A high efficient protocol for soybean root transformation by Agrobacterium rhizogenes and most stable reference genes for RT-qPCR analysis.

KEY MESSAGE: A 55% transformation efficiency was obtained by our optimized protocol; and we showed that GmELF1 - ß and GmELF1 - α are the most stable reference genes for expression analyses under this specific condition. Gene functional analyses are essential to the validation of results obtained from in silico and/or gene-prospecting studies. Genetic transformation methods that yield tissues of transient expression quickly have been of considerable interest to researchers. Agrobacterium rhizogenes-mediated transformation methods, which are employed to generate plants with transformed roots, have proven useful for the study of stress caused by root phytopathogens via gene overexpression and/or silencing. While some protocols have been adapted to soybean plants, transformation efficiencies remain limited; thus, few viable plants are available for performing bioassays. Furthermore, mRNA analyses that employ reverse transcription quantitative polymerase chain reactions (RT-qPCR) require the use of reference genes with stable expression levels across different organs, development steps and treatments. In the present study, an A. rhizogenes-mediated soybean root transformation approach was optimized. The method delivers significantly higher transformation efficiency levels and rates of transformed plant recovery, thus enhancing studies of soybean abiotic conditions or interactions between phytopathogens, such as nematodes. A 55% transformation efficiency was obtained following the addition of an acclimation step that involves hydroponics and different selection processes. The present study also validated the reference genes GmELF1-ß and GmELF1-α as the most stable to be used in RT-qPCR analysis in composite plants, mainly under nematode infection.

The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus.

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be approximately 6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.

When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

Epidermal growth factor receptors on cultured neoplastic human thyroid cells and effects of epidermal growth factor and thyroid-stimulating hormone on their growth.

Epidermal growth factor (EGF) receptors on primary-cultured human thyroid cells from 27 neoplasias (nine adenomas and 18 differentiated carcinomas) were analyzed and compared with those on the cultured nonneoplastic part of human thyroid cells. Total binding of 125I-EGF to the nonneoplastic part, adenoma, and carcinoma cells did not differ significantly. Scatchard analysis showed that the neoplastic human thyroid cells, like their adjacent nonneoplastic counterparts, consistently possessed EGF receptors with two components. In a paired study of five patients, the association constant of the carcinoma cells' high-affinity component (Ka1) was found to be significantly lower than that of adjacent nonneoplastic thyroid cells (P less than 0.05). Furthermore, a study of the cells from 18 carcinomas revealed that overall their Ka1s (4.15 +/- 0.82 x 10(9) M-1, mean +/- SEM) were significantly lower than those of adenoma cells (10.34 +/- 1.51 x 10(9) M-1, n = 9) and of nonneoplastic cells adjacent to them (8.32 +/- 0.84 x 10(9) M-1, n = 23). The difference in Ka1s for adenoma and nonneoplastic thyroid cells was not statistically significant. The number of receptor sites (Cmax) per cell was not significantly different in any of the three. Incorporation of [3H]thymidine (dThd) increased significantly in all kinds of thyroid cells examined following the addition of 10 nM EGF, and the paired study showed that the size of this increase was not significantly different in neoplastic and adjacent nonneoplastic cells. The addition of 300 microunits/ml of thyroid-stimulating hormone caused a significant increase in dThd incorporation by adenoma cells but not by carcinoma or nonneoplastic cells. Furthermore, combined treatment with EGF and thyroid-stimulating hormone additively promoted adenoma cell growth only. A close inverse relationship was observed between Ka1 and the stimulatory effect of EGF on the dThd uptake in both nonneoplastic thyroid cells and adenoma cells. Carcinoma cells also showed similar profiles, but Ka1 relative to dThd increases were much smaller than the other two.

Rapid detection of specific messenger RNAs in thyroid carcinomas by reverse transcription-PCR with degenerate primers: specific expression of oncofetal fibronectin messenger RNA in papillary carcinoma.

The search for mRNAs that are specifically expressed in cancer tissues is important for gene diagnosis and therapy. However, finding such mRNAs in human cancers is usually very difficult, both because of the limited volume of RNA obtainable from the tissues and the many technical difficulties of RNA analysis. To address these problems, the present study compared mRNA from thyroid cancer tissues with those from normal and benign tissues by reverse transcription-PCR using two degenerate primers. Amplified cDNAs were separated by electrophoresis with nondenaturing acrylamide gel, then three bands that are increased in cancer tissues were selected, reamplified by PCR, and cloned into T-vector. One of the bands was determined by sequencing analysis to be oncofetal fibronectin. The expression of oncofetal fibronectin mRNA in benign and malignant tissues was examined by Northern blot and reverse transcription-PCR using specific primers of its cDNA sequence, and its increased expression was observed only in papillary and anaplastic carcinomas. Thus, the present method rapidly detected specific mRNAs in cancer tissues, and one of these, oncofetal fibronectin mRNA, is a good target for gene diagnosis of papillary carcinoma.

Preoperative diagnosis of thyroid papillary and anaplastic carcinomas by real-time quantitative reverse transcription-polymerase chain reaction of oncofetal fibronectin messenger RNA.

The restricted expression of oncofetal fibronectin (onfFN) mRNA in thyroid papillary and anaplastic carcinomas was recently reported. In this study, we measured the copy number of onfFN mRNA in RNAs extracted from fine needle aspiration biopsies by real-time quantitative reverse transcription-PCR using thyroglobulin mRNA as an internal control. By measuring the onfFN:thyroglobulin mRNA ratio, preoperative aspirates from 31 papillary carcinomas and an anaplastic carcinoma can be distinguished from those from 5 adenomatous goiters, 5 follicular adenomas, and 4 follicular carcinomas. Thus, quantification of onfFN by real-time quantitative reverse transcription-PCR may be useful for the preoperative diagnosis of papillary and anaplastic carcinomas.

New approach for detection of amplification in cancer DNA using restriction landmark genomic scanning.

We developed a new approach for detecting the gene amplification of cancer DNAs with restriction landmark genomic scanning (RLGS). In cancer research, much effort has been made to find the amplified loci of cancer DNAs, because many lines of evidence indicate association between oncogene amplification and carcinogenesis. Conventionally, such gene amplification has been detected by using Southern hybridization with DNA probes. However, only the information of one locus can be obtained by one hybridization procedure, and analysis of many loci throughout the genome is too laborious and time consuming, even if only several candidate genes are investigated. On the other hand, the "in-gel renaturation method" was reported as another alternative for detection of amplified regions. However, even though this method is much improved, it is difficult to detect less than 7-fold amplification, which is often higher than the amplification of many cancer cases. To overcome these limitations and, in addition, to locate the amplified DNA two dimensionally, we applied RLGS for analysis of DNA amplification in cancer tissues, such as breast cancer (infiltrative tubuloadenocarcinoma), neuroblastoma, meningioma (endotheliomatous meningioma), and thyroid cancer (papillary adenocarcinoma). In some cases of breast cancer, several amplified spots located on the same amplicon were detected. In thyroid cancer, in which no amplification has yet been reported, low-grade amplification was also detected. In this report, we demonstrated that RLGS allows us to screen 2000-3000 restriction landmarks distributed on the genome simultaneously, and even low-grade amplification could be detected effectively. Thus, RLGS has proven to be a very useful method in detecting DNA amplification.

Accurate and objective preoperative diagnosis of thyroid papillary carcinomas by reverse transcription-PCR detection of oncofetal fibronectin messenger RNA in fine-needle aspiration biopsies.

Recently, the restricted expression of oncofetal fibronectin mRNA was reported in thyroid papillary and anaplastic carcinomas. In this study, by extracting RNA from the leftover cells inside the needles used for fine-needle aspiration biopsy, we establish a new method for gene diagnosis of these carcinomas without further invasiveness to the patient (aspiration biopsy-reverse transcription-PCR, ABRP). RNA was extracted from 177 fine-needle aspiration biopsies of thyroid nodules that were suspicious for malignancy, and then the gene diagnoses made by reverse transciption-PCR detection of oncofetal fibronectin mRNA were compared with cytological diagnoses. Thirty-five (94.6%) of 37 samples that were diagnosed as papillary or anaplastic carcinomas by cytological examination showed a positive result by gene diagnosis, whereas only 4 (3.7%) of 109 samples that were cytologically diagnosed negative for both carcinomas showed a positive result. Among all of the cases, 50 patients underwent surgery, and a histological diagnosis was consequently made. The sensitivity and specificity of this method were 96.9 and 100%, respectively. A combined examination using both genetic and cytological approaches may contribute to a more precise preoperative diagnosis of papillary and anaplastic carcinomas.

Cloning of the cDNA for a novel receptor tyrosine kinase, Sky, predominantly expressed in brain.

Based on homology to the tyrosine kinase domain of the chick erythroblastosis virus oncogene v-sea, we cloned a cDNA encoding a novel receptor tyrosine kinase from a human hepatoma HepG2 cDNA library. The encoded protein, which we termed 'Sky', contains an intracellular tyrosine kinase domain and a unique extracellular domain with two immunoglobulin (Ig)-like domains and two fibronectin type III (FN III) domains. The overall structure of Sky is homologous to the reported sequence of the oncogenic Axl/Ufo receptor tyrosine kinase. Phylogenetic analysis in the tyrosine kinase domain shows that Sky, Axl/Ufo, Ark, and v-Ryk form a sub-family distinct from other tyrosine kinases. Northern blot analysis revealed that sky mRNA is expressed predominantly in brain and faintly in tissues of other organs. As the combination of Ig-like and FN III domains is often observed in neural cell adhesion molecules and receptor protein tyrosine phosphatases, Sky may be involved in cell adhesion processes, particularly in the central nervous system.

Identification of a human cDNA encoding a novel protein kinase with two repeats of the LIM/double zinc finger motif.

By low-stringency screening of a human hepatoma HepG2 cell cDNA library, using the genomic fragment of chick c-sea receptor tyrosine kinase as a probe, we isolated overlapping cDNAs encoding a novel protein kinase, which we termed LIM-kinase (LIMK).* The predicted open reading frame encodes a 647-amino-acid polypeptide containing a putative protein kinase structure in the C-terminal half. In addition, LIMK has two repeats of cysteine-rich LIM/double zinc finger motif at the most N-terminus. To our knowledge, this is the first protein kinase seen to contain the LIM motif(s) in the molecule. Although the protein kinase domain of LIMK has highly conserved sequence elements of protein kinases, phylogenetic analysis revealed that LIMK cannot be classified into any subfamily of known protein kinases. Northern blot analysis revealed that the single species of LIMK mRNA of 3.3 kb was expressed in various human epithelial and hematopoietic cell lines. In rat tissues, LIMK mRNA was expressed in the brain, at the highest level. LIM is suggested to be involved in protein-protein interactions by binding to another LIM motif. As the LIM domain is frequently present in the homeodomain-containing transcriptional regulators and oncogenic nuclear proteins, LIMK may be involved in developmental or oncogenic processes through interactions with these LIM-containing proteins.

p53 induced by ionizing radiation mediates DNA end-jointing activity, but not apoptosis of thyroid cells.

To understand the effects of ionizing radiation on thyroid cells, we investigated the role of p53 in mediating apoptosis and in DNA repair following in vivo and in vitro irradiation of thyroid cells. In vitro exposure of human thyroid cells to ionizing radiation of up to 5-8 Gy failed to induce apoptosis in primary cells. The same results were obtained when the thyroid gland was irradiated in the intact rat. To explore the mechanism of failure of the wild-type p53 in inducing apoptosis in thyroid cells, we investigated the expression of apoptosis-related genes, bax, bcl-2 and fas/APO-1 following irradiation or induction of temperature-sensitive p53. The expression of Bax, Bcl-2 and Fas/APO-1 in human primary cultured thyroid cells did not change after irradiation. To further confirm the results, we established a clonal cell line (tsFRO) in which a temperature sensitive p53 (Val138) expression vector was stably transfected to a thyroid carcinoma cell line lacking endogenous p53. Incubation of tsFRO cells at the permissive temperature for three days, however, did not induce apoptosis although G1 arrest was noted. Although enhanced expression of the bax mRNA level was observed, the expression of Bax, Bcl-2 and Fas/APO-1 protein did not change by shifting tsFRO cells to permissive temperature as well as irradiated primary cells. Furthermore, DNA end-jointing ability was examined by transfection of linearized luciferase plasmid into tsFRO cells. Increased luciferase activity occurred when the cells were cultured at the permissive temperature, indicating that the wild-type p53 enhances DNA end-jointing activity. Our results indicate that the wild-type p53 does not lead to apoptosis but facilitates DNA end-jointing in thyroid cells. These results may reflect specific responses in thyroid cells following irradiation.

Rapidly progressive thyroid failure in Graves' disease after painful attack in the thyroid gland.

We studied a new type of Graves' disease: rapidly progressive thyroid failure after painful attack in the thyroid gland. Four women with the mean (+/- SD) age of 51 +/- 3.2 years had newly diagnosed hyperthyroid Graves' disease. A severe painful episode developed in the thyroid glands of two patients and permanent hypothyroidism occurred spontaneously within 2 or 3 months thereafter. Two to three episodes of pain developed in the thyroid glands of the other two patients during antithyroid drug therapy. There was a transient rise in serum thyrotropin level after each painful episode and permanent hypothyroidism developed 6 to 8 months after the initial painful attack. The clinical picture is characterized by moderate to severe pain in the thyroid gland with tenderness. Patients responded to steroid or anti-inflammatory therapy. During painful attack, increased or normal thyroid radioiodine uptake, elevated levels of C-reactive protein, and an elevated erythrocyte sedimentation rate were found, but there was no cytological evidence of subacute thyroiditis. After painful attack, serum thyroid stimulation antibody began to decrease in three of the patients while thyroid stimulation blocking antibody developed in one patient. This is a rapid and self-destructive process of the Graves' thyroid gland, which appears to be associated with painful attack in the thyroid gland.

Expression profile of receptor-type protein tyrosine kinase genes in the human thyroid.

Protein tyrosine kinases (PTKs) play a role in regulating the growth and differentiated functions of thyroid cells and are probably involved in tumorigenesis of papillary-type thyroid carcinoma. To better understand the roles of PTKs in the physiology and pathophysiology of the thyroid, we analyzed the expression profile of receptor-type PTKs in normal human thyroid tissues. Highly conserved regions in the catalytic domains of receptor-type PTKs were amplified by RT-PCR using degenerate oligonucleotide primers. Nucleotide sequencing of about 100 clones identified 21 PTKs, including 16 receptor type and 5 nonreceptor type; no novel PTK was identified. Insulin-like growth factor I receptor, platelet-derived growth factor receptor (PDGFR), TrkE, Axl, epidermal growth factor receptor, etc., appear to be the most abundant receptor-type PTKs in the thyroid; many of which (PDGFR, TrkE, Axl, etc.) have never previously been demonstrated to be expressed in the thyroid. The expression of messenger RNAs (mRNAs) for PDGFR, axl, and trkE in normal thyroid cells was confirmed by Northern blot analysis, and interestingly, the expression levels of PDGFR and trkE mRNAs were decreased in all three thyroid carcinoma cell lines examined (FRO, WRO, and NPA), whereas axl mRNA and protein were overexpressed in 2 of 3 thyroid carcinoma cell lines (FRO and WRO) compared with that in normal tissue. The axl gene was, however, neither amplified nor rearranged. The biological activity of the ligand for Axl, the product of growth arrest-specific gene 6 (Gas6), was then evaluated, demonstrating modest mitogenic activity in thyroid carcinoma cells overexpressing Axl. Furthermore, gas6 mRNA was expressed in FRO cells. Thus, we here identify a variety of PTKs expressed in the thyroid gland, many of which may participate in the regulation of thyroid cell function. Variable expression levels of some PTKs in normal and cancerous cells suggest that there may be an imbalance and disarray of phosphorylation events in thyroid carcinoma cells. Furthermore, Gas6 is identified as a novel growth factor for thyroid carcinoma cells overexpressing Axl receptor tyrosine kinase.

Thyrotropin regulates c-Jun N-terminal kinase (JNK) activity through two distinct signal pathways in human thyroid cells.

c-Jun N-terminal kinases (JNK) participate in cellular responses to mitogenic stimuli and environmental stresses. We investigated whether and how TSH, which promotes the proliferation and differentiation of thyroid cells, regulates JNK activity in primary cultured human thyroid cells. TSH stimulated JNK activity in cytosolic fractions of thyroid cells measured by in vitro kinase assay. A low concentration of TSH (10(-11) M) stimulated JNK activity but at a higher dose (10(-8)-10(-7) M), TSH suppressed JNK activity without any change of JNK protein level. Activation of JNK by TSH was also observed in CHO cells stably transfected with TSH receptor complementary DNA (cDNA), suggesting a ligand-receptor specific interaction. TSH stimulated JNK activity through a pertussis toxin-sensitive pathway. We next elucidated the signal transduction pathways in TSH-induced JNK activation by examining the involvement of four distinct intracellular signal molecules; protein kinase C (PKC), cAMP, Ca2+, and PI3-kinase. The stimulation of JNK by TSH was blocked by two PKC inhibitors and suppressed by 8-bromo-cAMP or forskolin. These findings demonstrate that TSH regulates JNK activity biphasically in human thyroid cells through an interaction between Gi-PKC and cAMP-PKA pathways.

Serum triiodothyronine to thyroxine ratio: a newly recognized predictor of the outcome of hyperthyroidism due to Graves' disease.

Patients with untreated hyperthyroidism due to Graves' disease have a proportionally greater increase in the serum T3 than in the T4 concentration and, therefore, have an elevation of the serum T3 to T4 ratio. The aim of this study was to investigate the alterations of the serum T3 to T4 ratio in relation to the outcome of antithyroid drug therapy. Of 47 patients with hyperthyroid Graves' disease, 37 patients had a serum T3 to T4 ratio greater than 20 ng/micrograms before therapy (normal range, 12-20; mean, 16.0). In 7 of 37 patients, serum T3 to T4 ratios remained high during a 2-yr course of antithyroid drug therapy, and in 6 of them (86%), hyperthyroidism recurred after cessation of drug therapy. In the remaining 30 patients, the initial high serum T3 to T4 ratios decreased to normal (less than 20) during treatment, and 15 of them (50%) had a remission of the disease after cessation of the drug. Of the 10 patients with initial serum T3 to T4 ratios within the normal range, this ratio remained normal during treatment, and 8 (80%) had a remission. Goiter size was larger in patients with high serum T3 to T4 ratios, and a reduction of goiter size occurred in some patients (57%) with decreasing serum T3 to T4 ratios. The serum T3 to T4 ratio is a simple and useful predictor of the outcome of antithyroid drug therapy in patients with Graves' disease. A ratio greater than 20 throughout therapy indicates that the likelihood of relapse is high, and a ratio below 20 either initially or during therapy is an indicator of prolonged remission.

Bromocriptine therapy for hyperthyroidism due to increased thyrotropin secretion.

We describe a patient with TSH-induced hyperthyroidism successfully treated with bromocriptine. A 25-yr-old woman was found to have hyperthyroidism due to excessive TSH secretion; no pituitary tumor was found. Her serum T4 level ranged between 21.9 and 25.9 micrograms/dl and that of T3 between 283 and 314 ng/dl. Serum TSH was between 5 and 9 microU/ml with an exaggerated response to TRH. Basal metabolic rate was +26 to +38%. Serum PRL was also elevated (79 ng/ml). Administration of bromocriptine for 4 months decreased serum TSH and PRL levels to normal with a concomitant fall in levels of serum T3 and T4. Regression of the clinical manifestations of hyperthyroidism occurred during bromocriptine drug therapy. These results suggest that reduction in hypothalamic dopaminergic tone may have contributed to the inappropriately increased TSH secretion in the patient.

Iodine content of serum thyroglobulin in normal individuals and patients with thyroid tumors.

We indirectly estimated the iodine content of serum thyroglobulin (TG) in normal individuals and patients with benign and malignant thyroid tumors. Because insufficient TG is present in the serum to perform chemical determinations, equilibrium density centrifugation was used to determine its density, a measure of TG iodine content. In five patients undergoing thyroidectomy, serum TG was compared to TG extracted from the nodules and TG from the surrounding normal thyroid tissue. The iodine content of the tumor TG was much less than that of normal TG in four of the five patients. In patients with benign and malignant nodules, the iodine content of serum TG was lower than that of normal TG, and it was similar in patients with benign and malignant disease. In normal individuals, serum TG was also poor in iodine, similar to the serum TG from the patients, and in the same position as TG with virtually no iodine. These findings are in accord with our report that serum TG in rats is nearly completely devoid of iodine. TG could enter the circulation either by secretion of newly synthesized TG or release of stored TG from the thyroid. The findings show that serum TG in normal individuals does not result from the release of preexisting TG. More likely, it arises from the secretion of poorly iodinated, newly synthesized molecules. Since the elevated serum TG found in patients with nodules also is poor in iodine, it must come directly from the tumor rather than from destruction of surrounding normal thyroid tissue.

Increased cyclic nucleotide phosphodiesterase (PDE) and calmodulin activities in soluble fraction of Graves' thyroid: analysis of increase in Ca+2 dependence of PDE activities.

Phosphodiesterase (PDE) and calmodulin (CaM) activities were studied in soluble fractions of normal and Graves' thyroid tissue. Normal and Graves' thyroid tissues were obtained at thyroid surgery. PDE activities were assayed with cAMP and cGMP as substrates. CaM activity was measured as the ability to activate bovine thyroid CaM-dependent PDE. cAMP and cGMP PDE activities were increased 1.5- and 2.2-fold above normal in Graves' thyroid, respectively. The major cause of the increase in enzyme activities was their higher Ca+2 dependence. CaM activity also was 1.6-fold increased in Graves' thyroid, although it probably does not contribute to the increase in the Ca+2 dependence of PDE activities because of its relative sufficiency vs. PDE in normal thyroid. Three forms of cAMP and cGMP PDE activities were eluted from Sephadex G-200 columns, with mol wt of 280,000, 140,000, and 80,000-100,000, respectively. The first peak had little or no CaM dependence, the second peak had moderate or dominant CaM dependence, and the third peak revealed weak or dominant CaM dependence. The increase in the CaM-dependent form of the third peak of PDE activity in Graves' thyroid tissue may explain the increase in Ca+2 dependence of PDE activities.

Monoclonal gammopathy in Hashimoto's thyroiditis and malignant lymphoma of the thyroid.

Serum protein electrophoresis was performed in 681 patients (43 men and 638 women) with an initial diagnosis of Hashimoto's thyroiditis between April and November 1983. All patients whose thyroid size was estimated to be greater than 50 g underwent biopsy; 1 man was found to have thyroid prelymphoma, and 13 patients (4 men and 9 women) were found to have malignant lymphoma of the thyroid. Monoclonal gammopathy (M-component) was demonstrated in 5 of 667 patients (0.7%) with Hashimoto's thyroiditis (1 man and 4 women), 1 man with thyroid prelymphoma, and 3 of the 13 patients (23.1%) with malignant lymphoma of the thyroid (2 men and 1 woman). Intracytoplasmic monoclonal immunoglobulin was found in the 1 thyroid prelymphoma and in all 3 malignant lymphoma of the thyroid in patients who had M-component in their serum, but not in thyroid tissue from any of the 5 patients with Hashimoto's thyroiditis who had M-component in their serum. Thus, the finding of monoclonal intracytoplasmic immunoglobulin in tissue sections permitted the diagnosis of malignant lymphoma of the thyroid or thyroid prelymphoma.

Function of DR-positive thyrocytes from patients with Graves' disease: quantitative analysis of thyroid peroxidase content by fluorescent photometry.

A considerable number of thyrocytes in patients with autoimmune thyroiditis ectopically express HLA-DR antigen. Furthermore, it has been reported that interferon-gamma-induced DR-positive thyrocytes in vitro secrete less thyroid hormone in response to TSH stimulation compared with DR-negative ones. However, the function of the intrinsically DR-positive thyrocytes is unknown. To evaluate their function, we stained by immunofluorescence for both DR antigen and thyroid peroxidase (TPO) in thyroid epithelial cells from patients with Graves' disease. We also measured the quantity of DR antigen and TPO using fluorescent photometry. The content of TPO was not significantly reduced in DR-positive thyrocytes compared with that in DR-negative thyrocytes. The TPO content is one measure of thyrocyte function. There was no significant difference between DR-positive and DR-negative thyrocytes. In conclusion, the function of DR-positive thyrocytes in vivo was not suppressed compared with that of DR-negative thyrocytes.