Inhibition of intrapituitary thyroxine to 3.5.3'-triiodothyronine conversion prevents the acute suppression of thyrotropin release by thyroxine in hypothyroid rats.

Iopanoic acid has been shown to block thyroxine (T4)-5'-monodeiodination in rat anterior pituitary in vitro. To test the hypothesis that the acute decrease in thyrotropin (TSH) after infusion of T4 into hypothyroid rats requires intrapituitary T4 to 3,5,3'-triiodothyroxine (T3) conversion, the effect of iopanoic acid treatment on the generation of nuclear T3 from intrapituitary conversion and the response to TSH were compared in control and iopanoic acid-treated animals. 5 mg/100 g body weight iopanoic acid given 24, 16, and 1.5 h before administration of 125I-T4 reduced the quantity of pituitary nuclear 125I-T3 from local (intrapituitary) T4 to T3 conversion by 60-100%. In association with inhibition of intrapituitary T4 to T3 conversion, there was an increase in the binding of 125I-T4 to the nuclear receptor of the pituitary but the total iodothyronine content of the nuclei was still less than half of the nuclear iodothyronine in control animals. Iopanoic acid did not affect the nuclear/plasma ratio of injected 131I-T3 in the same animals, but did appear to impair 131I-T3 clearance or reduce its distribution volume. Treatment with iopanoic acid did not reduce the quantity of nuclear 125I-T3 in the liver, kidney, or heart of the same animals more than expected from the changes in serum 125I-T3. In control hypo-thyroid animals pretreated with iopanoic acid, the mean TSH was not significantly decreased from the initial value by T4 injection. Iopanoic acid pretreatment did not interfere with the acute TSH response of chronically hypothyroid rats to 70 ng of T3/100 g body weight. These results establish that intrapituitary generations of T3 from T4 is required for the acute decrease in TSH which occurs after T4 infusion. The data also are consistent with the content that it is nuclear binding of the T3 generated from T4 which initiates the inhibition of TSH release.

Studies on the mechanism of 3,5,3'-triiodothyronine-induced suppression of secretagogue-induced thyrotropin release in vitro.

A double column perifusion procedure was used to study the feedback inhibition of L-T3 on TSH secretion from rat anterior pituitary fragments. Matching pituitary halves were pretreated with T3 (10(-7) M) for 2 h before exposure to 10(-8) M TRH, 59 mM K+, or 5 mM Ba2+ . TRH, high K+, and Ba2+ resulted in a 2-fold or greater stimulation of TSH release. T3 significantly inhibited the stimulation by these secretagogues to 0.77, 0.78, and 0.52 of control for TRH, high K+, and Ba2+, respectively. Neither rT3 (10(-7) M) nor T3 added together with TRH had an effect on TSH release by this secretagogue. Perifusion with 3.5 x 10(-5) M cycloheximide or 10(-6) M actinomycin D 1 h before and during T3 administration led to greater TSH release with TRH than in the presence of T3 alone. Neither protein synthetic inhibitor increased TRH responsiveness of pituitary fragments when perifused alone. When cycloheximide was perifused in a similar protocol before high K+ or Ba2+, there again was a significant decrease in the T3-induced inhibition of TSH release by these secretagogues. Cycloheximide alone did not increase TSH release in response to high K+ or Ba2+, eliminating this as a possible explanation for the enhanced TSH response when antibiotic was present with T3. These results indicate that the in vitro effect of T3 on secretagogue-induced TSH release can be blocked by an inhibitor of protein synthesis. The inhibitory effect of T3 on high K+- and Ba2+-induced TSH release suggests that the site of the acute T3 inhibition of TSH release may be subsequent to TRH interaction with its receptor.

Monoclonal antibodies to human chorionic gonadotropin: implications for antigenic mapping, immunoradiometric assays, and clinical applications.

Monoclonal antibodies to intact hCG and free beta-subunit of hCG permit the recognition of different individual antigenic sites on the hCG molecule. At least seven different epitopes may be recognized on the native molecule and a further two on the free beta-subunit. These antibodies were used in a mouse Leydig cell bioassay system to compare the degree of inhibition of hCG-induced testosterone production. Two antibodies were particularly potent at inhibiting hCG action, suggesting that they bind near to the receptor-recognition site on the hCG molecular. One antibody had little effect on biological action and was presumably binding distant from the biologically active site on the hCG. Combinations of monoclonal antibodies in immunoradiometric assays were used to develop highly sensitive and specific assays to intact hCG, free beta-subunit of hCG, and beta-subunit as part of intact hCG. Using these assays it was possible to detect 0.1 ng/ml hCG in the presence of high levels of LH. In 106 serum samples from pregnant woman free beta-subunit was considerably higher in samples with low concentrations of intact hCG, suggesting that free beta-subunit is not a limiting factor in placental production of intact hCG in early pregnancy. Comparison of urinary to serum ratios of hCG and free beta-subunit using specific immunoradiometric assays showed a good correlation for intact hCG but not for free beta subunit which was present in very high concentrations in urine.

A rapid, sensitive two-site immunometric assay for TSH using monoclonal antibodies: investigation of factors affecting optimisation.

Twenty-three monoclonal antibodies specific for human thyrotropin (TSH) have been prepared and characterised. Four different epitopes have been identified, 3 of which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. Only 1 epitope is expressed on free TSH beta-subunit. The best antibodies have affinities for TSH approaching 10(11) M-1 and cross-reactions with lutropin and chorionic gonadotropin of 0.2% or less. A 2-site immunoradiometric assay for TSH was established using 2 monoclonal antibodies, one of which was absorbed to plastic tubes and the other labelled with 125I. Several parameters affecting assay performance were investigated, including conditions for adsorption of antibodies to various plastics, selection of antibody combinations, concentration of labelled antibody and incubation protocol. The optimised assay covered a working range of 1-60 mU/1 (0.17-10 ng/ml) TSH in a 4 h, single incubation protocol, with no significant interference from other glycoprotein hormones at their maximum physiological or pathological concentrations.

Red cell-labelled monoclonal antibodies for assay of human chorionic gonadotropin and luteinising hormone by reverse passive haemagglutination.

The assay of human chorionic gonadotropin and luteinising hormone by reverse passive haemagglutination reaction, using monoclonal antibodies coupled to red cells, is described. Quantitation is achieved by end-point determination for serial dilutions of standard or sample, the haemagglutination reaction being observed after settling under gravity for 90 min. Red cell-labelled antibodies were stabilised with glutaraldehyde without loss of sensitivity and allowing long term storage. Various antibody combinations were assessed, and the best combination under optimum conditions gave a positive haemagglutination reaction down to 0.2 ng/ml with HCG.

Effect of TRH and dopamine on cyclic AMP levels in enriched mammotroph and thyrotroph cells.

Populations of normal anterior pituitary cells enriched in thyrotrophs or mammotrophs prepared by velocity sedimentation were used to investigate the effect of modulators of TSH and prolactin secretion on cyclic AMP accumulation. In both thyrotroph-enriched and mammotroph-enriched fractions, IBMX increased cyclic AMP accumulation. In the presence of IBMX, TRH invoked an increase in cyclic AMP suggesting that TRH modulates cyclic AMP accumulation in both of these cell types from normal pituitary glands. In the mammotroph-rich fraction, dopamine inhibited the increase in cyclic AMP induced by TRH. In contrast however, in the thyrotroph-enriched fraction dopamine lowered neither cyclic AMP concentration nor TSH secretion. Thus the inhibiting effect of dopamine on cyclic AMP appears to be specific for prolactin-secreting cells.

Ionic modulation of flow resistance in an immobilized proteoglycan model of the trabecular meshwork.

Resistance to aqueous humor outflow, which is essential for the maintenance of normal intraocular pressures, is thought to reside within the final few layers of the trabecular meshwork, adjacent to Schlemm's canal. Trabecular proteoglycans, with their extensive, negatively charged glycosaminoglycan (GAG) side-chains, are thought to contribute most of this outflow resistance. We hypothesize that one mechanism by which trabecular cells can regulate aqueous outflow is through modulation of the ionic extracellular microenvironment at or near their surfaces within the outflow pathway. To examine this possibility, we have developed an experimental model of the trabecular extracellular matrix. In this model, a large proteoglycan, aggrecan, is attached covalently by its core protein to the matrix of a tortuous-pore membrane. When viewed in scanning electron micrographs, an interesting similarity between the flow model and the human trabecular meshwork is observed. The fluid flow rate through the model membranes, measured by constant-pressure perfusion, decreases in a saturable, dose-dependent manner in response to increased proteoglycan binding. The proteoglycan is tightly bound, as judged by retention of radiolabeled proteoglycan, under the perfusion conditions used. Degradation of the GAGs without removal of the proteoglycan core protein, achieved by treatment with the enzyme chondroitinase AC, return flow rates to values near those measured through control membranes. Increasing the sodium chloride concentration in the perfusate produces a dose-dependent increase in flow rate, which plateaus by 100 mM. Increasing the calcium concentration produces a similar response, although the plateau is reached by 10 mM. Maximum flow is observed near physiological pH, declining at values above or below neutrality. We conclude that this membrane model with the immobilized proteoglycans is a useful system for evaluating potential roles of trabecular proteoglycans in modulating aqueous humor outflow, free from contributions of trabecular cellular dynamics. These studies support our hypothesis that trabecular cells can modulate flow by changing the ionic microenvironment of the extracellular matrices near their surfaces within the flow channels.

Administration of an insulin powder to the lungs of cynomolgus monkeys using a Penn Century insufflator.

A powder formulation of live-attenuated measles vaccine is being developed for administration to the lungs. The safety and efficacy of the powder will be assessed by insufflation into cynomolgus monkeys. A Penn Century insufflator has been evaluated for powder dosing to the monkeys using an insulin formulation having similar physicochemical characteristics to the vaccine powder. Insulin pharmacokinetics were compared following dosing by powder insufflation, solution instillation into the trachea and subcutaneous injection. The insulin dosed to the lungs and trachea was more rapidly absorbed than that administered subcutaneously. Insulin bioavailability was greater from the inhaled powder than from the instilled solution. The findings confirm that the Penn Century device is suitable for vaccine powder dosing to the deep lung.

Hydroxyapatite nano and microparticles: correlation of particle properties with cytotoxicity and biostability.

Synthetic colloid and gel hydroxyapatite (HA) nanoparticles (NPs) were spray dried to form microparticles (MPs). These are intended for use as slow release vaccine vectors. The physico-chemical properties of gel and colloid NPs and MPs were compared to those of HA obtained commercially. Their cytotoxicity to human monocytes'-derived macrophages (HMMs) was assessed in vitro using a range of techniques. These included the MTT assay, LDH leakage and a confocal based live-dead cell assay. Cytotoxicity differed significantly between preparations, with the suspended gel preparation being the most toxic (31-500 microg/ml). Other preparations were also toxic but only at higher concentrations (>250 microg/ml). Transmission electron microscopy (TEM) and stereology showed variable cellular uptake and subsequent dissolution of the various forms of HA. We have demonstrated that HA particle toxicity varied considerably and that it was related to their physico-chemical properties. Cell death correlated strongly with particle load. The intracellular dissolution of particles as a function of time in HMM suggests that increased cytoplasmic calcium load is likely to be the cause of cell death. Some HA NPs eluded the phagocytic pathway and a few were even seen to enter the nuclei through nuclear pores.

Stochastic models for toxicant-stressed populations.

We obtain conditions for the existence of an invariant distribution on (0, infinity) for stochastic growth models of Ito type. We interpret the results in the case where the intrinsic growth rate is adjusted to account for the impact of a toxicant on the population. Comparisons with related results for ODE models by Hallam et al. are given, and consequences of taking the Stratonovich interpretation for the stochastic models are mentioned.

Top predator persistence in differential equation models of food chains: the effects of omnivory and external forcing of lower trophic levels.

Persistence criteria are given for the highest trophic level predator in ordinary differential equation models of food chains exhibiting arbitrary omnivory and external supplementation of food source or an intermediate predator. The results are expressed in terms of inequalities involving the bounds on the intrinsic growth and interaction rates. Whether omnivory or external forcing enhances persistence is discussed, particularly for the examples of three-, four-, and five-link Lotka-Volterra food chains.