Influence of thyroid hormones and transforming growth factor-ß1 on cystatin C concentrations.

Serum cystatin C concentrations are reported to increase in the hyperthyroid state. Serum concentrations of cystatin C and transforming growth factor-ß1 (TGF-ß1) were measured in patients with thyroid dysfunction, and the effects of 3,5,3'-tri-iodothyronine (T(3)) and TGF-ß1 on cystatin C production in human hepatoblastoma (Hep G2) cells were studied. Serum concentrations of cystatin C and TGF-ß1 were significantly higher in patients with Graves' disease compared with control subjects. Significantly positive correlations were observed between thyroid hormones and cystatin C, thyroid hormones and TGF-ß1, and TGF-ß1 and cystatin C in patients with thyroid dysfunction. Serum concentrations of cystatin C and TGF-ß1 decreased after treatment for hyperthyroidism. Cystatin C mRNA levels and cystatin C secretion were increased by T(3) and TGF-ß1 in cultured Hep G2 cells. These results suggest that serum cystatin C concentrations increase in patients with hyperthyroidism. The mechanisms for this may involve elevation of serum TGF-ß1 levels and the stimulatory effects of T(3) and TGF-ß1 on cystatin C production.

Novel inactivating missense mutations in the thyrotropin receptor gene in Japanese children with resistance to thyrotropin.

We describe Japanese siblings with resistance to thyrotropin (TSH) who are compound heterozygotes for two novel mutations in the TSH receptor gene. The affected siblings had increased serum TSH, normal serum thyroid hormones, and normal positioned but slightly hypoplastic thyroid glands. The mutated paternal allele has the substitution of His (CAC) in place of Arg (CGC) at codon 450 (R450H) of the TSH receptor. The mutated maternal allele has the substitution of Ser (AGT) in place of Gly (GGT) at codon 498 (G498S) of the TSH receptor. COS-7 cells transfected with the R450H mutant exhibited a slightly decreased TSH binding and a slightly decreased cyclic adenosine monophosphate (cAMP) response to TSH, whereas cells transfected with the G498S mutant exhibited a markedly decreased TSH binding and a markedly decreased cAMP response to TSH. Flow immunocytofluorometry analysis demonstrated that the G498S mutant resulted in extremely low expression at the cell surface as compared with the wild type receptor and the R450H mutant, in spite of a normal intracellular synthesis. The present cases are the first Japanese patients with TSH resistance in whom mutations in the TSH receptor gene have been identified. These novel mutations may contribute to understanding of the struc-ture-function relationship of the TSH receptor.

Expression of thyrotropin receptors in rat thymus.

Thymic hyperplasia is associated with Graves' disease. It has been demonstrated that thyrotropin receptors are expressed in human thymus, and thymic thyrotropin receptors are suggested to be involved in the pathophysiology of Graves' disease. We have studied whether thyrotropin receptors are expressed in rat thymic tissue. Thyrotropin receptor mRNA was demonstrated in 5-day-old, 10-day-old, 20-day-old and adult rat thymus by reverse transcription polymerase chain reaction. Thyrotropin receptor mRNA was also demonstrated in cultured rat thymic epithelial cells. Thyrotropin stimulated cyclic AMP production in cultured rat thymic epithelial cells, suggesting the expression of functional thyrotropin receptors. The present results indicate that thyrotropin receptors are expressed in rat thymus.

Expression and regulation of type II iodothyronine deiodinase in human thyroid gland.

We have studied the expression of type II iodothyronine deiodinase (DII) in human thyroid tumors and cultured human thyroid cells to elucidate the mechanisms involved in the regulation of DII expression in human thyroid gland. Three cases with hyperfunctioning thyroid adenoma, including a case that showed an activating mutation of G(s)alpha with a constitutive activation of cAMP production in cultured cells, and six cases with papillary thyroid carcinoma were analyzed in the present study. Free T(3) was increased, whereas free T(4) was within the normal range in all patients with hyperfunctioning thyroid adenoma. Thyroid tumor tissue and surrounding nontumor tissue were obtained at the time of surgery, and DII expression was compared between tumor tissue and nontumor tissue in each case. Northern analysis demonstrated the presence of DII messenger RNA (mRNA) approximately 7.5 kb in size in all of the tumor and nontumor tissues. DII mRNA and DII activity in hyperfunctioning thyroid adenoma were significantly increased compared with those in nontumor tissue in each case. In contrast, DII mRNA and DII activity in papillary thyroid carcinoma were decreased compared with those in nontumor tissue in each case. DII mRNA and DII activity in cultured human thyroid cells were significantly stimulated by TSH in a dose-dependent manner. The promoter activity of the human DII gene including the complete cAMP response element, transfected to cultured human thyroid cells, was stimulated by (Bu)(2)cAMP. In summary, these results suggest that DII expression in human thyroid gland is regulated at the transcriptional level through the TSH receptor-G(s)alpha-cAMP regulatory cascade, which may be related to the increase in circulating T(3) level in patients with Graves' disease and hyperfunctioning thyroid adenoma.

Notch signaling suppresses IgH gene expression in chicken B cells: implication in spatially restricted expression of Serrate2/Notch1 in the bursa of Fabricius.

The bursa of Fabricius is a central organ for chicken B cell development and provides an essential microenvironment for expansion of the B cell pool and for generation of a diversified B cell repertoire. We report here that genes encoding the Notch family of transmembrane proteins, key regulators of cell fate determination in development, are differentially expressed in the bursa of Fabricius: Notch1 is expressed in medullary B cells located close to the basement membrane-associated epithelium (BMAE). In contrast, a Notch ligand, Serrate2, is expressed exclusively in the BMAE, which surrounds bursal medulla. A basic helix-loop-helix-type transcription factor, Hairy1, a downstream target of Notch signaling, is expressed in the bursa coordinately with Notch1 and Serrate2 and an immature B cell line, TLT1, which expresses both Notch1 and Serrate2. Furthermore, stable expression of a constitutively active form of chicken Notch1 or Notch2 in a B cell line results in a down-regulation of surface IgM expression, which is accompanied by the reduction of IgH gene transcripts. Transient reporter assay with the human IgH gene intronic enhancer reveals that an active form of Notch1 inhibits the IgH enhancer activity in chicken B cells, suggesting that Notch-mediated signals suppress the IgH gene expression via influencing the IgH intronic enhancer. These findings raise the possibility that the local activation of Notch1 in a subset of B cells by Serrate2 expressed in BMAE may influence the cell fate decision that is involved in B cell differentiation and selection inside the bursa.

Thyrotropin receptors in brown adipose tissue: thyrotropin stimulates type II iodothyronine deiodinase and uncoupling protein-1 in brown adipocytes.

It has been demonstrated that TSH receptors are expressed not only in thyroid gland but also in extrathyroidal tissues. Brown adipose tissue of guinea pig has been reported to express TSH receptor messenger RNA (mRNA), but the physiological roles of TSH receptors in brown adipose tissue have not been understood. We studied the expression and function of TSH receptors in rat brown adipose tissue and cultured rat brown adipocytes. Northern analysis demonstrated the expression of TSH receptor mRNA in rat brown adipose tissue and cultured rat brown adipocytes. TSH receptor mRNA in rat brown adipose tissue was decreased by cold exposure of the rat, and its mRNA in cultured rat brown adipocytes was also decreased by incubation with TSH or (Bu)(2)cAMP. TSH increased the intracellular cAMP concentration in cultured rat brown adipocytes in a dose dependent manner. Type II iodothyronine deiodinase mRNA, its activity, and uncoupling protein-1 mRNA in cultured rat brown adipocytes were significantly increased by incubation with TSH in a dose-dependent manner. These results suggest the expression of functional TSH receptors in brown adipose tissue, which may be involved in regulation of the expression of type II iodothyronine deiodinase and uncoupling protein-1.

A case of exaggerated mosquito-bite hypersensitivity with Epstein-Barr virus-positive inflammatory cells in the bite lesion.

We describe a unique patient with mosquito-bite hypersensitivity who had extremely high titres of Epstein-Barr virus antibodies. For many years he developed intractable ulcers on the sites of mosquito-bite. Epstein-Barr virus infection was detected in almost all inflammatory cells in the ulcers and in the peripheral blood lymphocytes by using in situ hybridization to Epstein-Barr virus-encoded small ribonucleic acids and by polymerase chain reaction to Epstein-Barr virus DNA. The inflammatory cells in the ulcers were positive for T-cell marker. Our results suggest that the Epstein-Barr virus infection in T cells may participate in the pathogenesis of exaggerated mosquito hypersensitivity and in delayed healing of ulcers on the sites of mosquito-bite.

Application of representational difference analysis to genomic fragments of Marek's disease virus.

A rapid and simple method for isolation of DNA fragments of Marek's disease virus (MDV) based on representational difference analysis (RDA) was developed. Multiple viral DNA fragments, the sizes of which were restricted to 0.3 to 3.5 kbp, were simultaneously amplified after subtraction of chicken DNA from BamHI-, BglII-, EcoRI-, HindIII-, or XhoI-digested DNA fragments of MDV-infected cells. Nucleotide sequence of two RDA-derived fragments coincided with the sequence determined from direct sequencing of the MDV genome. We detected an interstrain difference in the size of restriction enzyme-digested fragments on agarose gel. This method was used on a single feather pulp to generate sufficient MDV DNA for cloning.

Expression of type II iodothyronine deiodinase in brain tumors.

Type II iodothyronine deiodinase (DII) messenger ribonucleic acid (mRNA) and its activity have been demonstrated in human normal brain. Although DII activity has been demonstrated in brain tumors, expression of DII mRNA has not been studied in these tumors. To investigate the mechanisms involved in the expression of DII activity in brain tumors, we studied DII mRNA and DII activity in astrocytoma (two cases), glioblastoma (three cases), and oligodendroglioma (one case). DII mRNA, the size of which was indistinguishable from that in control cerebral cortical tissue, was demonstrated in all of the brain tumors tested, although the intensity of the hybridization signal showed wide variation among the tumors. DII activity was also detected in all tumors. DII mRNA and DII activity were highest in the tissue from oligodendroglioma. A significantly positive correlation was observed between DII mRNA and DII activity in these tumors (r = 0.94; P < 0.01), suggesting that DII expression in brain tumors is regulated at the pretranslational level. The present results demonstrate, for the first time, that DII mRNA as well as DII activity are expressed in brain tumors, and that DII mRNA is significantly correlated with DII activity in those tissues.

Cell cycle arrest and apoptosis induced by Notch1 in B cells.

Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Recently, we found that Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxP-mediated inducible expression system. Remarkably, the active Notch1 caused growth suppression of the cells, accompanied by a cell cycle inhibition at the G(1) phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induced the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G(1) cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1.

Anti-viral and anti-tumor effects induced by an attenuated Marek's disease virus in CD4- or CD8-deficient chickens.

By vaccination with an attenuated Marek's disease virus (MDV), strain CVI988, chickens are protected from the development of T cell lymphoma caused by an oncogenic MDV. To clarify the role of T lymphocyte subsets in the protection mechanisms of this vaccine, vaccinated chickens were depleted of T cell subsets by neonatal thymectomy and injections of monoclonal antibodies specific to chicken CD4 and CD8 molecules, and then challenged with an oncogenic MDV, strain Md5. The MDV titers rescued from CD8(+) T cells, which are the main targets for latent infection and subsequent transformation by MDV, was much higher in the CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. However, the neonatal vaccination prevented lymphoma formation by strain Md5 even in either CD4(+) or CD8(+) T cell-depleted chickens. These results suggest that specific CD8(+) T cell responses induced by the MD vaccine play a crucial role in the prevention of MDV infection during the latent phase, but may not be essential for the prevention of lymphoma formation by an oncogenic MDV.

Re-isolation of Marek's disease virus from T cell subsets of vaccinated and non-vaccinated chickens.

To know the effect of Marek's disease (MD) vaccines, we analyzed the distribution of MD virus (MDV) among T cell subsets from chickens vaccinated or non-vaccinated with MD vaccine and subsequently challenged with a virulent MDV. The challenged MDV was reisolated preferentially from CD4+ T cells, and the average titers of challenged MDV rescued were significantly lower in vaccinated chickens compared to that of non-vaccinated chickens. In addition, it was also shown that different serotypes of MDV, CVI988 and SB-1, have remarkable difference in recovery rates of viruses from CD4+ and CD8+ T cells, though both CVI988 and SB-1 can reduce the infection rates of virulent MDV to splenocytes.

Lateralized memory deficits on the Wada test correlate with the side of lobectomy only for patients with unilateral medial temporal lobe epilepsy.

The aim of this study was to determine whether the predictive value of the intracarotid amobarbital test (IAT) for the side to be resected is applicable only to medial temporal lobe epilepsy and to investigate whether there are different patterns of memory performances on the IAT between patients with unilateral mesial temporal sclerosis (UMT group) and those without (non-UMT group). We studied 30 patients in the UMT group and 10 in the non-UMT group, who underwent pre-surgical evaluation for intractable temporal lobe epilepsy. Memory performances on the IAT was defined as the percentage of memory items presented during unilateral hemispheric anesthesia that was recognized after recovery. More than a 20% decline of the memory performance on the IAT compared with the memory performance on the pre-test was regarded as a memory deficit. Age at onset of epilepsy was significantly younger in the UMT than in the non-UMT group. Surgical outcome was significantly better in the UMT than in the non-UMT group. The lateralizing value of unilateral memory deficits on the IAT was statistically confirmed. There was a significant association between falsely lateralizing memory performances and the non-UMT group. Excluding the exceptional cases with right-sided language dominance in spite of right-sided lesions, the high incidence of the unilateral right-sided memory deficits in the non-UMT group was statistically significant. This study suggested that the excellent lateralizing value of the memory performances on the IAT is limited to patients with mesial temporal lobe epilepsy. IAT memory performances in patients without such lesions can be misleading, even if lateralized, because their memory status presumably reflects a natural lateralization of the memory organization which is independent of the epileptogenic focus.

Expression of bcl-2 and bcl-x genes in lymphocytes and tumor cell lines derived from MDV-infected chickens.

To characterize the molecular events involved in both apoptosis and transformation process induced by Marek's disease virus (MDV), the expressions of the bcl-2 and bcl-x genes, ones of the dominant apoptosis-regulating genes, in Marek's disease (MD) tumor cell lines and cells prepared from MDV-infected chickens were analyzed. The expression of bcl-2 was down-regulated in both CD4+ and CD8+ T cells prepared from MDV-infected chickens at 3 weeks p.i. No bcl-2 transcript was detected in MD tumor-derived MSB1 and MTB1 cell lines, which had been established from primary MD tumors. On the other hand, the bcl-xL transcript whose product can also inhibit apoptosis was expressed in cell lines derived from MD. By the treatment with phorbol 12-myristate 13-acetate (PMA) and ionomycin, normal CD4+ T cells were induced to express bcl-xS which can promote apoptosis, while bcl-xL was constitutively expressed in MD cell lines. Our results suggest that bcl-xL rather than bcl-2 might play an important role in the transformation process by MDV.

BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius.

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

Analysis of tumor suppressor gene p53 in chicken lymphoblastoid tumor cell lines and field tumors.

To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD), lymphoid leukosis, reticuloendotheliosis, and field tumors, some portions of p53 cDNA corresponding to core and C-terminal domains (nucleotide positions 277-1104 in the p53 open reading frame (ORF)) were sequenced. Several mutations were identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been reported as the site for transformation-activating mutations. Moreover, partial cDNA clones with a 122-bp deletion in the p53 ORF were identified in two cell lines, MSB1 and MTB1 derived from MD tumors. Southern blot analysis showed that no deletion occurred in the genome of p53 in MSB1, indicating that deletion occurred at the transcriptional level. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. These mutations of the p53 gene and deletion in the p53 transcript may be ones of molecular changes specific to the transformation induced by MD virus.

Pineal dermoid cyst developing 18 years after gross total removal of a pineal mature teratoma.

A 21-year-old male presented with a pineal dermoid cyst manifesting as headache and diplopia. He had undergone gross total removal of a pineal mature teratoma 18 years before and had done well until recently. Diffusion-weighted magnetic resonance imaging showed a region of high signal intensity due to a round mass in the pineal region and extending into the trigone of the right lateral ventricle. Subtotal excision of the tumor was achieved. Histological examination showed an epidermoid cyst consisting of keratinized stratified squamous epithelium and connective tissue. Intraoperative observation had detected black hairs, so the diagnosis was dermoid cyst. The dermoid cyst may have arisen from a microscopic remnant of the cyst wall of the original pineal mature teratoma. The regrowth of a dermoid cyst 18 years after gross total removal of a mature cystic teratoma in the pineal region is exceptional. However, careful follow-up of patients who undergo gross total removal of a pineal teratoma is recommended for a period more than that of the patient's age at surgery plus 9 months.

Retroviral sequence located in border region of short unique region and short terminal repeat of Md5 strain of Marek's disease virus type 1.

A 246-base pair (bp) retroviral sequence, which was homologous to a long terminal repeat of avian erythroblastosis virus (AEV), was detected and cloned from Md5 strain (Md5) of Marek's disease virus type 1 (MDV1) by representational difference analysis (RDA). The retroviral sequence was thought to be located in the border region of short unique region (U(s) and short terminal repeat (TRs), but did not exist in the border region of U(s) and the inverted short repeat (IRs) of the Md5 genome. A cloned fragment of the US/TRs border region of the Md5 genome showed a construction of U-E'-R-U'-E-TRs with the regions designated as follows: E, expanded TRs reported by Jones et al. [Proc. Natl. Acad. Sci. U.S.A. 90, 3855, 1993]; E', a partial copy of the expanded TRs; R, the retroviral sequence detected in Md5 genome; U, TRs-end sequence of U(s); U', a partial copy of TRs-end sequence of U(s). The sequence unit indicated as E'-R-U' was thought to be heterogeneously repeated in the Md5 genome. Since this retroviral sequence reportedly did not exist in the original stock of Md5, the retroviral sequence is thought to be inserted in the Md5 genome without experimental co-infection of avian cells with retrovirus and MDV1. These results suggest that RDA could be useful for the detection of retroviral sequences in the herpesvirus genome.