Tamio Ieiri

Activation of macrophages by lactoferrin: secretion of TNF‐α, IL‐8 and NO

When macrophages were cultured with lactoferrin, cytokines such as tumor necrosis factor (TNF‐α), interleukin 8 (IL‐8) and nitric oxide (NO) were secreted. Secretion of TNF‐α peaked at 6 h of incubation in the presence of lactoferrin and then declined. About 80% of the maximum secretion of IL‐8 was observed at 6 h of incubation. The concentration of IL‐8 in the culture medium remained almost constant between 24‐72 h. In contrast, no siginificant effect on NO secretion was observed at 6 h, but a significant effect was observed at 24 h and secretion gradually increased between 24‐72 h. The effects of lactoferrin on the secretion of TNF‐α, IL‐8 and NO were dose‐dependent and lactoferrin had a significant effect on secretion at concentrations greater than 10 mg/ml. The use of reverse transcription‐polymerase chain reaction (RT‐PCR) showed that the results obtained were consistant with the cytokine secretion results. It is concluded that lactoferrin activates macrophages which result in the secretion of TNF‐α, IL‐8 and NO.

A Novel Homozygous Missense Mutation of the Dual Oxidase 2 (DUOX2) Gene in an Adult Patient with Large Goiter

OBJECTIVE To describe the first adult case of large goiter associated with a novel R1110Q mutation in the dual oxidase 2 (DUOX2) gene. She was initially euthyroid, and developed hypothyroidism later in her forties. DUOX2 is an essential enzyme in iodine organification of thyroid hormone biosynthesis. Only infant cases of congenital hypothyroidism due to mutations of the DUOX2 gene have been reported. Biallelic mutation of DUOX2 is thought to lead to total iodine organification defect. PATIENTS AND MEASUREMENT: This 57-year-old woman became first aware of goiter around the age of 20 years. Since the goiter had enlarged gradually, she consulted us at the age of 32 years. Goiter was soft, and thyroid function was normal. Antithyroid antibodies were negative. Both physical and mental development was normal. Three of her nine siblings and her mother had large goiters. At the age of 44 years, thyroid function demonstrated subclinical hypothyroidism. She started to take levo-thyroxine at a dose of 100 mug/day to reduce goiter. At the age of 56 years, goiter size remained the same. The perchlorate discharge rate was 72.8%, suggesting partial iodine organification defect. Thus, thyroid peroxidase (TPO) gene and DUOX2 gene were analyzed. RESULTS There was no mutation in the TPO gene, but a novel homozygous mutation (R1110Q) in the DUOX2 gene was identified. The same heterozygous mutation was detected in her two sons and two grandchildren. This mutation was not detected in 104 control alleles and was located at a site differing from any other reported mutations in the DUOX2 gene. CONCLUSIONS This homozygous missense mutation can be associated with thyroid dysfunction and goiter formation of an enlarged thyroid gland.

Partial iodide organification defect caused by a novel mutation of the thyroid peroxidase gene in three siblings

background Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808–13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their fathers DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well‐conserved amino acids in the peroxidase superfamily, Gly533Cys‐ and Asp574/Leu575del‐TPOs were thought to be affected structurally or functionally. In expression studies using CHO‐Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild‐type TPO and had enzyme activity, although Gly533Cys‐TPO was slightly lower in efficiency of expression and more degenerative than wild‐type TPO.

Clinical significance of measurement of plasma annexin V concentration of patients in the emergency room

Annexin V, a calcium-binding protein, is widely present in various organs and tissues. In the present study, plasma annexin V concentration was measured in 158 patients who were brought to the emergency room, including 25 patients suffering from acute myocardial infarction (AMI), 14 with cerebrovascular disease, 11 with trauma of the extremities, 11 with severe trauma associated with visceral damage, and 35 with witnessed cardiac arrest. Annexin V concentration in normal healthy individuals (n=110) was 1.9+/-0.7 ng/ml. Annexin V concentration in AMI and cardiac arrest patients was 11.0+/-4.9 and 15.3+/-7.9 ng/ml, respectively, being significantly higher than that in patients with cerebrovascular disease (5.4+/-2.7 ng/ml). The value in severe trauma patients was 15.9+/-9.4 ng/ml, being significantly higher than that in patients with trauma of the extremities (5.6+/-1.2 ng/ml). Annexin V concentrations in the cardiac arrest and AMI patients who survived more than 24 h after admission were lower than those in patients who died within 24 h after the onset of symptoms. Annexin V content in the lungs and myocardium in normal rats was extremely high in comparison to that in brain and skeletal muscle. These results suggest that the high levels of plasma annexin V in patients with AMI, cardiac arrest and severe trauma reflect the severity of damage of the myocardium and/or other visceral organs, and measurement of plasma annexin V concentration may help to assess the prognosis of patients brought to the emergency room.

A novel compound heterozygous mutation in the thyroglobulin gene resulting in congenital goitrous hypothyroidism with high serum triiodothyronine levels

AbstractThyroglobulin abnormality is a rare cause of congenital hypothyroidism and only a limited number of mutations in the thyroglobulin gene have been reported. We analyzed the thyroglobulin gene in a patient with congenital goitrous hypothyroidism. This girl was identified with hyperthyrotropinemia in a neonatal mass-screening test. The patient had goiter, and her body weight gain was poor. Distal femoral epiphysis was absent on roentgenography. Her serum thyroxine level was low; however, her triiodothyronine level was high. Autoantibodies against triiodothyronine, thyroid peroxidase, and thyroglobulin were all negative. Her serum thyroglobulin level was undetectable. The thyroglobulin gene from the genomic DNA of the patient was analyzed by direct sequencing. Two novel heterozygous missense mutations, Cys1897Tyr (exon 31) and Arg2336Gln (exon 40), were found in the patient. The former mutation was derived from her mother, suggesting a compound heterozygous state. Normal triiodothyronine and low thyroxine concentrations are often observed in patients with thyroglobulin gene mutations. We considered that some patients with thyroglobulin abnormality might have high triiodothyronine levels. In cases of congenital goitrous hypothyroidism with normal-to-high triiodothyronine levels and low serum thyroglobulin levels, thyroglobulin abnormality should be considered.

Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease.

To confirm the significance of excretion of annexin V into the urine and the change of urinary annexin V concentration in kidney disease, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two monoclonal antibodies. Urinary annexin V concentration was measured in healthy individuals and patients with kidney and other diseases. Urinary annexin V did not change over a range of pH between 5.0 and 8.0, and was stable during the course of the study for 24 h at room temperature and for 8 days at 4 degrees C. The mean urinary annexin V concentration in 105 normal healthy individuals was 1.5+/-1.5 ng/ml, while that in patients with nephrotic syndrome and systemic lupus erythematosis (SLE) nephritis was 9.3+/-9.1 and 6.6+/-6.7 ng/ml, respectively, and that in IgA nephropathy and chronic renal failure was 2.6+/-2.1 and 1.3+/-0.7 ng/ml, respectively. Annexin level correlated with urinary protein concentration (r=0. 717), but not the serum creatinine concentration, blood urea nitrogen (BUN) and 24-h creatinine clearance. Mean urinary annexin V concentration in patients with ischemic heart disease, hypertension, and diabetes mellitus was 1.4+/-1.0, 1.4+/-1.1, and 1.7+/-1.3 ng/ml, respectively. In one case of relapsing nephrotic syndrome, the urinary annexin V concentration was markedly increased in the early phase after admission and then decreased. This patient later required hemodialysis. These results suggest that a high urinary annexin V concentration may be an indicator of acute renal injury related to the urinary protein level.

Localization of annexin V in rat normal kidney and experimental glomerulonephritis.

The localization of annexin V, a calcium binding protein, was immunochemically and immunohistologically studied in experimental rat glomerulonephritis using annexin V polyclonal antibody. Plasma and urinary annexin V levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Urinary annexin V level, which was correlated with urinary l-lactate dehydrogenase activity, N-acetyl-β-d-glucosaminidase activity and protein level, increased time-dependently after the injection of nephritogenic antigen (bovine glomerular basement membrane), progressively increasing to attain a peak level at 4 weeks of 51.5±11.3 ng/h. However, plasma annexin V level showed no increase during the study period. Normal kidneys showed strong staining for annexin V in distal tubules, being particularly strong in tubules of the inner stripe of the outer medulla, but could not be detected in proximal tubules. Annexin V was seen in visceral epithelial cells, Bowman’s capsule of the glomerulus, the vascular endothelium of arterioles and interlobular arteries, and vascular smooth muscle. In nephritis, the lumen of distal tubules and the luminal cell membrane were deeply stained, with leakage of annexin V being observed from tubular cells. In the present study, renal annexin V was markedly excreted into urine, and its urinary level reflected the severity of damage of renal tissue and the progression of nephritis. These changes of annexin V in the distal tubule and visceral epithelial cells may be of significance in cell injury of the kidney.

Subclinical hypothyroidism caused by a mutation of the thyrotropin receptor gene

The propositus (a girl) was the first child of non-consanguineous Japanese parents. She was born at 39 weeks of gestation following an uncomplicated pregnancy and delivery, with a weight of 2682 g. She was referred because of a TSH level of 15 mU/L on neonatal screening at day 5 of life. The family history revealed no thyroid disease. At recall examination at day 46 of life, a serum TSH concentration of 12.0 mU/L in conjunction with normal serum concentrations of T 4 (10.4 μ g/dL) and T 3 (181 ng/dL) were detected. She did not have a goiter nor abnormal physical findings. Though her serum thyroid hormone levels were within normal limits, she was followed up for hyperthyrotropinemia. At the age of 3 years, an excessive TSH response to intravenous administration of thyrotropin releasing hormone (TRH) was considered diagnostic of congenital primary hypothyroidism, and subsequently, she was treated with levothyroxine (L-T 4 ). Figure 1 shows the clinical course of the propositus until 13 years of age. Before treatment with L-T 4 , the serum TSH level fluctuated between 10 and 30 mU/L. It declined to within normal limits after treatment with L-T 4 . The serum T 3 and T 4 levels were almost within normal limits throughout the course. At the age of 8 years, she underwent 123 I scintigraphy and TRH testing after the discontinuation of L-T 4 treatment. 123 I scintigraphy showed a normal-sized eutopic thyroid gland. Her 24 h 123 I uptake value was 17%. The perchlorate discharge test was negative. After the intravenous administration of 300 μ g/m 2 of TRH, her serum TSH concentration increased from 18.8 mU/L to a peak of 93.7 mU/L, while serum T 3 did not increase from the basal level of 111 ng/dL to 100 ng/dL at 120 min. This study revealed unresponsiveness to TSH. In addition, baseline and 120, and 180 min thyroglobulin levels after TRH administration were 19, 19 and 18 ng/mL, respectively. Consequently, L-T 4 therapy was reinstalled and has not been discontinued since then. During the following 20 years, the physical and intellectual development of the patient was normal.

TTF-2 stimulates expression of 17 genes, including one novel thyroid-specific gene which might be involved in thyroid development

Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism, but its molecular pathophysiology is largely unknown. Our hypothesis that some genes downstream to thyroid transcription factor-2 (TTF-2) might be responsible for development of the thyroid prompted us to identify genes whose expression is stimulated by TTF-2. PCR products of cDNA clones obtained by a subtraction PCR method in TTF-2 expressing cell lines were screened with labeled cDNA by microarray analysis. We isolated 17 genes up-regulated by TTF-2, which were subsequently confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). One of them is a novel gene designated T1560 that showed a highly thyroid-specific expression pattern. Luciferase reporter assays showed that expression of all of the 14 genes tested was stimulated by both TTF-2 and TTF-1, another thyroid-specific transcription factor. Our results have important implications for understanding normal thyroid development as well as the molecular defects underlying thyroid dysgenesis.

Secretion of TNF‐α from macrophages following induction with a lignin derivative.

Macrophages derived from rat bone marrow were treated with macrophage colony stimulating factor (M‐CSF) to obtain a sufficient number of cells for the tumor necrosis factor (TNF‐α) assay. The present study has been designed to investigate whether the production of TNF‐α, which induces multinucleated giant cell formation, is regulated by polyanions such as lignin derivatives. ELISA for TNF‐α showed that the polyanion induced TNF‐α production by macrophages. The secretion of TNF‐α from the cells reached a maximum at 3‐6 h, and then showed a slight decline. Northern blotting of TNF‐α mRNA showed that the amount of TNF‐α reached a maximum within 1 h of macrophage culture in the presence of a lignin derivative. On the other hand, TNF‐α mRNA was undetectable in the control cells. It was concluded that stimuli such as that provided by lignin derivatives increases the amount of TNF‐α mRNA, which is then followed by translation of TNF‐α.