Expression Of TPO Research Articles

Overexpression of Calr Mutants Perturbs Developmental Hematopoiesis in Zebrafish Embryos

Introduction: Calreticulin (CALR) is a 46-kDa highly conserved, multicompartmental and multifunctional protein. CALR acts as a Ca2+ binding chaperone protein and participates in ensuring proper protein and glycoprotein folding in the endoplasmic reticulum. CALR mutations have been identified in about 30% of JAK2 and MPL unmutated essential thrombocythemia and primary myelofibrosis. Although the expression of CALR mutants resulted in pathogenic thrombocytosis in adult mice, whether CALR mutants may disrupt normal hematopoiesis during early development remains unknown. Here we aimed to evaluate the effects of mutant CALR during embryonic hematopoietic development using the in vivo zebrafish model.Methods: Full-length CALR wild-type,and CALR-del52 and CALR-ins5 mutants cDNAs were subcloned in the pCS2+ vector and a bicistronic pSYC-102 T2A vector, respectively. Capped CALR mRNAs from the above vectors were micro-injected into 1-2 cells stage wild-type AB strain, Tg(cd41:GFP) and Tg(fli1:EGFP) zebrafish embryos, respectively. cd41+ thrombocytes were counted at 3 and 5 days post fertilization (dpf), respectively. Gene expression of hematopoietic lineage-specific and cytokine and cytokine receptor genes were evaluated by quantitative reverse-transcription and real-time polymerase chain reaction (Q-PCR) from 1 to 3 dpf. Morpholino (MO) was used to knock down cytokine receptor genes mpl, epor and csf3r.Results: The expression of CALR proteins from the injection of 100 pg mRNA was confirmed by CALR N-terminal and mutant specific antibodies, respectively. Expression of both CALR-del52 and CALR-ins5 mutant mRNA significantly increased the numbers of hematopoietic stem and progenitor cells in the caudal hematopoietic tissue when compared with CALR-wt mRNA at 3 dpf. No obvious changes in the angiogenesis were visualized in CALR-wt and mutant CALR expressing embryos at 3 dpf in the fli1:EGFP line when compared with uninjected control. Mutant CALR-del52 significantly increased the number of cd41+ thrombocytes at 5 dpf (mean 162.5±4.1 per embryo) when compared to CALR-wt (mean 117.1±3.1 per embryo, P<0.001), mutant CALR-ins5 (mean 128.3±6.1 per embryo, P<0.001) and uninjected control (mean 136.7±3.0 per embryo, P<0.001), respectively.At 5 dpf, the number of cd41+ thrombocytes significantly decreased upon mpl MO knockdown (mean 43.6±4.9 per embryo) when compared to the control MO group (mean 123.5±5.9 per embryo, P<0.001) and the mutant CALR-del52 group (P<0.001). Importantly, co-injection of CALR-del52 mutant mRNA (mean 73.7±5.1 per embryo) can only partially reverse the knockdown effect of mpl MO. In contrast, the numbers of cd41+ thrombocytes did not decrease significantly upon epor MO or csf3r MO knocked-down compared with the control MO group. When CALR-del52 mutant mRNA was co-injected with epor or csf3r MOs, the numbers of cd41+ thrombocytes were comparable to those of CALR-del52-injected embryos.In Q-PCR experiments, the expression of cmyb, runx1 and scl significantly increased in only CALR-ins5 mutant group at 3 dpf when compared to 2 dpf. The expression of αeHb and βeHb significantly decreased in both CALR-del52 and CALR-ins5 mutant groups at 2 dpf but the expression of gata1 remained unchange. The effects of CALR-del52 and CALR-ins5 mutant groups on the expression of cytokine and cytokine receptor genes included the upregulation of mpl at 1 dpf followed by progressive downregulation at 2 dpf and 3 dpf but relatively stable expression of tpo and epo during early development. In the group of genes related to thrombopoiesis, the expression of emilin1a and nbeal2 was significantly downregulated in CALR-del52 group at 3 dpf when compared to 2 dpf.Conclusions: In this study, we showed that the expression of mutant CALR causes thrombocytosis through an mpl-dependent mechanism and perturbs developmental hematopoiesis in zebrafish embryos. DisclosuresNo relevant conflicts of interest to declare.

Impact of co-exposure with butachlor and triadimefon on thyroid endocrine system in larval zebrafish

IntroductionButachlor (BTL) and triadimefon (TDF), the widely used herbicide and fungicide, are unavoidable enter into the aquatic environment. However, there were limited study regarding to the joint toxicity of these two pesticides on fish at present. AimTo evaluate the potential thyroid-disrupting toxicity and exposed to different concentrations of BTL mixed with TDF. Materials and methodsZebrafish embryo (n=3) were exposed to 0.01 and 0.05 fold of LC50 from the acute joint toxicity test, of which 0.32mg/L (BTL) and 9.41mg/L (TDF) for single or mixture agents (BTL: 0.0064mg/L, 0.032mg/L; TDF: 0.1882mg/L, 0.9410mg/L; co-exposure: 0.0032mg/L BTL+0.0941mg/L TDF, 0.016mg/l BTL+0.4705mg/L TDF) after 10-day post-fertilization. Hatching, malformation, survival rates and thyroid hormones (THs), genes expression involved in HPT-axis of embryos were measured and detected in control and separately/co-exposure treatments. THs contents were evaluated by ELISA kit and the expression levels of genes were determined by RT-PCR. ResultsHatching, malformation and survival rates of embryos exposed to single BTL exhibited no statistically significant difference from the control besides decreased of high concentration in survival rates. Exposure to TDF reduced hatching, survival rate and increased malformation. The combined exposure to BTL and TDF resulted in greater adverse effects on embryonic development. BTL exposure significantly increased free T3 and T4 contents. Elevated free T3 content was also observed in the larvae exposed with single BTL. Co-exposure of the two pesticides caused greater enhanced of T3 and T4 levels. Furthermore, gene data showed BTL up-regulated the mRNA expression of tpo, tshβ, tg, ttr, dio2, TDF up-regulated the mRNA expression of tpo, trα, ttr, dio2 and down-regulated trβ gene. The mixture of the two pesticides caused up-regulation mRNA expression of trα, trβ, tg, ttr, dio2. ConclusionBTL and TDF resulted in adverse effects on zebrafish embryonic development and caused thyroid endocrine disruption, BTL and TDF have a synergistic effect on development and thyroid endocrine by enhanced level of thyroid hormone.

Enantioselectivity in Developmental Toxicity of rac-metalaxyl and R-metalaxyl in Zebrafish (Danio rerio) Embryo.

Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac-metalaxyl and R-metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac-metalaxyl and R-metalaxyl, and the LC50 s at 96 h are 416.41 (353.91, 499.29) mg · L(-1) and 320.650 (279.80, 363.46) mg · L(-1) , respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic-pituitary-gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic-pituitary-thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac-metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R-metalaxyl. And likewise, rac-metalaxyl only caused the upregulation of dio2, while R-metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489-494, 2016. © 2016 Wiley Periodicals, Inc.

Thyroid cell differentiation from murine induced pluripotent stem cells.

Here, we demonstrate the successful differentiation of induced pluripotent stem (iPS) cells into functional thyroid cells indicating the therapeutic potential of this approach when applied to individuals with thyroid deficiency. Using embryonic murine fibroblasts, we generated iPS cells with a single lentiviral "stem cell cassette" vector and then differentiated these iPS cells into thyroid cells after transfection with PAX8 and NKX2-1 by Activin A and TSH stimulation. The generated iPS cells expressed pluripotent stem cell markers as assessed using both reverse transcription quantitative PCRs and immunofluorescence staining with ~0.5% reprograming efficiency. Compared to control cells, the expression of thyroid-specific genes NIS, TSHR, Tg, and TPO were greatly enhanced in PAX8(+)NKX2-1(+) iPS cells after differentiation. On stimulation with TSH, these differentiated iPS cells were also capable of dose-dependent cAMP generation and radioiodine uptake indicative of functional thyroid epithelial cells. Furthermore, the cells formed three-dimensional follicles in culture, and "thyroid organoids" formed after PAX8(+)NKX2-1(+) iPS cells transplanted into nude mice, and all expressed Tg protein as judged immunohistochemically. Taken together, thyroid epithelial cells differentiated from iPS cells, which were themselves derived from murine fibroblasts, exhibited very similar properties to thyroid cells previously developed from traditional murine embryonic stem cells. Thyroid cells differentiated from iPS cells offer the opportunity to examine the detailed transcriptional regulation of thyroid cell differentiation and may provide a useful future source for individualized regenerative cell therapy.

Ca2 +-binding protein expression in primary human thyrocytes

We recently identified several Ca2+-binding proteins (CaBP) from the S100 and annexin family to be regulated by TSH in FRTL-5 cells. Here, we study the regulation of S100A4, S100A6 and ANXA2 in primary human thyrocytes (PHT) derived from surrounding tissues (ST), cold benign thyroid nodules (CTN) and autonomously functioning thyroid nodules (AFTN). We investigated the expression and regulation of CaBP and the effect of their expression on Ca2+ and TSHR signaling. We used an approach that accounts for the potential of an individual PHT culture to proliferate or to express thyroid differentiation features by assessing the expression of FOS and TPO. We found a strong correlation between the regulation of CaBP and the proliferation-associated transcription factor gene FOS. PKA and MEK1/2 were regulators of ANXA2 expression, while PI3-K and triiodothyronine were additionally involved in S100 regulation. The modulated expression of CaBP was reflected by changes in ATP-elicited Ca2+ signaling in PHT. S100A4 increased the ratio of subsequent Ca2+ responses and showed a Ca2+ buffering effect, while ANXA2 affected the first Ca2+ response to ATP. Overexpression of S100A4 led to a reduced activation of NFAT by TSH. Using S100A4 E33Q, D63N, F72Q and Y75K mutants we found that the effects of S100A4 expression on Ca2+ signaling are mediated by protein interaction. We present evidence that TSH has the ability to fine-tune Ca2+ signals through the regulation of CaBP expression. This represents a novel putative cross-regulating mechanism in thyrocytes that could affect thyrocyte signaling and physiology.

Thyroid Peroxidase Gene Expression Is Induced by Lipopolysaccharide Involving Nuclear Factor (NF)-κB p65 Subunit Phosphorylation

Thyroid peroxidase (TPO), a tissue-specific enzyme expressed in differentiated thyroid follicular cells, is a major antigen that has been linked to autoimmune thyroid disease. We have previously reported the functional expression of the lipopolysaccharide (LPS) receptor Toll-like receptor 4 on thyroid follicular cells. Here we investigated the effect of LPS in TPO expression and analyzed the mechanisms involved. We found a dose-dependent enhancement of TSH-induced TPO expression in response to LPS stimulation. EMSAs demonstrated that LPS treatment increased thyroid transcription factor-1 and -2 binding to the B and Z regions of TPO promoter, respectively. Moreover, LPS increased TSH-stimulated TPO promoter activity. Using bioinformatic analysis, we identified a conserved binding site for transcription nuclear factor-κB (NF-κB) in the TPO promoter. Chemical inhibition of NF-κB signaling and site-directed mutagenesis of the identified κB-cis-acting element abolished LPS stimulation. Furthermore, chromatin immunoprecipitation assays confirmed that TPO constitutes a novel NF-κB p65 subunit target gene in response to LPS. Additionally, our results indicate that p65 phosphorylation of serine 536 constitutes an essential step in the p65-dependent, LPS-induced transcriptional expression of TPO. In conclusion, here we demonstrated that LPS increases TPO expression, suggesting a novel mechanism involved in the regulation of a major thyroid autoantigen. Our results provide new insights into the potential effects of infectious processes on thyroid homeostasis.

Developmental regulation of gene expression in the thyroid gland of Xenopus laevis tadpoles

Thyroid hormones (TH) are the primary morphogen regulating amphibian metamorphosis. However, knowledge about molecular mechanisms regulating thyroid gland activity in anuran tadpoles is very scarce. In this study, we characterized gene expression profiles in thyroids of Xenopus laevis tadpoles during spontaneous metamorphosis. Using real-time PCR, elevated expression of slc5a5, tpo, tshr, and sar1a mRNAs was detected at late prometamorphic and climax stages. For dio2 and dio3 but not dio1, developmental regulation of thyroidal expression was evident from a strong up-regulation at late stages. Conversely, expression of the DNA replication markers mcm2 and pcna declined at climax stages. The presence of functional feedback mechanisms at premetamorphic stages was examined in two experiments. Stage 52 tadpoles were exposed for 72 h to 1.0 μg/l thyroxine (T4). This treatment caused reduced mRNA expression of slc5a5, tpo, and dio2, whereas no significant changes were detectable for tshr expression in thyroids and tshb expression in the pituitary. In another experiment, stage 46 tadpoles were treated with 20 mg/l sodium perchlorate (PER) for 5 and 10 days. Within this period of time, control tadpoles developed to stages 50 and 52, respectively. PER treatment resulted in up-regulation of slc5a5, tpo, and tshr mRNAs at both time points and increased dio2 mRNA expression at day 10. Effects of PER on thyroid histology were only apparent on day 10. Together, our analyses of thyroidal gene expression demonstrate a marked developmental regulation for functional markers of thyroid activity, two deiodinases as well as for DNA replication markers. Expression patterns detected in PER- and T4-treated tadpoles indicate that functional feedback signaling controlling thyroid activity is already active during premetamorphosis.

Expressions of RASSF1A, Galectin-3 and TPO mRNA in papillary thyroid carcinoma and their clinical significance

To investigate the mRNA expressions of RASSF1A, Galectin-3 and TPO in papillary thyroid carcinoma and some other thyroid benign lesions, and evaluate their diagnostic significance. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of RASSF1A, galectin-3 and TPO in the samples from 73 cases, including 23 cases with papillary thyroid cancer, 16 with nodular goiter, 29 with thyroid adenoma and 5 with Hashimoto's disease. A statistically significant difference in the mRNA expression of RASSF1A, Galectin-3 and TPO was observed between papillary thyroid carcinoma and follicular benign lesions (P<0.05). However, there was no significant difference among various kinds of benign lesions (P>0.05). A negative correlation of the expression of RASSF1A and Galectin-3 mRNA was found between thyroid benign lesions and malignant ones (P = 0.000). While the mRNA expression of RASSF1A and TPO was positively correlated between benign and malignant lesions (P = 0.028). Loss of expression of RASSF1A and TPO mRNA but high expression of Galectin-3 mRNA in papillary thyroid carcinoma are common. Therefore, the products of these three genes may be closely related to the development of thyroid papillary carcinoma, and may be used as useful markers in differential diagnosis of papillary thyroid carcinoma from the benign lesions. The results are more reliable if this detection method is used in combination with other techniques.

Retinoic acid receptor β2 re-expression and growth inhibition in thyroid carcinoma cell lines after 5-aza-2′-deoxycytidine treatment

The treatment of both undifferentiated and de-differentiated thyroid tumors, which are unresponsive to radioiodine, represents one of the biggest challenges for thyroidologists. The aim of the present study was to investigate in vitro the methylation status of retinoic acid receptors (RAR)beta2 promoter and the effect of the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) on 5 human thyroid cancer cell lines. The methylation status of RARbeta2 promoter was analyzed by methylation-specific PCR. The effect of 5-Aza-CdR on cell growth and apoptosis was evaluated by cell counting, enzymelinked immunosorbent assay tests and fluorescence-activated cell sorting analysis, while the effect on the expression of RAR and thyroid-specific genes was measured by qualitative and quantitative RT-PCR. Methylation of RARbeta2 promoter was present only in ARO cells. 5-Aza-CdR determined growth inhibition in all cell lines, probably due to apoptosis in WRO, NPA, and ARO cells, and to inhibition of DNA synthesis in TT cells. Treatment with 5-Aza-CdR induced the expression of RARbeta mRNA in ARO and FRO cells, a slight increase of the expression of Tg, TPO and thyroid trancription factor 1 (TTF-1) mRNA and the new expression of low levels of NIS in TT cells. A significant increase of TTF-1 mRNA in FRO cells was also observed. In this study we demonstrated that RARbeta2 promoter was methylated in ARO cell line. However, the 5-Aza-CdR treatment induced RARbetamRNA expression not only in ARO but also in FRO and TT cell lines, whose RARbeta2 promoter was unmethylated. A significant reduction of cell growth, but not cell re-differentiation, was also observed after 5-Aza-CdR treatment.

Glanzmann Thromboasthenia with Isolated Amegakaryocytic Thrombocytopenia.

Glanzmann thromboasthenia is an autosomal recessive bleeding disorder caused by a quantitative and/or qualitative defect of the platelet glycoprotein GPalfaIIb/betaIIIa (IIb/IIIa integrin ITGA2B-ITGB3) complex. We have studied a 24 years old patient (exon 2113 T-C substitution missense C705F/C674R) with refractory bleeding platelet count above 25.000, and a very prolonged bleeding time, extensive mucocutaneous petechiae, nosebleeds, and bleeding gums, marked spenomegaly, and conjunctival hemorrage with a prevalence of HLA-DRw2 and DRB1-0410 and a severely decreased platelet production. Immunophenotyping of lymphocytes showed a relative increase of the subset of gamma/delta T-cell receptor (TCR) positive, and CD4 CD8 negative T lymphocytes; expression of c-Mpl and TPO were down-regulated. The bone marrow examination revealed extremely scarce and small megakaryocytes (with a great reduction of CD34+ and CD41+ and a small fraction of CD42+) and with normal marrow cellularity regarding other cellular lines; chromosomal analysis was also normal, while there was an associated severe anaemia with elevated HbF without any history of alcohol use or drugs ingestion. The important observation in this case was the need of many platelet trasfusions, and of fresh frozen plasma (FFP); the functional consequences of this kind of therapy include the induction of abnormalities of the hormones or their receptors (TPO/c-Mpl) that can be decreased in these patients; in addition, platelet mass blocks the biologic effects of SDF-1/CXCR4 receptor and the degree of this inhibition is correlated inversely with platelet count, and supports the evidence that the thrombocytopenia may be related to the inhibition of transcription factors (GATA-1, NF-E2, TAL-1, ETS family), and it directly correlates with the excess of platelet transfusions as an inducible knock-out in MK differentiation. Moreover, this findings indicate that when the receptors (c-Mpl) is saturated, the JAK-2 activation is neutralized and subsequent phosphorylation of STAT-3 and STAT-5 are down-regulated. This patient responded to treatment with danazol 200 mg daily and desmopressin (DDAVP) 0.3 mg/kg, while an additional therapy with traxenamic acid and recombinant FVIIa reduced mucosal bleeding.

Variability of gene expression of „normal“ thyroid tissue

Studies examining gene expression profiling of thyroid tumors have used different kinds of „normal“ thyroid tissues for comparison. Most studies used histologically defined „normal“ tissues whereas some studies used functionally characterized tissues. A recent study demonstrated differences in the expression for NIS and apical iodide transporter between thyroid tissue with low and normal TSH (Bruno et al JCEM 2005). Therefore and since the identification of differentially regulated genes critically depends on the type of reference tissues (Eszlinger et al, JCEM 2006) we asked the question: are there broader differences in the gene expression patterns of the so called „normal“ surrounding tissues (ST) of AFTNs and CTNs which might be attributable to their different TSH levels? To answer this question we compared the gene expression profiles of 15 STs of AFTNs and 22 STs of CTNs. Principal component analysis and hierarchical clustering of all 12,625 probe sets showed a distinct separation of the two sub-groups. Statistical analysis revealed a significantly changed expression pattern of more than 300 genes (FDR<1%). Among them a decreased expression of thyroid specific genes such as TSHR, TPO, and PAX8 in the ST of AFTNs could be shown, whereas numerous ribosomal proteins are characterized by an increased expression. Moreover, in the STs of AFTNs a correlation between the pre-surgical TSH levels and the gene expression of DIO1, TPO, SIAT1, and GNAI1 could be observed (r>0.7, p<0.01). In conclusion, these data show strong differences in the gene expression patterns of so called „normal“ thyroid tissues which can be attributed to their different TSH exposure. Therefore, if no intra-individual reference tissue is used, a careful selection of the „normal“ reference tissue by pre-surgical assessment of the TSH levels and szintigraphy in iodine deficient areas should be performed in studies investigating the gene expression patterns of thyroid tumors.

Evidence for abnormal iodination of thyroglobulin in cold thyroid nodules

Using a proteomics approach we previously observed upregulation of proteins involved in the reduction of peroxide in cold thyroid nodules. Further we gained first evidence for an abnormal processing of thyroglobulin (Tg) in CTN since several differential isoforms of Tg combined with a drastically increased induction of the cysteine protease cathepsin B as well as upregulation of molecular chaperones were detected by proteome analysis in CTN compared to the surrounding normal thyroid tissue (ST). These results point to either (I) a defect in the integrity of Tg which might prevent the organification and coupling of iodide (I-) to from thyroid hormones or (II) to a principal defect in the uptake and transport of I- in CTN. To investigate the functional protein expression characteristics in CTNs in more detail, immunohistochemical analysis was performed in a series of 12 CTNs and corresponding normal thyroid tissues. The following proteins were studied: (1) proteins involved in intrathyroidal iodine metabolism (Pendrin, ThOX 1/2 and TPO, Tg, T4-Tg); (2) proteins involved in the antioxidative system (PDRX 5, GST-π,) and (3) proteins involved in Tg-processing (cathepsin B). We found (I) complete absence of T4-rich iodinated Tg in CTNs while overall Tg expression was not altered, (II) higher expression of ThOXs and TPO in CTNs compared to ST, (III) upregulation of the antioxidant enzyme PDRX 5 in CTNs and (VI) expression of pendrin depending on follicular size (upregulation only in small follicles in normal thyroid tissues). Taken together, this study confirms the data obtained by proteomics and further reinforce the hypothesis of an altered Tg function with most likely compensatory yet ineffective upregulation of the thyroid hormone synthesis apparatus in CTNs. Moreover, it is tempting to speculate that increased proliferation in combination with deranged abundant peroxide generation may contribute to somatic mutations leading to clonal CTNs.

CML Developed in a Japanese Family Transmitting a Novel Point Mutation in the Thrombopoietin Gene(TPO).

Introduction: Four families are reported to have hereditary thrombocythemia (HT) with a mutation in TPO. Their clinical manifestation is essentially thrombocytosis without leukemia. CML is one of myeloproliferative disorders, and shows leukocytosis and thrombocytosis associated with a proliferation of malignant clone originated from a hematopoietic stem cell (HSC). The incidence of CML is about 5 per 100,000 in Japan. Mutations of cytokine receptor including c-kit, flt-3 and G-CSF receptor are reported as a cause of AML. Especially flt-3 abnormalities are found in about 20% of AML. However, abnormality of c-mpl or TPO is not reported as a cause of leukemia. In this paper, we analyzed a CML case with novel point mutation in the TPO who still had thrombocytosis after cytogenetic complete response.Case: Japanese, 35 y.o., male, complained leukocytosis. He had a family history of thrombocytosis in 4 individuals over 3 generations. A physical examination revealed a moderate splenomegaly. Laboratory tests at the time of diagnosis were as follows; WBC 141,000/μl (blast 1.8%, promyelo 2.4%, myelo 20.0%, meta 8.2%, stab 24.2%, seg 22.2%, immature eosinophils 1.8%, eosinophil 3.6%, immature basophils 0.4%, basophils 10.4%, mono 1.0%, lymphocytes 4.0%, erythroblast 3%), PLT 641,000/μl and NAP score 53 (nl; 156–271). Bone marrow showed hypercellularity with the increased megakaryocytes(Meg), bcr-abl fusion mRNA positive, Ph1 chromosome positive. After 5 months treatment with STI571, most of clinical findings including karyotype and fusion mRNA turned to be normal, but thrombocyte(PLT) still showed more than 1,000,000/μl. At this time, serum TPO concentration was 8.14 f mole/ml (nl; 0.40 +/− 0.28 f mole/ml, mean +/− SD). Genetic analysis of TPO revealed novel point mutation at splicing donor site of 3′-end of the exon3. A point mutation at splicing donor site is reported to cause an exon-skipping and intron-retention, which induce a malfunction of a suppressive post-transcriptional and translational regulation, and consequent high-level expression of functional TPO protein.Discussion: TPO was cloned as a c-mpl ligand, which leads to the production of PLTs. Its receptor is a c-mpl proto-oncogene product, which is expressed not only in Meg, but also in HSC. Thus, TPO can stimulate HSC. The c-mpl transgenic mice are reported to have the increased Meg, its committed progenitor and PLT. Knockout mice of TPO presented not only the decreased Meg, but also multi-lineage committed progenitors. Thus, a modulation of c-mpl or its ligand function affects on both Meg and HSC. The c-mpl was cloned as a cellular homolog of a viral oncogene, v-mpl of myeloproliferative leukemia virus (MPL). The MPL causes myeloproliferative leukemia syndrome through v-mpl function in mice. Since v-mpl and c-mpl indicate high homology, it is possible that abnormal c-mpl function causes v-mpl like response. Through continuous stimulation of c-mpl signal, high TPO concentration may have induced a malignant transformation of HSC or supported a survival of an immature malignant clone in the present case. Improvement of thrombocytosis in CML is one of hematological responses to an anti-CML treatment such as STI571. In such a case who had good response other than thrombocytosis, an existence of HT might be considered. On the other hand, during following up HT family, occurrence of CML should be noted.

Thyroid Peroxidase Expression in Diseased Human Thyroid Glands.

Histoenzymologic studies of the human thyroid have been restricted to attempts to differentiate benign from malignant tumors and, to our knowledge, there is no systematic study on the distribution and behavior of enzymes for several types of lesions of the thyroid gland. The aim of the present study was to investigate thyroid peroxidase (IPO) expression in diseased thyroids, as well as to study the role of this enzyme as a parameter to define the biological behavior of thyroid lesions. A total of 1 17 neoplastic and nonneoplastic lesions of the thyroid were evaluated. The expression of TPO was determined by the analysis of at least 200 cells/specimen. A rate of 80% of positive cells was considered a threshold for TPO positivity. TPO expression was detected in all nonneoplastic lesions of thyroid as well as in 78.8% of the adenomas. We also observed positivity for TPO in 20% of malignant lesions. Therefore, these findings do not allow separation of benign and malignant lesions of the thyroid based in the expression of TPO.