Down-regulation of thyroxine-binding globulin messenger ribonucleic acid by 3,5,3'-triiodothyronine in human hepatoblastoma cells.

A sensitive [125I]-T4 binding assay was used to measure serum T4-binding globulin (TBG) in 60 individuals selected on the basis of their total circulating T3 concentrations, and a relationship between TBG and circulating thyroid hormone levels in humans was confirmed. There was a significant correlation between serum TBG and T3 or free T4 index. TBG secretion and TBG messenger ribonucleic acid (mRNA) production were studied with a continuous culture of the human hepatoblastoma cell line, HepG2. Cells were maintained in serum-free media for experimental manipulations. The addition of 100 nmol/L T3 to the cell medium resulted in a time-dependent down-regulation of TBG mRNA to 33 +/- 6% (+/- SD, n = 4) of untreated control levels by 24 h. Suppression of TBG mRNA was first detectable at 8 h (57% of untreated control levels). The effect of T3 was dose-responsive, with half-maximal suppression of TBG mRNA occurring at a bioavailable T3 concentration of approximately 30 pmol/L. The effect of T3 on TBG mRNA was not caused by a change in mRNA stability. Proteins secreted by HepG2 cells bound T4 with an affinity identical to that of normal circulating TBG. Cell secretion of TBG was parallel to total protein secretion and consistent with a TBG secretion rate of 50 ng/10(6) cells per day. Variations in the concentration of secreted binding protein in the presence of T3 corresponded to the changes observed in TBG mRNA. These data show that circulating TBG concentration is negatively correlated with total serum T3 in vivo. The corresponding down-regulation observed between TBG mRNA and secreted protein in HepG2 cells suggests that this effect is the result of the action of T3 on cellular TBG mRNA synthesis.

Stimulation of sex hormone-binding globulin mRNA and attenuation of corticosteroid-binding globulin mRNA by triiodothyronine in human hepatoma cells.

We examined the time course and dose response of the triiodothyronine (T3) effect on mRNAs for sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) in cells of the human hepatoma line HepG2. After 7 h of exposure to a saturating dose of T3, SHBG mRNA was unchanged but increased to 1.5 +/- 0.1 times the unstimulated control at 22 h. Maximal stimulation (2.3 +/- 0.6) was observed at 2-3 days. Corticosteroid-binding globulin mRNA was unchanged for 22 h after exposure to T3 but diminished thereafter to 64% by day 3. At 3-4 days of exposure, the changes in both SHBG mRNA and CBG mRNA were dose-responsive to the T3 concentration. For both mRNAs, half-maximal response occurred between 10 and 20 pmol/l bioavailable T3. Cortisol-binding proteins secreted by HepG2 cells after 3 days in culture also were T3 dose-responsive. No re-uptake of secreted CBG by the cells was observed, suggesting that the T3 effect on CBG secretion occurs during production of the mature protein. These data suggest that T3 stimulates the expression of the SHBG gene and attenuates the expression of the CBG gene. The effects of T3 on these genes are consistent with the increase in circulating SHBG and decrease in circulating CBG observed in hyperthyroidism. The HepG2 cells may be a useful human cell line in which to study the diversity of the molecular mechanisms of T3 action.

Characterisation of the host response to Plasmodium falciparum infection. I. Cerebral malaria.

Cerebral malaria is a major form of complicated malaria consequent upon cerebral damage associated with endothelial cell necrosis. We have used assays of Plasmodium falciparum growth inhibition in vitro to study serum inhibitory factors in patients with cerebral malaria. Serum from children with cerebral malaria inhibited parasite growth in a non-synchronised 72-hour assay to a greater extent than did sera from immune adults or asymptomatic children (p less than 0.001). The high level of non-specific inhibition of parasite growth was particularly evident when sera were tested against three P. falciparum isolates, and contrasted with the inhibitory effect of sera from non-malaria febrile controls. In this study, serum from patients with cerebral malaria was more inhibitory than serum from the other groups (p less than 0.001) and its between-isolate variation, when tested against a panel of P. falciparum isolates in growth assays, was significantly less than that of the other groups tested (p less than 0.005). These results are consistent with the hypothesis of toxin-induced endothelial cell damage, with the sequence of pathogenic events involving host-derived serum factors capable of damaging P. falciparum.

Characterisation of the host response to Plasmodium falciparum infection in acute non-complicated malaria.

In this study we have examined serum from patients with Plasmodium falciparum malaria, collected at the time of acute attack and 14 days later. We have also examined sequential samples of sera taken from children living in Madang Province, Papua New Guinea, an area endemic for malaria. The total amount of antibody directed against P. falciparum in acute phase sera was less than that found in matched controls. An in vitro assay measuring inhibition of penetration of uninfected erythrocytes by merozoites of P. falciparum revealed less inhibitory activity in acute phase sera than in matched controls. The longitudinal study of sera from village children demonstrated that non-specific inhibition of intracellular parasite growth was fairly stable, while merozoite inhibiting activity was unstable and varied with time. A cloned P. falciparum species has been used to directly demonstrate the specific growth enhancement by serum in vitro.

Analysis of patterns of growth inhibition of P. falciparum in synchronised cultures induced by serum from children and adults from Madang, Papua New Guinea.

In this study we have modified a micro Plasmodium falciparum in vitro growth inhibition assay to allow dissection of growth inhibition induced by test sera into two categories: inhibition of intracellular schizont growth and inhibition of uptake of merozoites into a second cohort of erythrocytes. This was achieved using morphology-controlled synchronised cultures with incubation times restricted to cover either ring to schizont development or schizont to ring development. Sera tested were obtained from a large prospective study of healthy residents of Madang, Papua New Guinea, who were carefully documented with respect to presence of malarial parasites in the blood, spleen size, age, sex and history of fever. In the ring to schizont assay sera from all the children tested inhibited parasitic growth by at least 20%, compared to only 10 of 39 adults tested (P less than 0.0005). In the schizont to ring assay 20 of 39 adult sera tested inhibited the uptake of 14C-isoleucine into P. falciparum protein compared to 3 of the 15 children's sera tested (P less than 0.01). These results are interpreted as reflecting a switch from non-specific mechanisms of resistance with increasing age.