Food intake regulation of circulating thyroid hormones in domestic fowl

The relationship between food intake and thyroid function has been investigated in immature domestic fowl. Starvation delayed, but did not suppress, the triiodothyronine (T3) response to intravenously administered thyrotropin-releasing hormone (10 micrograms/kg). This probably resulted from a suppression of monodeiodinase activity, since the conversion of thyroxine (T4) to T3 in thyroidectomised birds following an intramuscular injection of T4 (10 micrograms/kg) was markedly reduced by starvation. Starvation, for 24 or 48 hr, lowered the circulating T3 level but increased the T4 concentration. When fasted birds were refed the T4 concentration was initially enhanced but subsequently declined as the T3 concentration progressively increased. The accompanying decline in the T4:T3 ratio in fasted-refed birds indicated that the rise in the T3 level resulted from the peripheral monodeiodination of T4. The increase in T3 concentration could be induced solely by carbohydrate; the intraperitoneal administration of glucose (2.0 g/kg) to fasted birds resulting in a slight, transient rise in the T3 concentration and a fall in the T4:T3 ratio. The generation of T3 was also energy dependent, in that the magnitude of the T3 response of fasted birds to refeeding was proportional to the amount of food consumed and to the metabolisable energy (ME) content of the diet. Moreover, when exogenous T4 (100 micrograms/kg) was intramuscularly administered to thyroidectomised birds fed a diet with a high ME content, the conversion of T4 to T4 was greater than that in birds fed a diet of lower ME content. These results demonstrate that nutritional stimuli are involved in the regulation of thyroid function in birds, particularly in the peripheral generation of T3.     

INHIBITION OF CONVERSION OF THYROXINE TO TRIIODOTHYRONINE IN PATIENTS WITH SEVERE CHRONIC ILLNESS

Many clinically euthyroid patients with severe, chronic, non-thyroidal illnesses (i.e. sick euthyroid patients) have very low circulating concentrations of total and absolute free triiodothyronine (T3), low-normal concentrations of total thyroxine (T4), elevated concentrations of absolute free T4, and circulating concentrations of thyrotrophin (TSH) that are either normal or subnormal. This study was undertaken to elucidate the mechanism of the low circulating T3 concentrations. The disappearance rate of ¹²⁵I-T3 from the circulation of five representative sick euthyroid patients, was studied and found to be slower, but not significantly so, compared with three control subjects, thus excluding an increased destruction rate as the cause of the low T3 levels. A selective decrease of T3 secretion from the thyroid gland of these patients was also excluded by the results of TSH stimulation tests. Inhibition of extra-thyroidal conversion of T4 to T3 was suggested by studies of the thyroid function in a hypothyroid woman with a Grade IV lymphoma on T4 replacement therapy. When the lymphoma was in remission, her circulating T3 concentration was 2–55 nmol/1 but when it relapsed it fell to 0–55 nmol/1. The T4 concentrations were 124–7 nmol/1 and 126 nmol/1 respectively. Decreased monodeiodination of T4 to T3 in sick euthyroid patients was confirmed by paper chromatography of extracted serum obtained 48 h after an i.v. injection of ¹²⁵I-T4 into two severely ill patients from the intensive therapy unit and a control subject. Peaks of radioactivity corresponding to ¹²⁵I-T4 and ¹²⁵I-T3 were detected in the control subject, but only a single peak corresponding to ¹²⁵I-T4 was detected in the ill patients.     

Divergent effects of 6-propylthiouracil on 3,3',5'-triiodothyronine (RT3) serum levels and in man

The effect of 6-propylthiouracil (PTU) on the peripheral conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (reverse T3 rT3) was investigated by assessments of the concentrations of T4 T3 and rT3 in peripheral venous blood from T4-treated healthy volunteers given PTU, 4 X 150 mg daily. Within on day of PTU administration, serum T3 concentrations were reduced, and those of rT3 enhanced. These deviations lasted as long as PTU was given (five days), and there was a rapid return towards normal within one day after PTU administration ceased. It seems probable that, in man, PTU can evoke a diversion of T4 monodeiodination, less being converted to the metabolically active T3 and more to the metabolically inactive rT3. It is ppossible that the rapidity whereby PTU can reduce T3 levels can offer an advantage in the treatment of hyperthyroidism.     

Recognition of cell surface GD3 by monoclonal antibody anti‐6C2 in rheumatoid arthritis synovial fluid: Expression on human T cells with transendothelial migratory activity

Objective

We have previously reported that the anti‐6C2 monoclonal antibody (mAb) defines a subset of human CD4+ memory T cells. The present study sought to determine the nature of the 6C2 molecule and the function associated with 6C2+ T cells, and to examine whether this T cell subset is involved in the pathophysiology of rheumatoid arthritis (RA).

Methods

Cytofluorographic analysis was performed for identification of T cell surface molecules displaying a distribution similar to that of the 6C2 molecule. T cells in the synovial fluid of RA patients were examined for expression of the 6C2 molecule. Transendothelial migratory activity was assessed by assay using monolayers of human endothelial cells. Specific reactivity of the anti‐6C2 mAb was determined by immunoblotting on gangliosides separated by thin‐layer chromatography, and flow cytometric analysis of the cells transfected with complementary DNA (cDNA) was performed for determination of the glycosyltransferases involved in biosynthesis of the gangliosides.

Results

On human peripheral T cells, the 6C2 molecule was distributed, by and large, in a pattern similar to that of CDw60, or O‐acetyl‐GD3. The majority (>70%) of synovial fluid T cells from patients with RA were found to be 6C2 positive, and those 6C2+ T cells exhibited a transendothelial migratory capacity that was inhibited by pretreatment of T cells with anti‐6C2 mAb. Moreover, treatment of T cells with neuraminidase resulted in a loss of 6C2 expression as well as a reduction in the transendothelial migratory activity. Anti‐6C2 mAb reacted specifically with GD3, but not with O‐acetyl‐GD3. The reactivity of anti‐6C2 mAb was induced on the cell surface only by transfection with cDNA for GD3 synthase.

Conclusion

The 6C2 molecule is a disialoganglioside, GD3, and is present on a subset of T cells with transendothelial migratory capacity. The 6C2/GD3 molecules, as well as 6C2/GD3+ T cells, appear to play a role in T cell migration and in the inflammation of RA.

     

Thyroid hormones and lipolysis in physically trained rats

In rats a single bout of exercise resulted in increased triiodothyronine (T3), thyroxine (T4), and triiodothyronine/reverse triiodothyronine ($\text{T3}\text{rT3}$) ratio 20 hr after exercise. The effect of norepinephrine on lipolysis in vitro was potentiated.In trained rats no changes were found in T4, T3 or rT3 concentrations. The $\text{T3}\text{rT3}$ ratio as well as basal and stimulated TSH concentrations decreased in comparison with sedentary, freely eating rats. Moderate food restriction to produce a body weight similar to that of trained animals caused no changes in T4, T3 or rT3 concentrations but caused a decrease in $\text{T3}\text{rT3}$ and in TSH levels. Training and moderate food restriction groups were not different. T3 in vitro caused a potentiation of catecholamine induced lipolysis in trained and food-restricted animals. With aging the serum concentration of T3 decreased and that of rT3 increased.Acute and chronic exercise both exert an effect on peripheral hormonal responses of lipolysis, while they have different and opposite effects on thyroid hormone concentrations. Physical training seems to have effects in this regard similar to those of moderate energy intake restriction. The results suggest that changes in peripheral effects of thyroid hormones during training should attract more attention.     

Effects of surgical stress and corticotrophin on the peripheral metabolism of thyroid hormones in rabbits.

The effect of surgical stress and ACTH injection on the peripheral monodeiodination of thyroxine (T4) was studied in the rabbit. These stimuli resulted in a switch from the peripheral formation of tri-iodothyronine (T3) to reverse T3 in normal rabbits and in rabbits whose thyroidal secretion was suppressed by administration of T4. This is analogous to the situation in man. These changes were not due to alterations in the serum binding capacity for thyroid hormones.     

Premigratory increase in circulating triiodothyronine/thyroxine ratio in the red-headed bunting (Emberiza bruniceps)

The circulating triiodothyronine/thyroxine ($\text{T}{}_3\text{T}{}_4$) ratio in the red-headed bunting significantly increased prior to migration. Marked extrathyroidal conversion of T₄ to T₃ also occurred. The possibility that migratory disposition in this bird may be related to an increase in serum concentration of T₃ is, therefore, raised.     

Effects of exogenous prolactin on ovarian growth and fattening in the redheaded bunting, Emberiza bruniceps

Subcutaneous daily injections of ovine prolactin (PRL) inhibit photoperiodic induction of gonads and fattening in the female redheaded bunting (Emberiza bruniceps). However, birds respond to the same photoperiod (15L:9D) after withdrawal of the exogenous PRL. Further, PRL injections given in late hours during the subjective days induce ovarian regression in the photostimulated females. The results suggest that the PRL has an inhibitory role on gonadal photoperiodic responses in redheaded buntings, and extend our understanding of the regulatory function of PRL in migratory birds.     

Abatacept (CTLA‐4IG) treatment reduces the migratory capacity of monocytes in patients with rheumatoid arthritis

Objective

The binding of abatacept (CTLA‐4Ig) to the B7 ligands CD80 and CD86 prevents the engagement of CD28 on T cells and thereby prevents effector T cell activation. In addition, a direct effect of CTLA‐4Ig on antigen‐presenting cells (APCs) could contribute to the therapeutic effect. To further elucidate the mechanism of CTLA‐4Ig, we performed phenotype and functional analyses of APCs in patients with rheumatoid arthritis (RA) before and after the initiation of CTLA‐4Ig therapy.

Methods

Peripheral blood mononuclear cells were analyzed before and at 2 and 4 weeks after the initiation of CTLA‐4Ig therapy. Proportions of APCs were determined by flow cytometry. CD14+ monocytes were further analyzed for the expression of costimulatory and adhesion molecules and for their transendothelial migratory capacity in vitro. In addition, CD14+ monocytes from healthy controls were analyzed for their migratory and spreading capacity.

Results

Proportions and absolute numbers of monocytes were significantly increased in RA patients treated with CTLA‐4Ig. The expression of several adhesion molecules was significantly diminished. In addition, monocytes displayed a significant reduction in their endothelial adhesion and transendothelial migratory capacity upon treatment with CTLA‐4Ig. Likewise, isolated monocytes from healthy controls revealed a significant reduction in their migratory and spreading activity after preincubation with CTLA‐4Ig or anti‐CD80 and anti‐CD86 antibodies.

Conclusion

We describe direct effects of CTLA‐4Ig therapy on phenotype and functional characteristics of monocytes in RA patients that might interfere with the migration of monocytes to the synovial tissue. This additional mechanism of CTLA‐4Ig might contribute to the beneficial effects of CTLA‐4Ig treatment in RA patients.

     

Induction of type II T4-5'-monodeiodinase activity in brown adipose tissue in fasted mice

In order to study the effect of starvation on brown adipose tissue (BAT) type II 5'-monodeiodinating activity (5'MDI), type II 5'MDI was measured in vitro in the presence of 20 mM dithiothreitol, 1 mM propylthiouracil, 2 nM thyroxine (T4) and appropriate amounts of 600 X g infranatant of BAT from fed control or 3 day fasted mice, with or without daily T4 replacement (1.2 micrograms/100 g bw) during starvation. I- released from 125I-T4 was measured by ion-exchange column chromatography. Activity of BAT 5'MDI was markedly elevated in the 3 day fasted group (133 +/- 28 fmol I-/h per mg protein vs. 26 +/- 6.4; p less than 0.05). Kinetic studies using BAT infranatant suggested that fasting-induced activity is associated with a similar change in the Vmax, but no demonstrable change in apparent Km of T4 monodeiodination. T4 replacement during fasting, which normalized both serum T4 and T3 in fed and 3 day fasted groups, did not stop the increase of BAT 5'MDI in the fasted group (p less than 0.01). The data suggest that: (1) the fasting-induced increase in BAT 5'MDI is due mainly to the changes in capacity rather than the affinity of the enzyme, and (2) the fasting-induced increase in BAT 5'MDI is not mediated entirely through changes in serum thyroid hormone concentration.     

Enhanced thyroxine metabolism in hexachlorobenzene-intoxicated rats

The effect of hexachlorobenzene (HCB) (1 g/kg bw) administration for 4 weeks, on thyroxine (T4) and triiodothyronine (T3) metabolism was studied in Wistar rats. The effect on serum binding of T4 has also been studied. Animals were injected with a tracer dose of either labeled hormone and by examining serum L-125I-T4 and L-125-I-T3, kinetics of radiolabeled hormones metabolism were calculated. The T4 metabolic clearance (MCI) as well as the distribution space, were increased by 6 fold. Decreased serum T4 levels result from an increase both in deiodinative and fecal disposal in HCB-treated rats. 125I-T3 metabolism was slightly affected. The enhanced peripheral disposition of thyroxine appears to lead to increased thyroid function, as measured by augmented TSH serum levels and 125I-thyroidal uptake. Serum binding of T4 was not affected.     

THYROID HORMONE LEVELS DURING GLUCOSE TOLERANCE TEST IN EUTHYROID SUBJECTS

Twelve normal, healthy, clinically and biochemically proven euthyroid volunteer subjects (age 19-35) were administered a standard glucose tolerance test (100 g glucose orally) and thyroxine (T4), triiodothyronine (T3) and reverse triiodothyronine (rT3) levels were determined by specific radioimmunoassays to determine the acute effect of glucose and insulin on peripheral monodeiodination of thyroxine. The fasting levels of T4, T3, rT3 and free thyroxine were 82.4 nmol/l, 1.7 nmol/l, 0.52 nmol/l, and 0.22 nmol/l, respectively, and these levels were unchanged during the 3 hours post-glucose load. The rise and fall of glucose and insulin levels were typical of the standard responses normally observed in the glucose tolerance test. The variations in insulin and glucose levels were not correlated with thyroid hormone concentrations at any interval during the test. It is therefore concluded that dietary glucose does not acutely cause shifts in peripheral monodeiodination of thyroxine in healthy euthyroid subjects.     

Imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node

Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8⁺ T cells and play critical roles in cytotoxic T-cell–mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8⁺ T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4⁺ T-cell activation by CD11b⁺ DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103^hi) DCs, which immigrate from the skin, and resident (CD8α^hi) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8⁺ T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8⁺ T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8⁺ T-cell activation.Dendritic cells (DCs) play critical roles in shaping T-cell responses as they present antigenic peptides and provide the requisite costimulatory signals for T-cell activation (1). In primary immune responses, naive T cells receive these antigen and costimulatory signals by physically interacting with DCs in secondary lymphoid tissues such as lymph nodes (LNs). Recent advances in intravital imaging techniques using two-photon excitation fluorescent microscopy reveal the dynamic DC and T-cell behaviors during T-cell activation. When naive T cells encounter DCs presenting sufficient amounts of antigenic peptides, they reduce their motility and attach to the DCs for prolonged periods of time, ranging from 10 min to hours (2–4). A limitation of previous imaging studies was the inability to visually distinguish endogenous DC subpopulations that are distinct from one another in phenotype and function (5–7). This has clouded the ability of the studies to define the relative contributions of the different DC subsets in T-cell activation.Cytotoxic T cells, which play critical roles in the defense against microorganisms and cancer, are generated from CD8⁺ T cells through interactions with relatively small populations of DCs. In LNs, it is the CD8α⁺CD103⁺ class of DCs that is capable of cross-presenting exogenous antigen-derived peptides on major histocompatibility complex (MHC) class I, making them important for CD8⁺ T-cell responses. Other DC subsets, including CD11b⁺ DCs, are mainly involved in CD4⁺ T-cell responses (5, 8, 9). Among CD8α⁺CD103⁺ DCs, CD8α^hiCD103^int DCs are a LN-resident population (hereafter called LN-resident DCs or CD8α^hi DCs) whereas CD8α^intCD103^hi DCs are a migratory subset derived from peripheral tissues (hereafter called migratory DCs or CD103^hi DCs). The migratory DCs constantly immigrate from the skin to the skin-draining LNs (SDLNs). Upon activation by innate stimuli, e.g., double-stranded RNAs, the skin migratory DCs increase their rate of immigration to the SDLNs (10). The first compelling evidence for the differential distribution of distinct DC populations in LNs was reported a decade ago (11). Until recently, however, the distribution of CD8α^hi DCs and CD103^hi DCs could not be studied because of the difficulty in histologically identifying these cells in LNs. Computational processing of multicolor histological images suggested that these cross-presenting DC subsets were preferentially accumulated in deep areas of the T-cell zone in the SDLNs (12). Other studies directly visualized both DC subsets by histological analyses of chemokine (C motif) receptor 1 (XCR1) expression, which is selectively and highly expressed in both LN-resident DCs and migratory DCs (13–16). These studies have demonstrated the differential localization of cross-presenting DCs and other types of DCs in the SDLNs.Despite the above advances, there remain many open questions concerning the interactions of DCs and T cells. For example, it is not known whether the activation of CD8⁺ T cells and CD4⁺ T cells in the SDLNs is spatially coordinated according to the differential localization of cross-presenting DCs and other DCs. Moreover, little is known about the dynamics of migratory DCs in the SDLNs. In addition, it is difficult to compare the contributions of LN-resident DCs and migratory DCs to CD8⁺ T-cell activation in the SDLNs. Only somewhat indirect assays have been used; for example, an ex vivo assay using DCs sorted from the SDLNs has been used to study the events of herpes simplex virus type 1 (HSV-1) infection (17). For soluble protein vaccination, the DC subsets that most contribute to CD8⁺ T-cell activation in the SDLNs remain unknown. Soluble protein antigens administered s.c. are delivered to the SDLNs not only by skin DCs but also via lymph flow in lymphatic sinuses and LN conduits, permitting DCs in the SDLNs to sample them (18, 19). Mechanical disruption of LNs for the ex vivo assay may artificially expose irrelevant DC subsets to soluble protein antigens. Thus, it is important to establish the in vivo assay system to evaluate the differential roles for the migratory and LN-resident DC subsets in CD8⁺ T-cell activation.In this study, we have used direct imaging approaches to determine the locations for activation of CD8⁺ T cells and CD4⁺ T cells in the SDLNs. Using newly generated reporter mice expressing a photochromic fluorescent protein in DCs that express the XCR1 gene (Xcr1), we have developed a method to distinctively visualize the LN-resident DCs and migratory DCs in the SDLNs. Intravital microscopy has been used to analyze cognate interactions of the DC subsets with CD8⁺ T cells after immunization with soluble antigen. Our results provide insights into the DC inter- and intratissue migration dynamics, which are associated with in vivo contributions of different DC subsets to CD8⁺ T-cell activation.     

The inner ring (5-) monodeiodination of thyroxine (T4) in cerebral cortex during fetal, neonatal, and adult life

Inner ring (-5) monodeiodination of T4 was studied by incubating T4 (approximately 0.26 mumol/L) with rat cerebral cortical homogenate (approximately 4 mg protein) in the presence of dithiothreitol (up to 400 mmol/L) and quantifying the amount of the product, rT3, by a specific radioimmunoassay. The production of rT3 was dependent on duration of incubation (up to 2 hours), amount of tissue protein (up to 8 mg), temperature (optimal at 37 degrees C) and pH (optimal, 7.0) of the incubation mixture and the concentration of DTT (maximally stimulated at 400 mmol/L). The apparent Km and Vmax of the T4-inner ring monodeiodinating activity were 36 nmol/L and 1.75 pmol/mg protein/h, respectively. The activity was inhibited by T3 and 3,5-T2, but not by 3'5'-T2, PTU, methimazole, sodium salicylate, or 8-anilino-I-naphthalene sulfonic acid. Ipodate weakly inhibited T4-to-rT3 monodeiodination. Hyperthyroidism induced by T4 (100 micrograms/d IP X 3 days), T3 (80 micrograms/d IP X 3 days) or DIMIT (45 micrograms/d IP X 3 days) significantly stimulated T4-to-rT3 conversion; DIMIT was the most potent agent. Hypothyroidism inhibited T4-to-rT3 converting activity in cerebral cortex. Fasting for three days had no appreciable effect on T4-to-rT3 conversion in cerebral cortex. Cerebral cortical T4 5-deiodinase activity in the pregnant rat at term was about 50% of that in the adult nonpregnant rat, whereas that in the fetus was about three-fold higher than that in the nonpregnant adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of mechanisms mediating uptake and efflux of thyroid hormones in the human choriocarcinoma cell line, JAR

We compared the specificities of transport mechanisms for uptake and efflux of thyroid hormones in cells of the human choriocarcinoma cell line, JAR, to determine whether triiodothyronine (T3), thyroxine (T4) and reverse T3 (rT3) are carried by the same transport mechanism. Uptake of 125I-T3, 125I-T4 and 125I-rT3 was saturable and stereospecific, but not specific for T3, T4 and rT3, as unlabelled L-stereoisomers of the thyroid hormones inhibited uptake of each of the radiolabelled hormones. Efflux of 125I-T3 was also saturable and stereospecific and was inhibited by T4 and rT3. Efflux of 125I-T4 or 125I-rT3 was, in contrast, not significantly inhibited by any of the unlabelled thyroid hormones tested. A range of compounds known to interfere with receptor-mediated thyroid hormone uptake in cells inhibited uptake of 125I-T3 and 125I-rT3, but not 125I-T4. We conclude that in JAR cells uptake and efflux of 125I-T3 are mediated by saturable and stereospecific membrane transport processes. In contrast, the uptake, but not the efflux, of 125I-T4 and 125I-rT3 is saturable and stereospecific, indicating that uptake and efflux of T4 and rT3 in JAR cells occur by different mechanisms. These results suggest that in JAR cells thyroid hormones may be transported by at least two types of transporters: a low affinity iodothyronine transporter (Michaelis constant, Km, around 1 microM) which interacts with T3, T4 and rT3, but not amino acids, and an amino acid transporter which takes up T3, but not T4 or rT3. Efflux of T4 and rT3 appears to occur by passive diffusion in these cells.