Anti-thyrotropin (TSH) receptor autoantibodies (TRAbs) have been known to be involved in Graves' disease. To understand the molecular mechanism for pathogenesis of TSAbs in Graves' disease, we ...isolated and reconstituted the Ig genes of EBV-transformed B cell clones producing monoclonal thyroid stimulating Ab (TSAb) obtained from patients with Graves' disease. The V region genes of Ig heavy (H) and light (L) chains of two TSAb clones, IgG clone B6B7 and IgM clone 101-2, were isolated by the PCR. Nucleotide sequencing analysis revealed that germ-line VH and VK segments widely used for autoantibodies including the previously isolated TRAbs were utilized in the two clones. A significant number of somatic mutations were found in V regions of both clones, indicating the involvement of somatic mutations for the TSAb specificity. Reconstituted Ig H and L chain genes of the two clones were stably introduced into myeloma cells for IgG1 production. IgGs purified from cultured supernatants of both transfectants exhibited significant TSAb activities, while they did not inhibit TSH binding to the receptor. The successful expression of recombinant TSAbs in eukaryotic cells will provide opportunities to apply them to various pathophysiologic, diagnostic and therapeutic investigations in autoimmune thyroid diseases.
Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, ...strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 95% confidence interval, 1.4 to 3.9; Kumamoto: odds ratio, 2.4 95% confidence interval, 1.4 to 4.0). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.
In a transgenic mouse model of autoimmune haemolytic anaemia, CD5+ B lymphocytes localized in the peritoneal cavity are shown to play an important role in the onset of autoimmune disease. The authors ...have examined whether CD5+ B cells are present in the peritoneal cavity of 12 human individuals with non‐invasive gastrointestinal tumours and found that in humans CD5+ B cells preferentially lodge in the peritoneal cavity as compared to the peripheral blood and spleen while the numbers of the peritoneal B lymphocytes in humans are much lower than in mice and vary widely between individuals.
Ghrelin, an endogenous ligand for the GH secretagogue receptor, induces GH secretion, food intake, and positive energy balance. Although ghrelin exhibits a variety of hormonal actions, the mechanisms ...regulating ghrelin expression and secretion remain unclear. To understand regulation of human ghrelin gene expression, we examined the genomic structure of approximately 5,000 bp of the 5′-flanking region of the human ghrelin gene. We performed rapid amplification of cDNA ends to estimate transcriptional start sites, indicating that there are two transcriptional initiation sites within the human ghrelin gene. Both transcripts were equally expressed in the human stomach, whereas the longer transcript was mainly expressed in a human medullary thyroid carcinoma (TT) cell line. Functional analysis using promoter-reporter constructs containing the 5′-flanking region of the gene indicated that the sequence residing within the −349 to −193 region is necessary for human ghrelin promoter function in TT cells. Within this region existed several consensus sequences for a number of transactivating regulatory proteins, including an E-box site. Destruction of this site decreased to 40% of the promoter activity. The upstream region of the promoter has two additional putative E-box sites, and sitedirected mutagenesis suggested that these are also involved in promoter activation. Electrophoretic mobility shift assays demonstrated that the upstream stimulatory factor specifically bound to these E-box elements. These results suggest a potential role for upstream stimulatory factor transcription factors in the regulation of human ghrelin expression.
Autoimmune thyroid disease (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT), is caused by multiple genetic and environmental factors. The clinical and immunological features of ...GD and HT are distinct; however, there are multiplex families with both GD and HT, and cases in which GD evolves into HT. Thus, there may be specific susceptibility loci for GD or HT, and common loci controlling the susceptibility to both GD and HT may exist. A genome-wide analysis of data on 123 Japanese sib-pairs affected with AITD was made in which GD- or HT-affected sib-pairs (ASPs) were studied to detect GD- or HT-specific susceptibility loci, and all AITD-ASPs were used to detect AITD-common susceptibility loci. Our study revealed 19 regions on 14 chromosomes (1, 2, 3, 5, 6, 8, 9, 10, 11, 12, 13, 15, 18 and 22) where the multipoint maximum LOD score (MLS) was >1. Especially, chromosome 5q31-q33 yielded suggestive evidence for linkage to AITD as a whole, with an MLS of 3.14 at D5S436, and chromosome 8q23-q24 yielded suggestive evidence for linkage to HT, with an MLS of 3.77 at D8S272. These observations suggest the presence of an AITD susceptibility locus at 5q31-q33 and a HT susceptibility locus at 8q23-q24.
Background. It has been indicated that T-box 21 (TBX21) and H 2.0-like homeobox (HLX) are transcription factors related to the differentiation of T helper 1 cells, whereas GATA-binding protein 3 is ...the master transcription factor of T helper 2 cells. Methods. We genotyped − 1514T/C (rs17250932) and − 1993T/C (rs4794067) polymorphisms of TBX21, − 742C/G polymorphism (rs2184658) of HLX and − 1420G/A polymorphism (rs1269486) of GATA3 in genomic DNA samples from Japanese patients; 51 patients with severe Hashimoto's disease (HD), 39 with mild HD, 66 with intractable Graves' disease (GD), in whom remission was difficult to induce, 47 with GD in remission and 79 healthy volunteers. Results. The T alleles of the TBX21 − 1514T/C and − 1993T/C polymorphisms were more frequent in patients with intractable GD than in those with GD in remission. Among individuals with the TBX21 − 1993TT genotype, the G allele of HLX − 742C/G polymorphism, which correlates with low HLX expression, was more frequent in patients with intractable GD than in those with GD in remission. Conclusions. Functional polymorphisms in TBX21 are associated with the development of autoimmune thyroid diseases and prognosis of GD, and a functional polymorphism in HLX in combination with the TBX21 polymorphism is also associated with the prognosis of GD.
Ghrelin not only strongly stimulates GH secretion, but is also involved in energy homeostasis by stimulating food intake and promoting adiposity through a GH-independent mechanism. These effects of ...ghrelin may play an important role in the pathophysiology of inflammatory wasting syndrome, in which both the somatotropic axis and energy balance are altered. In this study we investigated plasma ghrelin concentrations after lipopolysaccharide (LPS) administration to rats, a model of the wasting syndrome and critical illness. In addition, the therapeutic potential of the antiwasting effects of ghrelin was explored using LPS-injected rats. A single LPS injection suppressed plasma ghrelin levels 6 and 12 h later. Maximal reduction was observed 12 h after LPS injection, in a dose-dependent manner. In contrast, plasma ghrelin levels were elevated after repeated LPS injections on d 2 and 5. Peripheral administration of ghrelin twice daily (10 nmol/rat) for 5 d increased body weight gain in repeated LPS-injected rats. Furthermore, both adipose tissue weight and plasma leptin concentrations were increased after ghrelin administration in these rats. In conclusion, plasma ghrelin levels are altered in LPS-injected rats, and ghrelin treatment may provide a new therapeutic approach to the wasting syndrome and critical illness.
We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ...ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, β cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism.
Objective
Ghrelin is a brain-gut peptide that is mainly secreted from gastric endocrine cells (X/A like cells). In addition to promoting growth-hormone release and appetite, ghrelin also affects ...gastric motility and secretion. Circulating ghrelin levels are related to appetite and energy balance. Functional dyspepsia (FD) is a disorder characterized by the presence of chronic or recurrent symptoms of upper abdominal pain or discomfort. Although no known specific organic abnormalities are present in FD, abnormalities in gastrointestinal motility and sensitivity are thought to play a role in a substantial subgroup of patients. In addition, some patients also suffer from anorexia and body-weight loss. To investigate the role of ghrelin in the pathophysiology of FD, circulating ghrelin levels in affected patients were measured. Material and methods
Eighteen Japanese female patients with functional dyspepsia and 18 healthy volunteers were recruited for the study. Acylated and desacyl forms of ghrelin were measured using commercially available enzyme-linked immunosorbent assay kits. Results
Although plasma levels of acylated or desacyl ghrelin were not significantly different between healthy subjects and FD patients, plasma acylated, but not desacyl ghrelin, levels were correlated with a subjective symptom score in FD patients. In addition, the ratio of acylated to desacyl ghrelin (A/D ratio) was correlated strongly with acylated, but not desacyl, ghrelin levels. Conclusions
The correlation of circulating acylated ghrelin levels with the subjective symptom score and the A/D ratio in FD patients suggest that acylated ghrelin may play a role in the pathophysiology of FD.
Two synthetic peptides, P354-14 (amino acid nos. 354 to 367) and P338-16 (nos. 338 to 353), corresponding to the partial amino acid sequences of the hTSH receptor structure were studied for their ...ability to bind specifically serum IgGs from patients with Graves' disease and to inhibit thyroid stimulating TSH receptor antibody (TSH-R SAb) activity. IgG binding was measured by an ELISA using sera from 102 Graves', 20 Hashimoto patients, and 9 normal subjects. Both peptides showed significantly increased IgG binding of Graves' sera compared with those of Hashimoto and normal sera. There was a significant correlation (r = 0.529) between the amount of IgG bound by the two peptides, but neither of these values correlated well with their TSH-R SAb activity nor thyrotropin-binding inhibitor TSH receptor antibody (TSH-R IAb) activity. TSH-R SAb inhibiting effects of these peptides were then analysed by measuring TSH-R SAb activity after incubation with the peptides. Among eight Graves' IgGs tested the TSH-R SAb activity of three was inhibited by both peptides, two were inhibited only by P354-14 and three were not affected by either. These TSH-R SAb inhibiting effects were dose-dependent and reproducible. To confirm these findings, a peptide-sepharose gel affinity absorption study was performed. Eleven Graves' IgGs were applied to both peptide gels and the TSH-R SAb activity of the unabsorbed fraction was measured. The TSH-R SAb activity of five IgGs was strongly absorbed only by P354-14 and five others were absorbed by both peptides to an almost similar extent.
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