Serum ferritin and isoferritins are tools for diagnosis of active adult Still's disease.

OBJECTIVE: Still's disease is an acute systemic inflammatory disorder. There are no pathognomonic symptoms or specific laboratory abnormalities. Serum ferritin concentration in rheumatoid arthritis together with some plasma glycoproteins such as alpha 2-glycoprotein and C-reactive protein are part of the response to inflammation. Ferritin in plasma is glycosylated and the sialoglycosylated forms increase its microheterogeneity. Our purpose was to confirm in a large series that high values of ferritin can be found in adult Still's disease (ASD) and to see if a specific isoferritin can be isolated in this disease compared with the other systemic diseases. METHOD: Thirty-one sera were investigated from 11 men and 9 women with ASD and compared with 27 sera from 27 patients with systemic diseases. We studied the course of one case of ASD for 15 months. Serum ferritin was determined by immunoenzymology (Abbott Ferrizin). The isoferritins were investigated by isoelectric focussing and the percentage of glycosylation by affinity for concanavalin A (Con-A). RESULTS: In patients with active ASD, the ferritin levels were higher than in patients with inactive ASD or other systemic diseases: p < 0.001. The glycoforms of ferritin were basic and the proportion of ferritin bound to Con-A was lower than other ASD: p < 0.001. CONCLUSIONS: Serum ferritin levels have a diagnostic value for acute ASD. The study of sialylation and abnormalities in the glycosylation of ferritin helps to discriminate ASD from arthritis or other systemic diseases. In conclusion, the glycoform of isoferritins and the percentage of glycosylation offers an additional tool for the diagnosis of Still's disease.

Comparative effects of diabetes and monensin on the lectin asialoglycoprotein receptor: biosynthesis, structure and function in rats.

The hepatic asialoglycoprotein receptor is the first studied mammalian lectin. Modulations in vivo by diabetes and in vitro by the carboxylic ionophore monensin gave rise to similar apparent alterations on its biosynthesis, structure and ligand binding capacity. In normal rats, the receptor (whether purified by ligand or antibody-affinity chromatography) presented a similar pattern in SDS-PAGE analysis, with a major 42-kDa band and two minor ones (49 and 52-54 kDa). In diabetic rats, a new 38-kDa band appeared, but only after antibody-affinity purification. In vitro biosynthesis of the receptor by normal hepatocytes in the presence of 35S-methionine showed that this 38-kDa band was present at the end of a 30-min pulse but decreased during a 180-min chase, in association with an increase in the major 42-kDa band. In diabetic cells, this evolution was retarded. Using a 30-min pulse followed by a 120-min chase in the presence of 100 microM monensin, we showed that this carboxylic ionophore had similar effects on diabetes, leading to a delay in the maturation process of the 42-kDa band and the persistent emergence of the 38-kDa species. Allowing incubation in the presence of 25 or 100 microM monensin, we observed a decrease in the number of ligand binding sites both at the surface (40%) and within the cell (28%). In hepatocytes from diabetic rats, monensin showed no additional effect on the partial diabetes-induced inactivation.

Plasma free amino acid profiles and nutrition proteins in chronic renal failure; effect of dialysis treatment.

A well preserved nutritional status is beneficial in chronically uremic patients for slowing the pace of deterioration of renal function, and delaying the need for dialysis therapy. The purpose of this study was to assess the nutritional profile of 10 patients in a steady state of advanced CRF, and of 15 patients with terminal renal failure immediately prior to their first hemodialysis session (J0), and 7, 14, 45, 60, days post start of dialysis. Patients were 18 to 65 years old with total plasma proteins ≥ 60g/1. Plasma concentrations of amino acids, nutrition proteins, apolipoproteins A1, and B were evaluated. Non inflammatory reaction was evaluated by determination of alpha-1-acid glycoprotein, and C reactive protein. The data (mean ± 1 SD) were compared with mean values of 15 healthy individuals.

A macrophage-derived factor induced by alpha 1-acid glycoprotein that inhibits IL-1 comitogenic activity.

After exposure to a concanavalin A (Con A)-unreactive variant of alpha 1-acid glycoprotein (AGP), macrophages released an inhibitor of interleukin-1 (IL-1) proliferative activity in the thymocyte comitogenic assay. This effect was observed with AGP concentrations above 100 micrograms/ml in the macrophage supernatant and would appear to be mediated by the macrophages, since native AGP had no activity on thymocyte proliferation. Preliminary physicochemical characterization showed that the factor was partially resistant to heating, undialyzable, and eluted with an apparent molecular mass of 50-100 kDa when subjected to Sephacryl S-200 chromatography. Murine IL-1 and human (h) recombinant (r) IL-1 were affected by this factor to the same extent. IL-1 and IL-2 co-induced thymocyte proliferation, which is mitogen-independent, was also inhibited, whereas hrIL-2 activity was not suppressed when assayed in thymocytes with PHA at a submitogenic concentration or in CTLL cells. The factor did not interfere with TNF alpha or hrIL-6 activity when tested against their specific cell line. These data indicate that the inhibitor may act specifically against IL-1 activity and further elucidate the possible role of AGP in the modulation of IL-1 activity via the secretion of an inhibitor.

Prevalence of tri- and tetraantennary glycans of human alpha 1-acid glycoprotein in release of macrophage inhibitor of interleukin-1 activity.

Based on the affinity for concanavalin A (Con A), human alpha 1-acid glycoprotein (AGP) can be separated by chromatography on Con A-Sepharose gel into three variants: Con A unreactive AGP, Con A weakly reactive AGP, and Con A strongly reactive AGP. When exposed to native AGP or to its glycan variants, murine peritoneal macrophages released a factor that inhibited the interleukin-1 (IL-1) proliferative activity as measured in terms of the thymocyte comitogenic assay. Con A unreactive AGP, which contains tri- and tetraantennary glycans and no biantennae, proved to be more effective than Con A weakly and Con A strongly reactive variants, which contain one and two diantennary glycans, respectively. The inhibitory effect was not a function of the negative charge related to the sialyl residues and was not mediated by the mannosyl-fucosyl receptor.

Action of ornithine alpha ketoglutarate on DNA synthesis by human fibroblasts.

Ornithine alpha ketoglutarate (OKG) is largely used in clinical nutrition for its anabolic effects. However, the mechanism of its action remains questionable. We investigated the effect of OKG on the rate of DNA synthesis in human fibroblasts. The in vitro experimental procedure required to demonstrate in cell culture the anabolic effects of OKG observed in vivo was found to be glutamine-free and serum-poor medium with sparse cells. In these conditions, OKG induced a significant increase in [3H]thymidine incorporation compared to untreated control cells. This effect was dose-dependent and was observed in all the cultures tested. Taken individually, the two constituents of OKG, i.e. alpha KG and Orn, also showed a stimulatory effect, but did not demonstrate a dose-dependent response. Concomitant analysis of extracellular aminoacids showed in alpha KG-treated cultures an increase in glutamate and a decrease in aspartate, suggesting a cellular transamination of alpha KG. Glutamine, which is the preferential energetic substrate of fibroblasts, can be produced from glutamate and might play a role in the action of OKG. Moreover, OKG induced a rise in the cellular polyamine content. This, in association with the inhibitory effect on OKG action of difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suggests a link between the polyamine biosynthesis pathway and the anabolic effect of OKG.

Effects of human recombinant IL-1 beta on rheumatoid and non rheumatoid human synovial cell growth.

The growth of adherent synovial cells passaged once was studied in response to human recombinant interleukin 1 (hr IL-1) beta. Human synovial cell cultures were established from tissues obtained during therapeutic joint surgery for patients with rheumatoid arthritis (rheumatoid synovial cells, RSC) or non inflammatory rheumatic diseases (non rheumatoid synovial cells, NRSC). The effect of IL-1 beta (0.1 to 10 ng/ml) on the time course of proliferation showed that values for DNA synthesis and cell numbers in RSC cultures were higher than in NRSC cultures. Similarly, untreated control RSC cultures grew more quickly than NRSC. These results demonstrate that RSC, which are continuously stimulated by IL-1 beta produced in the rheumatoid pannus in vivo, have a higher capacity for proliferation than NRSC but are less responsive to IL-1 beta. A dose-response curve of proliferation was established 72 hrs. after the addition of IL-1 to the medium. The stimulating effect of IL-1 beta (0.001 to 10 ng/ml) was dose-dependent in both RSC and NRSC and reached a plateau at 10 ng/ml; the response of NRSC was stronger than that of RCS.

Comparative proliferation of non-rheumatoid and rheumatoid human synovial cells.

While normal synovial membrane cells have a very long doubling time, rheumatoid arthritis increases cell turn-over leading to the formation of a pannus. We studied the comparative proliferative behaviour in culture of synoviocytes of rheumatoid (RA) and non-rheumatoid (NR) origin in order to evaluate the usefulness of this model to investigate the drugs used in the treatment of inflammatory diseases. First-passage cultures of cells from patients with clinically defined non-inflammatory joint disease or rheumatoid arthritis were observed for 8 days. In the presence of various combinations of supplemented media, 3H-thymidine incorporation, protein content and cell density were assessed. In addition to a relationship between the fetal calf serum (FCS) concentration and cell growth, it was found that RA cells proliferated more rapidly than NR cells. In 1% FCS, protein content and cell density increased in RA cultures whereas NR synoviocytes accumulated in the quiescent phase. In 5% and 10% FCS, RA cells responded more strongly than NR in terms of protein and DNA synthesis and cell division. After 48 hours of relative FCS deprivation, NR cells abruptly started to proliferate; the response of RA cultures was delayed, but the synoviocytes quickly reached preconfluence.

Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition.

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.

Increased affinity to concanavalin A and enhanced secretion of alpha 1-acid glycoprotein by hepatocytes isolated from turpentine-treated rats.

Hepatocytes were isolated from adult rats at various times after subcutaneous injection of turpentine (1 ml). The affinity to concanavalin A (Con A) of alpha 1-acid glycoprotein (AGP) and the intracellular content and rate of secretion of AGP and albumin were evaluated over a period of 19 days. Inflamed hepatocytes secreted mainly the Con A-reactive form of AGP whereas control hepatocytes secreted a higher amount of the Con A-non-reactive form. The intracellular content and rate of secretion of AGP by inflamed hepatocytes increased markedly whereas those of albumin decreased. However, when the residence time (ratio of intracellular content to rate of secretion) was evaluated, it appeared that the efficiency of secretion of both proteins was higher than in control hepatocytes. The changes in the affinity of AGP to Con A and in the secretion of AGP and albumin were reversible. These findings indicate that acute inflammation leads to posttranslational alterations during the intracellular transit of these secretory proteins.

Fate of enterally administered ornithine in healthy animals: interactions with alpha-ketoglutarate.

In order to improve our understanding of the metabolic interactions between alpha-ketoglutarate (alpha KG) and ornithine (Orn), which constitute the two parts of ornithine alpha-ketoglutarate (OKG) used as an adjuvant in enteral nutrition, we have investigated the plasma appearance and tissue distribution (qualitative and quantitative) of enterally administered 14C-Orn and 14C-alpha KG in healthy mice and rats. The influence of unlabelled alpha KG or Orn on 14C-Orn or 14C-alpha KG metabolism, respectively, was also studied. Unlabelled alpha KG was able to reduce strongly the rate of intestinal absorption of 14C-Orn, whereas the inverse was not true. This alpha KG-induced loss in plasma radioactivity after a load of Orn was associated with a decrease of radioactivity in tissue with no modification of the qualitative distribution in organs. In this study, a direct interaction between alpha KG and Orn was demonstrated at the intestinal level. The mechanisms involved in this phenomenon probably involve the regulation of metabolic conversions among alpha KG, Glu, pyrroline-5-carboxylate, and Orn. This is of importance in the therapeutic use of ornithine salts in clinical nutrition.

Human alpha 1-acid glycoprotein-exposed macrophages release interleukin 1 inhibitory activity.

The concanavalin A-unreactive variants of alpha 1-acid glycoprotein introduced in culture medium of monocytes/macrophages induces the inhibition of thymocyte proliferative activity of interleukin 1. LPS or LPS receptors were not involved in the activity of alpha 1-acid glycoprotein on macrophages. alpha 1-acid glycoprotein did not show any direct effect on thymocytes whereas the monocyte/macrophage-supernatant inhibited the interleukin 1 proliferative effect. The activity of tumor necrosis factor and interleukin 2 was not altered by the alpha 1-acid glycoprotein-macrophage supernatant.

Sensitivity of cultured fibroblasts to human, bovine and porcine insulins.

We have studied the effects of human, bovine and porcine insulin on sugar transport by cultured chicken embryo fibroblast monolayers. For a 30 min. association time, human and bovine insulin at a concentration of 5.10(-8) M stimulated 2-deoxy-D-glucose uptake. (respectively by an average 58 p.cent and 55 p.cent over basal). Porcine insulin was less potent since a concentration of 5.10(-7) M was necessary to obtain similar stimulation. Moreover, the maximal effect of porcine insulin occur only after 60 min. association time instead of 30 min. for the other peptides. The differences between the effects of insulin from different sources is related to species-dependent differences in their structure.

Alterations in the carbohydrate moiety of alpha-1-acid glycoprotein purified from human cirrhotic ascitic fluid.

The carbohydrate analysis of alpha 1-AGPc purified from cirrhotic ascitic fluid was performed by immunoaffinity chromatography. It showed a large increase in the fucosyl molar ratio and sugar content (47%). The molar ratio of the oligosaccharides which were released by hydrazinolysis and fractionated by high-performance liquid chromatography confirms the marked increase in fucosyl residues in each fraction. A shift towards fractions with a high degree of branching was also observed. Moreover, the studies of sugar molar ratios and methylation of the tetrasialylated fraction indicated the simultaneous presence of sialyl and fucosyl residues on one of the outer branches.

Effect of insulin on aminoisobutyric acid uptake by human non-rheumatoid and rheumatoid synovial cells.

The biological sensitivity of cultured non-rheumatoid human synovial cells (NRSCs) and rheumatoid synovial cells (RSCs) was examined in terms of the ability of insulin to stimulate the uptake of alpha-aminoisobutyrate (AIB). NRSCs, like numerous fibroblastic lines, were sensitive to physiological concentrations of the hormone: half-maximal stimulation was obtained with (4 X 10(-10) M) insulin, while maximum transport was found with a 60-90 min association time. On the contrary, although the basal transport was similar in RSCs, insulin was totally unable to accelerate AIB transport in these cells. Inflammatory processes lead to an insulin resistance which most likely involves a post-receptor step at the cellular level.

Behavior of aldehyde moieties involved in the activation of suppressor cells by sodium periodate.

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.

Modulation of insulin action on 2-deoxyglucose uptake by chloroquine in chick embryo fibroblasts.

Blazar et al. recently found that chloroquine therapy decreased intravenous insulin requirements in a case of extreme insulin resistance. However, no relationship has been shown to exist between insulin degradation and the stimulation of glucose uptake. In this study we investigate the action of insulin on glucose uptake by the ability of this hormone to stimulate 2-deoxyglucose. The effect on alpha-aminoisobutyrate uptake, which is known to be insulin sensitive, is also investigated. Cell-associated 125I-labeled insulin and trichloroacetic acid-soluble and -precipitable substances were measured in parallel. Chloroquine increased insulin-stimulated uptake of 2-deoxyglucose and alpha-aminoisobutyrate. Three hours were required for this effect to appear, and it did not depend on DNA synthesis. Chloroquine also increased cell-associated insulin and slightly decreased the percentage of trichloroacetic acid-soluble products. Methylamine affected neither nutrient uptake processes nor insulin binding and degradation; however, it did abolish the effect of chloroquine on these parameters. These data suggest that in chick embryo fibroblasts a relationship may exist between the increase in undegraded cell-associated insulin and the ability of the hormone to stimulate sugar and amino acid uptake.