Variations in otolith patterns, sizes and body morphometrics of jack mackerel Trachurus japonicus juveniles.

Variations in otolith patterns, sizes and body morphometrics of jack mackerel Trachurus japonicus juveniles were investigated. Under transmitted light, translucent (W(t)) and opaque otoliths (W(o)) were detected in juveniles collected from Wakasa Bay between July 2005 and April 2006, whereas only opaque otoliths (G(o)) were detected in Goto-nada Sea individuals between May and June 2006. Three groups of juveniles were distinguished based on differences in hatch season, otolith size and growth history, and body morphometrics. As T. japonicus has different spawning seasons according to spawning grounds, each group was estimated to hatch in different waters. Juveniles with W(t) otoliths were considered to have stayed in coastal habitat longer, as the hatch area was estimated to be near Wakasa Bay. Juveniles with W(o) and G(o) otoliths appear to recruit to coastal waters at larger size, since their hatch areas were estimated to be far from each collection area. Larger otoliths of W(t) were attributed to otolith accretion after the second growth flexion, which was observed only for W(t) . Standard length of W(t) fish at the second otolith growth flexion was estimated to correspond to recruitment size to coastal rocky reefs in Wakasa Bay. Body morphometrics were correlated with otolith size after removing body size effect, suggesting that morphological variations of T. japonicus juveniles were also associated with the timing of recruitment to coastal habitat.

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Effect of interleukin-1 (IL-1) on thyroid hormone metabolism in mice: stimulation by IL-1 of iodothyronine 5'-deiodinating activity (type I) in the liver.

To elucidate the mechanism by which the low T3 and low T4 syndrome occurs in patients with infection, recombinant human interleukin-1 (IL-1) was administered to mice, and their thyroid hormone metabolism was studied. Continuous sc infusion of IL-1 alpha or IL-1 beta at a dose of 0.015-1 microgram/day for 3 days decreased food intake and serum T4, T3, and rT3 concentrations in a dose-dependent manner. In pair-fed control (PFC) mice, serum T4 and T3 also decreased, but rT3 was reciprocally increased. The T3/T4 ratio was greater in IL-1-treated mice than in PFC mice. Although food intake was decreased by 65% in IL-1-treated mice (1 microgram/day) compared with that in fed control mice, type I iodothyronine 5'-deiodinating activity in liver was significantly increased compared with that in fed control mice. Furthermore, the T3 and T4 responses to TSH were greatly diminished in IL-1-treated mice. These findings suggest that IL-1 directly inhibited the effect of TSH on the thyroid gland and decreased the serum concentrations of T4 and T3, and that an increase in type I iodothyronine 5'-deiodinating activity in livers of IL-1-treated mice may account for the greater T3/T4 ratio and lower serum rT3 concentration than those in PFC mice. Since tumor necrosis factor-alpha has a similar effect, we speculate that both cytokines may be synergistically involved in the altered thyroid hormone metabolism in mice (decreased serum T4, T3, and rT3 concentrations) and hypercatabolism in a febrile state.

Parathyroid hormone-related protein and interleukin-1 alpha synergistically stimulate bone resorption in vitro and increase the serum calcium concentration in mice in vivo.

To elucidate the mechanism of humoral hypercalcemia elicited by human esophageal carcinoma cells (EC-GI), which constitutively produced interleukin-1 alpha (IL-1 alpha) and PTH-like factor, the effects of IL-1 alpha and PTH-related protein (PTH-rP) on bone resorption in vitro and on serum calcium concentrations in vivo were investigated. Nude mice transplanted with EC-GI cells invariably developed hypercalcemia, although their urinary cAMP excretion remained within the normal range. IL-1 alpha or PTH-rP-(1-34) stimulated 45Ca release from prelabeled fetal mouse forearm bones in a concentration-dependent manner, and when combined, IL-1 alpha and PTH-rP-(1-34) synergistically stimulated bone resorption in vitro. Injection of PTH-rP-(1-34) into mice three times a day for 2 days increased the serum calcium concentration in a dose-dependent manner. Continuous infusion of IL-1 alpha occasionally increased the serum calcium concentration. Simultaneous administration of IL-1 alpha at rates of 1-2.7 micrograms/day and PTH-rP-(1-34) at doses of 15-30 micrograms/day synergistically increased the serum calcium concentration in vivo. These findings suggest that PTH-rP and IL-1 alpha produced by the tumor cells were synergistically responsible for the humoral hypercalcemia observed in both the original patient and the tumor-bearing nude mice, and that at least two bone-resorbing factors [PTH-rP and another nonadenylate cyclase-stimulating bone-resorbing factor(s)] are active in patients with malignancy-associated hypercalcemia, in whom nephrogenous cAMP excretion is neither increased nor decreased.

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles.

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

Reversible inhibition by interferons alpha and beta of 125I incorporation and thyroid hormone release by human thyroid follicles in vitro.

When interferons (IFN-alpha-2a, IFN-alpha-2b, natural IFN-alpha and IFN-beta) were cultured with human thyroid follicles, each IFN inhibited TSH-induced thyroid function (125I incorporation and release of 125I-T4) in a concentration-dependent manner. The minimal inhibitory effect was exerted at 1-10 U/ml. However, the inhibitory effect was reversible, and no inhibitory effect was detected when IFNs were removed from the medium within 12 h. These in vitro findings suggest that each IFN directly inhibits human thyroid function at clinically attainable levels. However, since the inhibitory effect was reversible, IFN therapy would provoke only subtle thyroid dysfunction in a majority of patients without preexisting thyroid autoimmunity.

Thyroglobulin release-stimulating activity in immunoglobulin G from patients with Graves' disease studied by human thyroid cells in vitro.

The ability of TSH or immunoglobulin G (G-IgG) from untreated patients with hyperthyroidism due to Graves' disease to stimulate thyroglobulin (Tg) release from human thyroid cells was studied. Thyroid tissue obtained from antithyroid drug-treated Graves' hyperthyroid patients was dispersed enzymatically and cultured in monolayers; medium was changed every 3 days. The cultured cells initially released large but declining amounts of Tg, independent of the presence of TSH (approximately 5 micrograms/dish on day 3 and approximately 1.5 micrograms/dish on day 6). After 6 days, TSH had a dose-dependent stimulatory effect on Tg release, and the peak response occurred on day 15. G-IgG-induced Tg release was found on the 12th day of culture and was maximal on day 18. Thyroid cells cultured for 12 days in the absence of TSH responded to TSH and G-IgG in a time- and dose-dependent fashion. Using 12-day cultures, Tg release-stimulating activity (Tg-RSA) was tested using 5 mg/ml (7.5 mg/dish) G-IgGs from 20 patients and 72-h incubation. The Tg-RSA of individual patients varied. However, significant correlations were found between Tg-RSA values and serum Tg concentrations or Tg-RSA and thyroid-stimulating immunoglobulin activities. No correlation was found between Tg-RSA and TSH binding inhibitor immunoglobulin activities. These results suggest that Tg-RSA can be an indicator of abnormal IgG of hyperthyroid Graves' patients. Whether the activity is identical with thyroid-stimulating activity remains to be clarified.

Potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125I incorporation and de novo synthesized thyroid hormone release in human thyroid follicles.

Using a highly sensitive bioassay for TSH, in which human thyroid follicles incorporate 125I and release de novo synthesized thyroid hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than purified hCG. Asialo-hCG purified from a patient with choriocarcinoma had very potent TSH-like activity (468 microU hTSH/mg). Submaximal doses of bTSH and hCG variants produced additive stimulation of thyroid function. Furthermore, the thyrotropic effect of hCG was inhibited by anti-TSH receptor antibody obtained from patients with myxedema. These in vitro findings suggest that although hCG is reported to exert potent cAMP-stimulating activity on rat thyroid-like cells (FRTL-5) and Chinese hamster ovary cells transfected with hTSH receptor complementary DNA (0.092-0.72 microU hTSH/U hCG), the thyrotropic activity induced by authentic hCG in human thyroid follicles is too weak to cause hyperthyroidism in normal pregnancy. However, hCG produced by some trophoblastic tumors, particularly asialo-hCG, has potent thyrotropic activity sufficient to cause clinically overt hyperthyroidism when produced excessively.

Inhibition of 125I organification and thyroid hormone release by interleukin-1, tumor necrosis factor-alpha, and interferon-gamma in human thyrocytes in suspension culture.

To elucidate the mechanism of decreased 131I uptake by the thyroid gland in patients with subacute thyroiditis and painless thyroiditis, human thyroid follicles were cultured with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and/or interferon-gamma (IFN gamma), and the effects of these cytokines on thyroid function were studied in vitro. When human thyrocytes were cultured in RPMI-1640 medium containing 0.5% fetal calf serum and TSH for 5-8 days, the cells incorporated 125I, synthesized de novo [125I]iodotyrosines and [125I]iodothyronines, and secreted [125I]T4 and [125I]T3 into the medium. IL-1 alpha and IL-1 beta inhibited 125I incorporation and [125I]iodothyronine release in a concentration-dependent manner. The minimal inhibitory effect was detected at 10 pg/ml. Electron microscopic examination revealed a marked decrease in lysosome formation in IL-1-treated thyrocytes. TNF alpha and IFN gamma also inhibited thyroid function in a concentration-dependent manner. Furthermore, when thyrocytes were cultured with IL-1, TNF alpha and IFN gamma, these cytokines more than additively inhibited thyroid function. Although the main mechanism of 131I uptake suppression in the thyroid gland in subacute thyroiditis is due to cellular damage and suppression of TSH release, our present findings suggest that IL-1, TNF alpha, and IFN gamma produced in the inflammatory process within the thyroid gland further inhibit iodine incorporation and at least partly account for the decreased 131I uptake by the thyroid gland in destruction-induced hyperthyroidism.

Risk factors associated with postoperative persistent hypertension in patients with primary aldosteronism.

BACKGROUND: Unilateral adrenalectomy was performed in 63 patients with primary aldosteronism. During a mean follow-up time of 4.1 years, none of the patients showed recurrence of hyperaldosteronism. However, 24 patients (38%) had persistent hypertension. The purpose of this study was to determine factors responsible for postoperative persistent hypertension. METHODS: A stepwise multivariate logistic regression analysis was performed to assess the combined predictive effects of the clinicopathologic variables. RESULTS: Age, sex, and pathologic findings were the best predictive factors of postoperative persistent hypertension. For a patient aged 50 years or more, the odds of persisting hypertension are 10.6:1, compared with those of a patient under 40 years of age. A male patient appears to have a greater chance of hypertension than a female patient; the odds ratio is 5.9:1. Persistent hypertension develops in patients with multiple adenomas or with an adenoma associated with macronodules more frequently than in those patients with a solitary adenoma; the odds ratio is 8.1. CONCLUSIONS: This study suggests that early surgical intervention at a younger age results in a more favorable outcome for patients with primary aldosteronism. The presence of macronodules in association with an adenoma is a cautious predictor of persistent hypertension after adrenalectomy.

Amiodarone-induced thyrotoxicosis associated with thyrotropin receptor antibody.

Since amiodarone was first marketed in 1992 in Japan, the incidence of amiodarone-induced thyrotoxicosis (AIT) has been increasing. Among 12 thyrotoxic patients, a patient with arrhythmogenic right ventricular dysplasia, who had been taking amiodarone for 4 years, developed thyrotoxicosis with subacute onset, accompanied by transiently positive thyrotropin (TSH) receptor antibody (TRAb), or thyrotropin-binding inhibiting immunoglobulin (TBII). The immunoglobulin G (IgG) obtained from the TRAb-positive serum of the patient elicited no thyroid hormone-releasing activity in cultured human thyroid follicles, whereas all IgGs obtained from untreated Graves' disease elicited positive results. These in vitro findings and clinical course suggest that TRAb/TBII without thyroid-stimulating activity may develop in patients with amiodarone-induced destructive thyroiditis, as reported in patients with destructive thyroiditis, such as subacute and silent thyroiditis.

Amiodarone stimulates interleukin-6 production in cultured human thyrocytes, exerting cytotoxic effects on thyroid follicles in suspension culture.

To investigate whether amiodarone increases interleukin-6 (IL-6) production in thyrocytes, human follicles obtained from subtotally thyroidectomized patients with Graves' disease were cultured in serum-free medium supplemented with various concentrations of bovine thyrotropin (bTSH) and amiodarone. The follicles gradually formed monolayer cells and secreted triiodothyronine (T3), thyroglobulin (Tg), and IL-6 for at least 14 days. TSH dose-dependently increased T3 and Tg but not IL-6 levels in the conditioned medium. Amiodarone exerted no significant effect on T3, Tg, or IL-6 concentrations at 0.1-1 microM. In contrast, at 10-20 microM, it decreased T3 and Tg, but increased IL-6 levels, and these changes were accompanied by increased expression of IL-6 mRNA. Amiodarone-induced IL-6 production was inhibited by prednisolone at 10(-7) M. Electron microscopic examination revealed that the thyroid follicles in the suspension culture remained intact at 1 microM, but that cytotoxic effects (decreased microvilli and increased onion-like inclusion bodies) occurred at higher concentrations (10-25 microM). These in vitro findings indicate that amiodarone does not impair thyroid function at clinically attainable serum levels (1 microM), but exerts cytotoxic effect by inducing the production of a proinflammatory cytokine (IL-6) at higher concentrations. Because amiodarone-induced IL-6 production was inhibited by prednisolone, it is reasonable to administer glucocorticoids to patients with amiodarone-induced destructive thyrotoxicosis (type II).

Immunoglobulins of untreated Graves' patients with or without thyrotropin receptor antibody (determined by porcine thyrocytes) universally elicit potent thyroid hormone-releasing activity in cultured human thyroid follicles.

Thyrotropin receptor antibody (TRAb), comprising thyrotropin binding inhibitor immunoglobulin (TBII) and thyroid-stimulating antibody (TSAb), both of which are conventionally determined using porcine thyrocytes in Japan, is not always positive in patients with untreated Graves' disease. To elucidate whether immunoglobulin G (IgG) obtained from TBII/TSAb-positive (+) or negative (-) Graves' disease patients are responsible for hyperthyroidism, we investigated the thyroid hormone-releasing activity (THRA) of these IgGs in human thyroid follicles in suspension culture, in which bovine thyrotropin (bTSH) is detectable even at 0.1 microU/mL. Human thyroid follicles, obtained from Graves' disease patients by subtotal thyroidectomy, were cultured in serum-free F-12/RPMI-1640 medium supplemented with bTSH or purified Graves' IgGs. After preculturing for 3 days, 125I was added, and after an additional 3 days of culture, 1251 incorporated into the thyroid follicles and organic 125I released into the culture medium (mainly 1251 -T4 + 125I-T3) were counted. Seventy TBII(+)/TSAb( + )-, 3 TBII( + )/TSAb( - )-, and 3 TBII( - )/TSAb( + )- patients with untreated Graves' disease were all positive for THRA, which became undetectable in spontaneous remission obtained after several years of medical treatment. The THRA was equivalent to 0.8-230 microU/mL bTSH. Furthermore, 2 TBII(-)/TSAb(-) patients were significantly positive for THRA. This TBII(-)/TSAb(-)IgG stimulated human thyrocytes to produce cyclic adenosine monophosphate (cAMP), and this was partially inhibited by antihuman IgG antibody. The THRA induced by TBII(+)/TSAb(+) IgGs as well as TBII(-)/TSAb(-) IgG was inhibited by blocking-type TRAb obtained from TBII(+) patients with myxedema. There was a significant correlation between THRA and TSAb. These in vitro findings suggest that all IgGs obtained from untreated Graves' patients (n = 78) elicit potent THRA in human thyroid follicles in suspension culture. Because the TBII(-)/TSAb(-) IgGs can stimulate cAMP production in human but not in porcine thyrocytes, they probably recognize epitope(s) of TSH-binding sites specific to the human thyrotropin (hTSH) receptor. Furthermore, we have demonstrated that the thyroid gland of hyperthyroid Graves' patients is stimulated by IgG(s) equivalent to at least 0.8 microU/mL bTSH (about 5 microU/mL hTSH) in vitro.

Pathogenesis of autoimmune hypothyroidism induced by lymphokine-activated killer (LAK) cell therapy: in vitro inhibition of human thyroid function by interleukin-2 in the presence of autologous intrathyroidal lymphocytes.

Lymphokine-activated killer (LAK) cell therapy frequently results in primary hypothyroidism. To elucidate the responsible mechanism, we investigated the effects of interleukin-2 (IL-2) on thyroid function of cultured human thyroid follicles in the presence or absence of autologous thyroid-derived lymphoid cells (TDLC). Human thyroid follicles, obtained by subtotal thyroidectomy from patients with Graves' disease, were cultured in serum-free medium containing bTSH and various concentrations of human IL-2, with or without TDLC. After 5 days of culture, 125I was added, and after an additional 3 days of culture, 125I incorporated into thyroid follicles and organic 125I (125I-T4 + 125I-T3) released into the culture medium were estimated. In the absence of TDLC, IL-2 did not affect bTSH-induced thyroid function. In the presence of TDLC, however, IL-2 inhibited the bTSH-stimulated thyroid function in a concentration-dependent manner. The minimum IL-2 concentration required to inhibit thyroid function was 1 U/ml. At 100 U/ml, IL-2 inhibited thyroid function completely. IL-2 increased the concentration of IFN-gamma in the culture medium conditioned by TDLC but not by thyroid follicles. When both were cocultured, IL-2 more significantly increased the concentration of IFN-gamma to an extent sufficient for inhibiting thyroid function per se. Furthermore, the addition of anti-IFN-gamma antibody to the medium partially restored the IL-2-inhibited thyroid function. In summary, by coculturing human thyroid follicles and autologous intrathyroidal lymphocytes with IL-2, it was possible to reproduce the thyroid dysfunction associated with LAK cell therapy. Our data indicate that IL-2 inhibits thyroid function, at least partly, by stimulating production of IFN-gamma, a potent inhibitory cytokine for thyroid cells.

[Unfavorable characteristics in patients with early postoperative recurrence of Graves' disease after subtotal thyroidectomy].

Unfavorable characteristics of early postoperative recurrence of hyperthyroidism were studied in patients with Graves' disease after subtotal thyroidectomy. We examined 338 patients who underwent subtotal thyroidectomy during a 9-year period from 1981 to 1989. Thyroid functional status was determined biochemically and the following variables were collected and divided into two categories for each patient: sex, age at operation, indication for surgery, serum titer of antimicrosomal hemagglutination antibody, weight of resected thyroid tissue and size of remnant thyroid tissue relative to body surface area. Early postoperative recurrence was defined as relapse of the disease within one year after surgery, necessitating medication or radioiodine therapy. Logistic regression analysis disclosed that the most important characteristics were age < 20 yr, relatively large thyroid remnant and weight of resected thyroid tissue > or = 100g, the odds ratios for these variables being 20.6, 16.7 and 3.4, respectively (p < 0.05). Although candidates for surgical treatment of Graves' disease are likely to be younger and to have a large goiter, these characteristics are unfavorable, since they predict the early recurrence of hyperthyroidism. One alternative is to leave a smaller than usual thyroid remnant thyroid in these patients, even if the risk of postoperative hypothyroidism is high.