characterization of this antibody showed it to be predominantly a complement-fixing IgG1 preciptin. No evidence of tolerance was seen; previously sensitized calves responded to reintroduction of a soya diet with marked increases in antibody levels. The soya antigen was shown to be resistant to proteolysis and, to a lesser degree, to the microbial action of rumen fluid. Biopsy studies showed that the feeding of soya protein resulted in morphological disturbances to the villi and lamina propria of the intestine. Physiological studies by Thirty-Vella loop perfusion in the pig showed that soya protein solutions resulted in significant inhibition of flow rates. The effect was only observed after previous sensitization with the soya antigen. This study shows the necessity of applying immunological criteria to the quality control of soya bean processing in order to ensure that the sensitizing agent is eliminated and the nutritional qualities of soya protein concentrates are optimized.

The pig synovium, II. Some properties of isolated intimal cells.

I. The effect of trypsinization. Certain physiological and immunological properties of isolated intimal synoviocytes were studied in various in vitro systems. Suspensions of the synoviocytes were obtained by incubating sheets of joint capsule in a solution of trypsin and then scraping off the intimal cells with a small knife. Synovium was examined immediately after trypsinization, but before scraping, by light and electron microscopy. The intimal cells had withdrawn many of their long processes, acquired lamelliform pseudopodia, and somewhat resembled cells of the A-type (Barland et al. 1962). To test the viability of the trypsinized synovium, fragments of trypsinized joint capsule were explanted in organ culture with the intima in contact with a Millipore substrate (Fell et al. 1976). After two days the intimal synoviocytes had become branched and, as in untreated control explants, now resembled B-cells. II. Phagocytosis and opsonic adherence. In a freshly prepared suspension of synoviocytes scraped from trypsinized synovial tissue many cells were still branched, but during 1 1/4 hours' incubation in serum-containing medium the majority had withdrawn their processes and become rounded. Cells in the branched form had little capacity for phagocytosis, but most of those in the rounded form were actively phagocytic. After suspensions of intimal cells had been incubated with opsonized sheep erythrocytes, most of the rounded, but none of the few remaining branched cells, had formed rosettes. Intimal cells from scraped synovial tissue were maintained in Sykes-Moore chambers for periods of up to 48 hours. Although the young pig synovium contains only a small proportion of macrophage-like (A-type) cells, in the cultures the cell population consisted of cells indistinguishable from macrophages, with a few small colonies of typical fibroblasts. In marked contrast to the fibroblasts, the macrophage-like cells in the Sykes-Moore cultures were highly phagocytic and formed conspicuous opsonic rosettes. Excessive phagocytosis inhibited subsequent rosetting by the macrophage-like cells. III. The effect of antiserum, with and without complement, on synovial cells. In view of earlier work by Fell & Barratt (1973) the effect of rabbit antiserum to pig erythrocytes (AS) with and without serum complement (C') on intimal synoviocytes in a Sykes-Moore chamber was investigated. AS+C' either lysed the macrophage-like cells or caused them to fuse into multinucleate giant cells. In the presence of AS without C', the macrophage-like cells formed large lakes of multinucleated cytoplasm. Fibroblasts in the same cultures were much more resistant to lysis by AS+C' and did not form multinucleate giant cells when exposed to AS either with or without C'.

The pig synovium. I. The intact synovium in vivo and in organ culture.

1. The normal synovium of the metacarpophalangeal joints of young pigs was examined by light and electron microscopy with special reference to the superficial layer (intima). 2. Cells of the macrophage-like or A-type (Barland et al. 1962) constituted only a small proportion of the intimal synoviocytes; the majority were of the intermediate and B-types. 3. Synovial villi were explanted on Millipore filters and maintained as organ cultures. The intimal cells in contact with the Millipore formed long branched processes which penetrated deeply into the substrate; these cells, which had a very well-developed endoplasmic reticulum, resembled those of the B-type. The synoviocytes at the upper (free) surface of the villus withdrew their long processes, acquired lamelliform pseudopodia, and their endoplasmic reticulum regressed; they were similar in appearance to the A-type. 4. In the organ cultures the highly branched cells (B-type) next to the Millipore were less phagocytic than the rounded cells (A-type) at the free surface of the villus.

The capacity of pig articular cartilage in organ culture to regenerate after breakdown induced by complement-sufficient antiserum to pig erythrocytes.

Explants of pig articular cartilage including invading marrow and subchondral bone (together='invasion zone') were cultivated for 10 or 14 days in complement-sufficient rabbit antiserum to pig erythrocytes (AS+C'), and then transferred to heat-inactivated normal rabbit serum (NRS) for a period of recovery. In AS+C' the cartilage matrix lost first proteoglycan and then collagen, but the cells remained viable. The degradation of collagen was accompanied by a fibroblastic transformation of the chodrocytes, first seen in the region immediately above the invasion zone. Immunohistochemical studies showed that after cultivation in AS+C', IgG antibodies entered areas in which the matrix was depleted of proteoglycan and reacted strongly with the majority of chondrocytes; those that had under-gone fibroblastic transformation exhibited little or no reaction. The degree of recovery in NRS depended on the extent to which the matrix had broken down in AS+C'. If degradation of collagen was confined to the region immediately above the invasion zone, and elsewhere only proteoglycan had been lost, new metachromatic material was regenerated in the non-calcified cartilage, and the fibroblast-like chondrocytes resumed their normal appearance and regained their reactivity with the IgG antibodies of AS; new cartilage and chondroid tissue appeared in the cavities of the invasion zone. If degradation of collagen and fibroblastic transformation of chondrocytes had spread throughout the cartilage, breakdown continued in NRS and cartilage disappeared completely above the invasion zone; new cartilage waw sometimes formed in the cavities of the invasione zone.

Breakdown of proteoglycan and collagen induced in pig articular cartilage in organ culture.

Explants of articular cartilage from young pigs were maintained in organ culture for 10--16 days, and degradation of matrix was induced by retinol or complement-sufficient antiserum. The percentage breakdown of proteoglycan and collagen (as hydroxyproline release) was measured. The response of the cartilage depended on whether or not the explants were cut so as to include some of the invading marrow ('invasion zone'). In media containing retinol, cartilage lost up to three-quarters of its proteoglycan whether the invasion zone was present or not, but very little of its collagen unless this region was included. In the presence of complement-sufficient anti-serum, however, cartilage without the invasion zone was virtually unaffected, but both proteoglycan and hydroxyproline were released when invasion zone was included; here proteoglycan release began almost immediately, but there was a time-lag of 6--8 days before a substantial amount of hydroxyproline appeared in the medium. Histological examination of sample explants from the experiments supported the biochemical findings. The possible significance of the results in relation to rheumatoid arthritis is discussed.

Role of the chondrocytes in the breakdown of pig articular cartilage induced by complement-sufficient antiserum to pig erythrocytes.

Previous work showed that in organ culture pig articular cartilage is not affected by complement-sufficient antiserum to pig erythrocytes (AS plus C') unless it is associated with soft connective tissue. In the presence of synovial tissue the matrix becomes depleted of proteoglycan and may finally disintegrate. Immunoglobulin fails to enter normal cartilage matrix, but penetrates depleted matrix and reacts with the surfaces of the chondrocytes. The present experiments were made to see whether the breakdown of partially depleted cartilage matrix would progress in AS plus C' after removal of the synovial explant. Affronted explants of articular cartilage and synovial tissue were cultivated in AS plus C' for 10 days (primary cultures). The synovial tissue was then removed and the isolated cartilage maintained for a further period either in AS plus or C' or in one of a variety of control media (SECONDARY CULTURES). The behavior of the isolated cartilage during the secondary culture period depended on the degree of breakdown attained at the end of the primary period. If only proteoglycan but not collagen had been seriously depleted, degradation of the matrix did not progress in AS plus C' and slight regeneration of metachromatic materal sometimes took place; regneration of matrix was greater after transfer to control medium. If collagen as well as proteoglycan had been destrobyed, the chondrocytes assumed a fibroblastic form, their surfaces no longer reacted with IgG antibody, and in secondary culture they failed to regenerate new matrix.