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Context: Thyroglobulin (Tg) measurement is a major tool for the follow-up of differentiated thyroid cancer (DTC) patients; however, in patients who do not undergo radioactive iodine (RAI) ablation, normal ultrasensitive Tg levels measured under levothyroxine treatment (usTg/l-T4) are not well defined., Objective and Design: This single-center retrospective study assessed usTg/l-T4 level in 86 consecutive patients treated with total thyroidectomy without RAI ablation for low-risk DTC (n=77) or for tumors of uncertain malignant potential (TUMP) (n=9)., Results: DTCS were classified as PT1, PT2, and PT3 in 75, 1, and 1 case respectively and PN0, PN1, and PNX in 40, 6, and 31 respectively. following surgery, ten patients had TG antibodieS (TGAB). Among those without TGAB, the first USTG/L-T4 determination obtained at a mean time of 9 months after surgery was 0.1NG/ML in 62% of cases, 0.3NG/ML in 82% of cases, 1NG/ML in 91%, and 2NG/ML in 96% of cases. after a median follow-up of 2.5 years (range: 0.6-7.2 years), one patient had persistent disease with an usTg/l-T4 at 11 ng/ml and an abnormal neck ultrasonography (US) and two patients had usTg/l-T4 level >2 ng/ml (3.9 and 4.9 ng/ml) with a normal neck US. Within the first 2 years following total thyroidectomy without RAI ablation, usTg/l-T4 level is ≤2 ng/ml in 96% of the cases., Conclusion: After total thyroidectomy, sensitive serum Tg/l-T4 level is ≤2 ng/ml in most patients and can be used for patient follow-up.

Objective: The progression of medullary thyroid cancer is difficult to assess with imaging modalities; we studied the interest of calcitonin and carcinoembryonic antigen (CEA) doubling times and of Ki-67 labeling and mitotic index (MI)., Patients and Methods: Fifty-five consecutive medullary thyroid carcinoma (MTC) patients with elevated calcitonin levels underwent repeated imaging studies in order to assess tumor burden and progression status. We looked for relationships between tumor burden and levels of calcitonin and CEA and between progression status according to the response evaluation criteria in solid tumors (RECIST) and calcitonin and CEA doubling times, and Ki-67 labeling and MI., Results: The calcitonin and CEA levels were correlated with tumor burden. Ten patients with calcitonin levels below 816 pg/ml had no imaged tumor foci. Among the 45 patients with imaged tumor foci, 19 had stable disease and 26 had progressive disease, according to the RECIST. The calcitonin and CEA doubling times were strongly related to disease progression, with very few overlaps: 94% of patients with doubling times shorter than 25 months had progressive disease and 86% of patients with doubling times longer than 24 months had stable disease. Ki-67 labeling and MI were not significantly associated with disease progression., Conclusion: For MTC patients, the doubling times of both calcitonin and CEA are efficient tools for assessing tumor progression.

Background: Serum thyroglobulin (Tg) is the marker of differentiated thyroid cancer after initial treatment and TSH stimulation increases its sensitivity for the diagnosis of recurrent disease., Aim: The goal of the study is to compare the diagnostic values of seven methods for serum Tg measurement for detecting recurrent disease both during L-T4 treatment and after TSH stimulation., Methods: Thyroid cancer patients who had no evidence of persistent disease after initial treatment (total thyroidectomy and radioiodine ablation) were studied at 3 months on L-T4 treatment (Tg1) and then at 9-12 months after withdrawal or recombinant human TSH stimulation (Tg2). Sera with anti-Tg antibodies or with an abnormal recovery test result were excluded from Tg analysis with the corresponding assay. The results of serum Tg determination were compared to the clinical status of the patient at the end of follow-up., Results: Thirty recurrences were detected among 944 patients. A control 131I total body scan had a low sensitivity, a low specificity, and a low clinical impact. Assuming a common cutoff for all Tg assays at 0.9 ng/ml, sensitivity ranged from 19-40% and 68-76% and specificity ranged from 92-97% and 81-91% for Tg 1 and Tg2, respectively. Using assays with a functional sensitivity at 0.2-0.3 ng/ml, sensitivity was 54-63% and specificity was 89% for Tg1. Using the two methods with a lowest functional sensitivity at 0.02 and 0.11 ng/ml resulted in a higher sensitivity for Tg1 (81% and 78%), but at the expense of a loss of specificity (42% and 63%); finally, for these two methods, using an optimized functional sensitivity according to receiver operating characteristic curves at 0.22 and 0.27 ng/ml resulted in a sensitivity at 65% and specificity at 85-87% for Tg1., Conclusion: Using an assay with a lower functional sensitivity may give an earlier indication of the presence of Tg in the serum on L-T4 treatment and may be used to study the trend in serum Tg without performing any TSH stimulation. Serum Tg determination obtained after TSH stimulation still permits a more reliable assessment of cure and patient's reassurance.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chimera (TFO-P) can specifically recognise its DNA target at physiological salt and pH conditions. Bound to the double-stranded target DNA in a promoter region, the TFO-P is able to activate gene expression. Our results suggest that this type of molecule may prove useful in the design of new tools for artificial modulation of gene expression.

Recent observations based on immunohistochemistry and RT-PCR demonstrate that early placenta insulin-like peptide (EPIL), encoded by the INSL4 gene, is present in the placenta during gestation. In the present study, we report on the development of a specific immunoassay, entirely based on two monoclonal anti-peptide antibodies (mAbs), and its use for the detection of pro-EPIL forms in biological fluids during pregnancy. One mAb directed against the C-connecting peptide was used to capture pro-EPIL forms and their binding was revealed by a radiolabeled anti-A chain mAb as the indicator. A composite synthetic peptide, encompassing the C- and A-domains, was utilized as the standard. Under these experimental conditions, the assay displays a sensitivity limit of 2 ng/mL. Pro-EPIL molecular forms were detected in both amniotic fluid and maternal serum of pregnant women. At 12 to 16 weeks of pregnancy, the pro-EPIL level was higher in amniotic fluid (246 +/- 50.8 ng/mL) than in maternal serum (5 +/- 2.0 ng/mL). As gestation advanced, so the concentration of pro-EPIL forms decreased in amniotic fluid while its level increased in maternal serum. Interestingly, in amniotic fluid, the pattern of pro-EPIL concentration during pregnancy is very similar to that observed for human chorionic gonadotropin (hCG) and its free subunits. The pattern of serum pro-EPIL concentration is similar to that of the free alpha-subunit. Together with our previous immunohistochemical observations, these results indicate that pro-EPIL is preferentially secreted by cytotrophoblasts in amniotic fluid and that the biosynthesis of hCG subunits and EPIL may be regulated by common pathways. Overall, our observations strongly suggest that EPIL may play a critical physiological role during embryonic and foetal development.

Screening was performed in 130 consecutive patients with apparently sporadic neuroendocrine tumors (NET) to assess the prevalence of multiple endocrine neoplasia type 1 (MEN1) and hormonal production. Screening for MEN1 included measurement of serum calcium and PTH [PTH-(1-84)], gastrin, PRL, and insulin-like growth factor type I (IGF-I) levels. MEN1 genetic testing was performed in patients with two components of the MEN1 syndrome. Screening for hormonal production included measurement of serum neuron-specific enolase (NSE), calcitonin (CT), glycoprotein alpha-subunit (GP alpha), hCG beta-subunit (free hCG beta), and somatostatin levels. Twenty-four-hour urinary free cortisol (UFC) and 5-hydroxyindolacetic acid (5-HIAA) determinations were also performed. Four patients had hyperparathyroidism, none of whom had pituitary or familial disease. Hyperprolactinemia was compatible with a pituitary disease in one patient. No acromegalic feature or any increase in IGF-I was found. Hypergastrinemia, compatible with an associated pancreatic NET, was found in one patient. Genetic screening of the MEN1 gene was performed in five of the six patients with two components of the MEN1 syndrome. A nonsense mutation (Arg108stop) was identified in the tumor of one patient. Elevated NSE, 5-HIAA, CT, GP alpha, free hCG beta, SMS, and nonsuppressible UFC were found in 47%, 46%, 14%, 19%, 12%, 3%, and 6% of NET patients, respectively. Production of CT, GP alpha, and free hCG beta was highly related to the primary site: all but two of these secretions originated in foregut NET. 5-HIAA secretion was found in 27% of foregut-derived and 85% of midgut-derived NET. In conclusion, MEN1 is a rare event in patients presenting with apparently sporadic NET. It occurred mainly in foregut NET and should be screened for by serum calcium and PTH-(1-84) measurements. Routine hormonal measurements should depend on the primary site. NSE, 5-HIAA, CT, and alphaGP should be routinely measured in foregut-derived NET; only serum NSE and 5-HIAA measurements are recommended in midgut-derived NET.

Antipeptide antibodies raised against the carboxyl-terminal region of the human sodium/iodide (Na+/I-) symporter (hNIS) were used to investigate by immunohistochemistry the presence and distribution of the hNIS protein in normal thyroid tissues, in some pathological nonneoplastic thyroid tissues, and in different histotypes of thyroid neoplasms. In normal thyroid tissue, staining of hNIS protein was heterogeneous and limited to a minority of follicular cells that were in close contact with capillary vessels. In positive cells, immunostaining was limited to the basolateral membrane. In contrast, in Graves' disease the majority of follicular cells expressed the hNIS protein. In autoimmune thyroiditis, the number of hNIS-positive cells, was similar to that found in normal tissue. These positive cells were found essentially close to lymphocytic infiltrates. This observation supports the concept of hNIS as an autoantigen. In diffuse nodular hyperplasia, hNIS staining was heterogeneous, but the number of hNIS-positive cells exceeded that found in normal tissue. In well differentiated follicular or papillary carcinoma, the number of hNIS-positive cells was significantly lower than in normal tissue. In poorly differentiated follicular carcinoma, the number ofhNIS-positive cells was less than that found in well differentiated carcinoma, or there were no positive cells. Interestingly, in all of these thyroid tissues, the number of follicular cells exhibiting TSH receptor (TSHR) immunoreactivity was greater than the number ofhNIS-positive cells. As hNIS expression appears to be related to TSHR stimulation, the decreased number of TSHR-positive cells in cancers may contribute to the reduced capacity of neoplastic cells to concentrate iodide. In one patient with a follicular cancer with an absence of hNIS immunostaining, the total body 131I scan showed no uptake in metastatic tissue. In three cancers with positive hNIS cells, the 131I scan showed uptake in lymph node metastases. This suggests that immunodetection of hNIS could predict radioiodine uptake in thyroid cancers.

Human lutropin (hLH) exhibits both carbohydrate and peptidic heterogeneities that affect its biological potency and the duration of its activity in vivo. Peptidic changes within the hLH beta-subunit are characterized as intrachain proteolytic nicking and carboxyl terminus heterogeneity. To date, the carboxyl terminus of hLHbeta appears to end at either position Gln114 or Gly117, as determined by sequencing of purified subunit. Furthermore, the complementary DNA for hLHbeta predicts a protein containing an additional peptidic stretch, which would make the beta-subunit 121 residues long. This extension may be responsible for the particular intracellular behavior of hLHbeta. To investigate the carboxyl terminus polymorphism of natural hLHbeta, monoclonal antipeptide antibodies were raised against a synthetic peptide mimicking the 104-119 portion of hLHbeta. One antibody, designated LHP09, was found to specifically react with the recombinant hLHbeta ending at position hLHbeta[Leu119] but not with other recombinant forms ending at [Ser116], [Phe120] or [Leu121]. Immunochemical analysis of hLH, either pituitary or urinary in origin, indicated that only pituitary hLH contains a Leu119-ending form of hLHbeta. Finally, immunohistochemical detection was performed using LHP09 and showed specific staining of a normal adult pituitary gland. These observations support the in vivo existence of intrapituitary molecular forms of hLHbeta ending at various positions between Gln114 and Leu121.

Recently, a new member of the insulin gene superfamily termed insulin-like 4 (INSL4) was identified in the human placenta and uterus. The present study investigated whether placenta translates INSL4 mRNA into a putative peptide named early placenta insulin-like (EPIL). Among antibodies elicited against the C chain of pro-EPIL, one antibody (AB7381) was specifically directed against the C chain 59-88 portion, and among those elicited against the A and B chains of EPIL, one antibody (Ab1661) was directed against the A chain 115-139 and the B chain 23-52 portions. Immunohistochemistry based on antibody 7381 to pro-EPIL and antibody 1661 to EPIL demonstrated that the cytotrophoblast from early placenta preferentially expresses the pro-EPIL peptide, whereas the EPIL peptide is expressed by both the cytotrophoblast and the syncytiotrophoblast. At term, the pro-EPIL peptide was detected in villous cytotrophoblast cells, whereas the EPIL peptide was not detected. Moreover, in vitro experiments performed on term placenta showed that the steady state levels of INLS-4 mRNA in the cytotrophoblast are 10 times (one log unit) lower than in the differentiated villous syncytiotrophoblast cells. Taken together, these findings reveal that expression of EPIL peptides in the villous cytotrophoblast is different from that displayed by the syncytiotrophoblast. Finally, these data are the first demonstration that INSL4 mRNA are translated into pro-EPIL and EPIL peptides.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

The present study was designed to characterize monoclonal antibodies (mAbs) specific for the free beta-subunit of hCG (free hCG beta), to develop two-site immunoradiometric assays (m-IRMAs) specific for free hCG beta, and to study the reactivities of various molecular forms of hCG beta in these assays. We attempted first to delineate the antigenic regions present specifically on the free hCG beta by studying the binding pattern of seven mAbs directed preferentially to hCG beta, designated FBT11, P8E, P10F, HB2, P5D, P5H, and INN-64. Competitive inhibition experiments performed by RIA demonstrated the specificity of these mAbs for the free hCG beta as noncross-reacting with the beta-subunit of human (h) LH beta. m-IRMAs were used to analyze the arrangement of epitopes on hCG beta. Experiments performed with the seven mAbs used either as capture antibodies or radiolabeled indicators confirmed the specificity of the seven mAbs for the free hCG beta, and that mAbs FBT11, P8E, and P10F bound to equine LH beta (eLH beta), but did not bind to a fragment of hCG beta called the beta-core fragment (beta CF). These antibodies defined an antigenic domain identified as A. In contrast, mAb HB2 bound neither to eLH beta (e beta) nor to beta CF and was directed to domain B (e beta negative, beta CF negative). Finally, mAbs P5D, P5H, and INN-64 bound to beta CF, but did not bind to eLH beta, and defined a third domain identified as C (e beta negative, beta CF positive). Collectively, these results demonstrate that at least six antigenic domains are present on the free hCG beta and that a limited set of amino acids was shared among these domains; domains A, B, and C are present only on the free beta-subunit, while three other domains recognized by mAbs FB19, FBT10, and 518B7 are present on both free hCG beta and hCG. Thus, most of the surface of the hCG beta appears to be antigenic and accessible to antibody binding. Three different m-IRMAs specific for free hCG beta were then constructed using either mAb FBT11 (domain A) or HB2 (domain B). The study of the reactivities of various molecular forms of hCG beta in these assays demonstrated that the recognition of hCG beta forms nicked at position 43 (beta 43), 44, and/or 51 (beta 44/51) varied between assays.(ABSTRACT TRUNCATED AT 400 WORDS)

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.

To improve our knowledge of the structural features of the alpha-subunit of hCG we have studied the antigenic site recognized by monoclonal antibody (MAb) ECG01 raised against equine CG (eCG) which binds to hormones and alpha-subunits from human and equine species. We have also delineated regions of hCG alpha comprising the epitope recognized by HT13 which was raised against hCG and binds to hCG and hCG alpha. To define the residues involved in the antigenic sites recognized by ECG01 and HT13, we have studied the reactivities of these two MAbs with native or chemically modified LH and CG with subunits from equine, human, or ovine (o) species or with synthetic peptides analogous to various portions of hCG alpha. We have also compared these reactivities with those displayed by MAbs AHT20 and FA 36, whose epitopes have been previously described; anti-hCG alpha MAb AHT20 is specific for the free alpha-subunits of various species and recognizes residues localized to the 36-41 region of hCG alpha, whereas antipeptide MAb FA36 binds to the 87-92 carboxyl-terminal part of hCG alpha. Our results show that the epitopes of HT13 and ECG01 are 1) probably discontinuous, as these MAbs did not bind to the reduced and S-carboxymethylated hCG alpha; and 2) constituted by residues borne on the 1-35 and 52-86 sequences, as they do recognize the hCG alpha core missing the 36-51 portion, yet do not recognize hCG alpha-(87-92) region recognized by FA36. The comparative studies performed with specific two-site immunoradiometric assays to determine the interspecies cross-reactivities of the MAbs allow us hypothetical assignment of residues on the primary structure of hCG alpha. The antigenic site recognized by ECG01 might include two to six amino acids, four of these residues being located at inverted places compared to those of oLH alpha (Asp6/Gly22 and Arg67/Lys75). These residues present important charged functional groups highly conserved among evolutionarily related variants, and it is likely that they are located on the surface of both the intact hormone and its alpha-subunit. Three peptidic portions of hCG alpha, 16-17, 64-66, and 73-76, respectively, might be involved in the epitope recognized by HT13, although we could not rule out the possibility that other residues were also involved in the antigenic site. These observations allow us to identify several residues as potentially constituting the epitopes recognized by two MAbs on both hCG and hCG alpha.