Polymorphisms in the TNFA and IL6 Genes Represent Risk Factors for Autoimmune Thyroid Disease

Background

Autoimmune thyroid disease (AITD) comprises diseases including Hashimoto''s thyroiditis and Graves'' disease, both characterized by reactivity to autoantigens causing, respectively, inflammatory destruction and autoimmune stimulation of the thyroid-stimulating hormone receptor. AITD is the most common thyroid disease and the leading form of autoimmune disease in women. Cytokines are key regulators of the immune and inflammatory responses; therefore, genetic variants at cytokine-encoding genes are potential risk factors for AITD.

Methods

Polymorphisms in the IL6-174 G/C (rs1800795), TNFA-308 G/A (rs1800629), IL1B-511 C/T (rs16944), and IFNGR1-56 T/C (rs2234711) genes were assessed in a case-control study comprising 420 Hashimoto''s thyroiditis patients, 111 Graves'' disease patients and 735 unrelated controls from Portugal. Genetic variants were discriminated by real-time PCR using TaqMan SNP genotyping assays.

Results

A significant association was found between the allele A in TNFA-308 G/A and Hashimoto''s thyroiditis, both in the dominant (OR = 1.82, CI = 1.37–2.43, p-value = 4.4×10⁻⁵) and log-additive (OR = 1.64, CI = 1.28–2.10, p-value = 8.2×10⁻⁵) models. The allele C in IL6-174 G/C is also associated with Hashimoto''s thyroiditis, however, only retained significance after multiple testing correction in the log-additive model (OR = 1.28, CI = 1.06–1.54, p-value = 8.9×10⁻³). The group with Graves'' disease also registered a higher frequency of the allele A in TNFA-308 G/A compared with controls both in the dominant (OR = 1.85, CI = 1.19–2.87, p-value = 7.0×10⁻³) and log-additive (OR = 1.69, CI = 1.17–2.44, p-value = 6.6×10⁻³) models. The risk for Hashimoto''s thyroiditis and Graves'' disease increases with the number of risk alleles (OR for two risk alleles is, respectively, 2.27 and 2.59).

Conclusions

This study reports significant associations of genetic variants in TNFA and IL6 with the risk for AITD, highlighting the relevance of polymorphisms in inflammation-related genes in the etiopathogenesis of AITD.     

Genetic Factors in Hashimoto's Struma

It has previously been established that there is a significant history of thyroid disorders in families of patients with Hashimoto''s struma or chronic thyroiditis. In the present study, 99 relatives of 20 patients with Hashimoto''s struma or chronic thyroiditis were studied with regard to the incidence of circulating thyroid autoantibodies; 42 of these 99 relatives were found to have such antibodies. Twenty of the 99 relatives were shown to have thyroid abnormalities (chiefly goitre); of this group of 20, antibodies were found in 12. In the remaining 79 persons (who had no clinical evidence of thyroid disease), 30 were found to have circulating antithyroid antibodies. The incidence of such antibodies among these relatives is very significantly greater than in the general population.From these and other similar studies, there is strong evidence favouring a genetic predisposition for Hashimoto''s struma and chronic thyroiditis. The mode of inheritance is not yet established, and the pathogenesis of the disease has not yet been elucidated.     

Hypothyroidism after Thyroidectomy for Graves's Disease: A Search for an Explanation

Out of 38 patients who had undergone subtotal thyroidectomy for Graves''s disease seven to 20 years previously 15 developed hypothyroidism. In these 15 patients autoantibodies against thyroid cytoplasm were significantly more frequent than in the 23 euthyroid patients, though there was no difference in the prevalence of autoantibodies against thyroglobulin. Histological examination of the thyroid tissue removed at operation showed that significantly more plasma cells and lymphoid follicles with germinal centres were present in patients who subsequently developed hypothyroidism than in those who remained euthyroid. No differences in the amount of lymphocytic infiltration were seen in hypothyroid and euthyroid patients.The results suggest that B lymphocytes play a part in the development of postoperative hypothyroidism in Graves''s disease. It is proposed that Graves''s disease and Hashimoto''s disease are different aspects of the same basic autoimmune process.     

Relationship of age-specific incidence rates to immunological aspects of Hashimoto's thyroiditis

Studies of the age-specific incidence rates of the appearance of Hashimoto''s thyroiditis indicate that this disorder appears at random in a genetically preselected population. Following an initial lag in the first few years of life, the disease appears at a constant rate thereafter in this population.The age-specific incidence rates were similar to those previously reported for Graves'' disease. Moreover, there is considerable evidence implicating cell-mediated immunity in both diseases, with the likelihood of cooperating humoral antibodies as well. It may be hypothesized that the two diseases are primarily due to genetic defects in immunological surveillance, which result in an inability to destroy or control a specific forbidden clone of thymicderived lymphocytes which may arise by normal random mutation. The T-lymphocyte interacts with its complementary antigen (on a hitherto normal thyroid cell), setting up a cell-mediated immune response; in addition it may cooperate with bursa-equivalent lymphocytes, which then produce humoral antibodies. It is possible that both cell-mediated immunity and humoral antibodies are necessary for the full expression of the disease.     

Measurement of autoantibodies against human eye muscle plasma membranes in Graves' ophthalmopathy.

Antibodies that reacted with plasma membranes of human eye muscle but showed no binding to plasma membranes of human skeletal muscle were identified in serum of patients with Graves'' ophthalmopathy. Rabbit antithyroglobulin serum at a dilution of 1 X 10(-3) to 20 X 10(-3) had no effect on the binding of these antibodies to eye muscle membrane antigens. There was no correlation between antihuman eye muscle plasma membrane antibodies and antihuman thyroid membrane antibodies or antibodies against thyroglobulin. It is suggested that specific antibodies against eye muscle membranes are present in Graves'' ophthalmopathy and that this disease might represent a distinct autoimmune disorder.     

Comparative thermodynamics of opioid receptor ligand interaction in the bovine adrenal medulla membranes—Evidence of opioid site heterogeneity

1. A marked dependence on temperature of agonist binding δ, μ and κ₁₋₃, opioid sites in the bovine adrenal medulla was observed, at the range of 0 to 37°C. These changes concern kinetic (k₁) and equilibrium constants (K_d), but not binding capacities (B_max).2. These dependences are different for each ligand and each opioid receptor, suggesting their molecular heterogeneity.3. The comparative thermodynamics indicates that the interaction of opioid agonists with their receptor is exergonic (ΔG° < 0) and entropy driven (ΔS° > 0).4. The comparison of Van't Hoff and Arrhenius plots indicates a discrete mechanism in the binding of each opioid receptor.     

Thermodynamic Parameters of Ligand-Receptor Interactions: Computation and Error Margins

Abstract

A semiempirical relationship describing the temperature function of ligand-receptor dissociation constants (K_d), derived from heat capacities of the system in equilibrium, is suggested for computation of the standard enthalpy (δH°) and standard entropy (δS°) changes in equilibrium. The use of the linear expression (called Gibbs-van't Hoff equation) may lead to inaccurate results when heat capacity C_p displays a considerable temperature dependence. The accuracy of K_d, δH° and δS° has been studied by simulation experiments. In the case of K_d, deviations of computed from “true” values are determined by both the accuracy of experimental data used for its estimation, and by the shape of the binding isotherm (for instance, by Hill coefficients or by the presence of low affinity sites). As a rule, if errors of bound ligand measurements are greater than 20 per cent, K_d estimates ought to be considered as less reliable. However, computations of δH° and δS° that use such K_d values, are more correct, probably due to an error compensation. The suggested nonlinear temperature function of K_d enables an estimate of the heat capacity of the system and its temperature dependence.     

Revised model of calcium and magnesium binding to the bacterial cell wall

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca²⁺, Region I has a K_A = (1.0 ± 0.2) × 10⁶ M^?1 and Region II has a K_A = (0.075 ± 0.058) × 10⁶ M^?1. For Mg²⁺, K_A1 = (1.5 ± 0.1) × 10⁶ and K_A2 = (0.17 ± 0.10) × 10⁶. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η₂, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca²⁺ and Mg²⁺ respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.     

Characterization of phenylalkylamine binding sites in insect (Periplaneta americana) nervous system and skeletal muscle membranes

This study has identified specific, stereoselective phenylalkylamine (PAA, (±)- [³H]verapamil) binding sites of low-affinity and high-density in cockroach (Periplaneta americana) nervous system and skeletal muscle membranes. Scatchard transformation of equilibrium binding data revealed a single population of binding sites in both tissues with dissociation constants (K_d) of 273 nM and 377 nM and binding capacities (B_max) of 23 pmol·mg protein^?1 and 37pmol·mg protein^?1 for cockroach nervous tissue and skeletal muscle membranes, respectively. The PAA binding site in cockroach nervous tissue membranes was found to be dihydropyridine (DHP)-insensitive, whereas the corresponding site in cockroach skeletal muscle membranes was DHP-sensitive. This property of a DHP-sensitive PAA receptor distinguishes the binding sites identified in cockroach skeletal muscle from those in cockroach nervous tissue and indicates that pharmacologically distinct putative Ca²⁺ channel subtypes are present in insect nerve and muscle. © 1993 Wiley-Liss, Inc.     

Differential Regulation of Transferrin Receptor Gene Expression in Human Hemopoietic Cells: Molecular and Cellular Aspects

Abstract

As we have shown earlier (-)¹²⁵lodocyanopindolol (¹²⁵ICYP) binding to β-adrenoceptors (β-AR) in human mononuclear leucocytes (MNL) yields evidence for the existence of high affinity (B_hiaff) and low affinity (B_loaff) binding sites. We studied the regulation of these 2 classes of binding sites during 240 min of (-)-epinephrine (EPI) infusion (0.1 μg/kg/min) (n=8) in male healthy volunteers. Saturation experiments were performed on MNL membranes with ¹²⁵ICYP over a large concentration range (1–550 pmol/l). Binding parameters were calculated by computer analysis assuming 2 classes of binding sites. We found a preinfusion value of 830±50 [sites/cell] (K_D=1.5±0.2 pmol/l) of B_hiaff binding sites and 5210±510 [sites/cell] (K_D=420±80 pmol/l) of B_loaff. During EPI infusion we observed biphasic modulation of the B_hiaff and an inverse modulation of the B_loaff. After 40 min of EPI B_hiaff increased to 1970±280 [sites/cell] (K_D=4.2±0.8 pmol/l), whereas B_loaff decreased to 2720±280 [sites/cell] (K_D=140±70 pmol/l); despite constant plasma epinephrine concentration (PEC) after 240 min of EPI B_hiaff changed to 1310±240 [sites/cell] (K_D=2.8±1.0 pmol/l) vs. 4370±760 [sites/cell] (K_D=190±100 pmol/l) B_loaff. These results suggest an interdependent inverse modulation of the 2 classes of binding sites for ¹²⁵ICYP on MNL during EPI infusion.     

Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding.

The characteristics of [³H]flunitrazepam binding to brain specific benzodiazepine receptors were determined at varying temperatures. The rates at which [³H]flunitrazepam associated with and dissociated from benzodiazepine receptors increased with increasing temperatures. The dissociation constant (K_D) also increased with increases in temperature. The (K_D) determined by Scatchard analyses of saturation isotherms showed a similar change with changes in temperature. The maximal binding capacity (Bmax) did not change with changes in temperature. The inhibitory constants of several benzodiazepines to inhibit [³H]flunitrazepam binding to brain were also higher at 37°C than at 0°C, suggesting that the binding affinity of all benzodiazepines to brain benzodiazepine receptors is lower at 37°C than at 0°C. Van't Hoff analysis of [³H]flunitrazepam binding to brain at different temperatures reveals two linear components to this relationship.     

甲状腺激素对适应性免疫细胞的作用

摘要：甲状腺激素（Thyroid hormones,THs）参与免疫功能的调节,在固有免疫和适应性免疫中发挥着重要作用。THs异常分泌所致的免疫功能失调被认为参与了格雷夫斯病和桥本甲状腺炎等自身免疫性疾病的发生发展。目前,THs在固有免疫细胞（中性粒细胞、巨噬细胞、树突状细胞、自然杀伤细胞、肥大细胞）中的作用已得到了较好的阐明,但THs对适应性免疫细胞（T淋巴细胞与B淋巴细胞）的影响等方面的研究仍未引起足够的重视。因此,本研究从适应性免疫细胞的角度出发,重点讨论了THs对这些细胞的发育、分化及功能等方面的影响,为进一步理解THs调节免疫功能的作用提供新视角。     

Uptake of high-density lipoprotein by Y-organs of the crab,cancer antennarius. II. formal characterization of receptor-mediation with isolated membranes

Y-organs are the ecdysial glands of crustaceans, responsible for synthesis and secretion of ecdysteroid hormones. For this purpose, the glands acquire cholesterol as obligate precursor entirely from circulating high-density lipoprotein (HDL). A preceding study provided evidence for the mechanism of acquisition: Y-organs take up cholesterol bound to HDL by an energy-requiring process, receptor-mediated absorptive endocytosis. The present study characterized the receptors involved utilizing isolated Y-organ membranes. HDL binding was saturable and specific; a dissociation constant (K_d) of 1.08 × 10^?7 M and a binding maximum at equilibrium (B_max) of 70 μg HDL protein/mg membrane protein, were obtained. Binding was decreased by protease and was dependent upon calcium. Y-organs are regulated negatively by a peptide hormone from the eystalks, molt-inhibiting hormone (MIH). Y-organ membranes from de-eyestalked crabs (MIH absent) exhibited the same K_d value as membranes from intact crabs, but a B_max 17% higher. Thus, MIH activity apparently does not change the binding affinity of HDL, but decreases the number of binding sites. These results agree with our previous findings that MIH depresses ecdysteroid synthesis in part by inhibiting cholesterol uptake. Generally, Y-organ cells appear to contain receptors for HDL that are of high affinity and high binding capacity, similar to the characteristics reported for the binding of insect HDL (vitellogenin) to fat bodies and oocytes. © 1995 Wiley-Liss, Inc.     

Binding of β1-adrenoreceptor antagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial nodeantagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial node

Specific β₁-adrenoreceptors antagonist [³H]CGP 26505 binding was characterized in rat cerebral cortex and heart sinus atrial node. In both tissues [³H]CGP 26505 binding was maximal at 25°C, it was specific, saturable and protein concentration dependent. Scatchard analysis of saturation isotherms of specific [³H]CGP 26505 binding in cerebral cortex showed that [³H]CGP 26505 binds a single class of high affinity sites with a dissociation constant (K_D) of 1±0.3 nM and a maximal number of binding sites (B_max) of 40±2 fmol/mg of protein. In sinus atrial node, [³H]-CGP 26505 binds a single class of high affinity sites (K_D=1.9±0.4 nM, B_max=28±2 fmol/mg of protein).     

Multiple [3H]-Oxytocin Binding Sites in Rat Myometrial Plasma Membranes

Abstract

The affinity spectrum method has been used to analyse binding isotherms for [³H]-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (K_d) of 0.6–1.5 × 10^?9, 0.4–1.0 × 10^?7 and 7 × 10^?6 mol/l were identified and their existence verified by cluster analysis based on similarities between K_d, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTPrS (guanosine-5′-0-(3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

Circulating prostacyclin and thromboxane in patients with graves' disease

We measured plasma levels of PGI₂ as 6-keto-prostaglandin F_la (6-keto-PGFla) and thromboxane A₂ (TXA₂) as thromboxane B₂ (TXB₂) in patients with Graves' disease and in normal subjects. The levels of plasma 6-keto-PGFla were significantly elevated and correlated with those of serum T₄ and T₃, respectively, in hyperthyroid patients with Graves' disease. Significant reduction of 6-keto-PGF_1a levels was observed after antithyroid drug therapy. In contrast, the levels of plasma TXB₂ were significantly lower in untreated patients with Graves disease than in normal subjects. These data suggest that an elevation of plasma 6-keto-PGF_1a may play some additional role in pathophysiology of Graves disease.     

N‐Heterocyclic‐based adsorbents for antibody purification—effect of ligand structure

A new set of ligands based on substituted pyridine and other N‐heterocyclic structures, possessing an aliphatic primary amino group tether and an exocyclic sulphur atom, has been prepared and immobilized onto epoxy‐activated matrices such as Sepharose 6 Fast Flow®. The derived adsorbents have been evaluated for their utility to capture and purify humanized monoclonal antibodies. Favourable binding properties were assessed from screening assays to determine optimal conditions for the capture and elution of the monoclonal antibodies. Static and dynamic binding experiments were employed to derive the equilibrium dissociation constants K_D's and binding capacities Q_max's. Typically, the K_D values were in the range of 2–5 μM and the Q_max values between 20 and 75 mg mAb/ml resin, depending on the stereo‐electronic properties of the substituent in the N‐heterocyclic ring structure. The effect of ligand structure on the selectivity of these adsorbents was also investigated, and criteria for their use in the purification of monoclonal antibodies from cell culture supernatants established. Copyright © 2014 John Wiley & Sons, Ltd.     

Physiological characteristics of inorganic nitrogen uptake by spatially separate algal communities in a nitrogendeficient lake

1. The physiological characteristics of nitrogen uptake by sublittoral and eulittoral (splash zone) epilithic periphyton as well as epipelic periphyton in N-deficient Castle Lake, California were determined by evaluating the half-saturation constants (K_t) and initial slopes (V_max/K_t) of uptake kinetics curves. These results were compared to similar studies of phytoplankton nitrogen uptake in this lake. 2. The strategies of nitrogen uptake differed among the various communities and were largely determined by the proximity of each to pools of available dissolved inorganic N (DIN). 3. The sublittoral algae did not have a high biological affinity for either NH₄ or NO₃ and depended on nitrogen fixation for their N supply. The eulittoral community showed an increased capacity for DIN uptake at low substrate concentration, but not as high as measured for the phytoplankton community. Epipelic algae live immediately adjacent to a large pool of interstitial sediment NH₄ and showed no physiological adaptations for surviving in a N-deficient environment. 4. K_t values for all benthic communities were approximately two orders of magnitude greater than ambient substrate levels. In contrast, the half-saturation constants for NH₄+ NO₃ uptake by phytoplankton were very similar to in situ levels of these nutrients.     

Bradykinin antagonists with dehydrophenylalanine analogues at position 5

Continuing the studies on structural requirements of bradykinin antagonists, it has been found that analogues with dehydrophenylalanine (ΔPhe) or its ring‒substituted analogues (ΔPhe(X)) at position 5 act as antagonists on guinea pig pulmonary artery, and on guinea pig ileum. Because both organs are considered to be bradykinin B₂receptor tissues, the analogues with ΔPhe or ΔPhe(X) at position 5, but without any replacement at position 7, seem to represent a new structural type of B₂receptor antagonist. All the analogues investigated act as partial antagonists; they inhibit the bradykinin‒induced contraction at low concentrations and act as agonists at higher concentrations. Ring substitutions by methyl groups or iodine reduce both the agonistic and antagonistic activity. Only substitution by fluorine gives a high potency. Incorporation of ΔPhe into different representative antagonists with key modifications at position 7 does not enhance the antagonist activity of the basic structures, with one exception. Only the combination of ΔPhe at position 5 with D Phe at position 7 increases the antagonistic potency on guinea pig ileum by about one order of magnitude. Radio‒ligand binding studies indicate the importance of position 5 for the discrimination of B₂receptor subtypes. The binding affinity to the low‒affinity binding site (K_L) was not significantly changed by replacement of Phe by ΔPhe. In contrast, ring‒methylation of ΔPhe results in clearly reduced binding to K_L. The affinity to the high‒affinity binding site (K_H) was almost unchanged by the replacement of Phe in position 5 by ΔPhe, whereas the analogue with 2‒methyl‒dehydrophenylalanine completely failed to detect the K_H‒site. The peptides were synthesized on the Wang‒resin according to the Fmoc/Bu^tstrategy using Mtr protection for the side chain of Arg. The dehydrophenylalanine analogues were prepared by a strategy involving PyBop couplings of the dipeptide unit Fmoc‒Gly‒ΔPhe(X)‒OH to resin‒bound fragments. © 1998 European Peptide Society and John Wiley & Sons, Ltd.