Anti-calcitonin Antibodies Research Articles

Development of Parafollicular Cells and their Relationship with Developing Thyroid Follicles in Human Foetuses.

The parafollicular cells or clear (C) cells in man are part of neuroendocrine system under Amine Precursor Uptake and Decarboxylation (APUD) cells. Their role in adults has been reputable but in foetus is still unclear. The present study was a baseline study endeavouring to describe the chronological development of the parafollicular cells with particular focus on its correlation with developing human thyroid follicles. The study was conducted on 10 aborted foetuses (14-28 weeks), procured from the Department of Obstetrics and Gynaecology, Lok Nayak Hospital, New Delhi, India. Serial sections of foetal thyroid gland were generated, stained with haematoxylin and eosin, and immunohistochemistry using the anticalcitonin antibody and examined qualitatively. In our study, the parafollicular cells were seen as earlier as by 14th week. They became morphologically and functionally mature by 16th week of gestation. The parafollicular cells were getting organized from scattering to parafollicular location then to a more localized area, i.e., intrafollicular along with the follicular development. As the follicles were enlarging, the intrafollicularly located parafollicular cells which was initially present in groups was getting displaced singly between the follicular cells in the same follicle. The sequential development pattern of the parafollicular cells in relation to developing thyroid follicles was established. This immunohistochemical study also concluded that the parafollicular cells might have higher character to play in the early gestational age such as regulation of ossification in the human foetus.

A specific Tween-80-Rhodamine S-MWNTs phosphorescent reagent for the detection of trace calcitonin

The present study proposed a simple sensitive and specific immunoassay for the quantification of calcitonin (CT) in human serum with water-soluble multi-walled carbon nanotubes (MWNTs). The COOH group of MWNTs could react with the NH group of rhodamine S (Rhod.S) molecules to form Rhod.S-MWNTs, which could emit room temperature phosphorescence (RTP) on acetate cellulose membrane (ACM) and react with Tween-80 to form micellar compound. Tween-80-Rhod.S-MWNTs (TRM), as a phosphorescent labelling reagent, could dramatically enhance the RTP signal of the system. The developed TRM phosphorescent reagent was used to label anti-calcitonin antibody (AbCT) to form the TRM-AbCT labelling product, which could take high specific immunoreaction with CT, and the ΔIp (= Ip2−Ip1, Ip2 and Ip1 were the phosphorescence intensity of the test solution and the blank sample, respectively) of the system was linear to the content of CT. Hence, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) was established for the determination of CT in human serum. This sensitive (limit of quantification (LOQ) was 8.0×10−14gmL−1), accurate, selective and precise method has been applied to determine CT in human serum and predict primary osteoporosis and fractures, with the results in good agreement with those obtained by chemiluminescence immunoassay (CLIA). Simultaneously, the structure of MWNTs was characterized with scanning electron microscopy (SEM) and infrared spectroscopy (IR), and the reaction mechanisms of both labelling AbCT with TRM and SSRTPIA for the determination of trace CT were discussed.

Synthesis, characterization and evaluation of bone targeting salmon calcitonin analogs in normal and osteoporotic rats

In order to assess the therapeutic efficacy of an antiresorptive drug with imparted bone targeting potential using bisphosphonate (BP) conjugation and an improved pharmacokinetic profile using PEGylation, we synthesized, characterized and evaluated in vivo efficacy of bone-targeting PEGylated salmon calcitonin (sCT) analog (sCT-PEG-BP). sCT-PEG-BP was compared with non-PEGylated bone targeting sCT analog (sCT-BP) and unmodified, commercially available sCT. sCT-PEG-BP conjugates were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis. The effect of PEG-BP or BP upon sCT secondary structure was examined by Circular Dichroism and sCT-PEG-BP was evaluated for in vitro bone mineral Hydroxyapatite (HA) binding ability and calcium salts specificity using a binding assay for bone HA and several calcium salts. Anti-calcitonin antibody binding ability of these analogs was determined using enzyme-linked immunosorbent assay (ELISA), by reacting bone targeting sCT analogs with calcium phosphate coated Osteologic® plates and detecting the bound sCT using anti-sCT antibody. Potential cytotoxicity of these compounds was evaluated in monocytic RAW 264.7 cells, and sCT bioactivity was evaluated using an in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. Finally, in vivo efficacy of each compound was evaluated by determining the plasma levels of calcium after s.c. administration in normal rats, and in a rat model of Osteoporosis, secondary to ovariectomy (OVX). In vivo micro-computed tomography (micro-CT) was used to temporally map and quantify alterations in bone volume and bone mineral density (BMD) in the same animals at 1, 4, 8 and 12weeks after OVX surgery. Sixteen 6week old virgin female rats underwent OVX surgery followed by the daily s.c. injection of 2.5IU/kg/day sCT or equivalent analogs, and compared to four sham-operated, placebo treated control rats. Our results showed the chemical coupling of PEG-BP or BP to sCT altered its secondary structure without altering its antibody binding ability. sCT analogs retained strong sCT bioactivity, were non-toxic to RAW 264.7 cells in culture and elicited a comparable hypocalcemic effect to that of unmodified sCT in normal rats. Compared to marketed unmodified sCT, sCT-PEG-BP showed significantly improved efficacy in terms of preserving bone volume, BMD and trabecular micro-architecture in osteoporotic rats at the initial dose tested. Bisphosphonate-mediated targeting of PEGylated sCT to bone represents a new class of targeted antiresorptive compounds that has not previously been attempted.

Retrospective review of 13 cases of pyriform sinus fistula

Purpose To review 13 cases of pyriform sinus fistula (PSF) and to summarize the clinical and histologic features. Materials and methods A retrospective review and immunohistochemical analysis of 13 cases of PSF treated in Sapporo Medical University Hospital from 1985 to 2005. This study includes 13 patients with PSF diagnosed by the existence of fistulous tract radiologically and/or pathologically. Fistulectomy was performed in all cases. Surgically obtained specimens were processed for immunohistochemistry with commercial anticalcitonin antibody. Results The patients consisted of 4 males and 9 females, and the ages ranged from 4 to 55 years. The fistula was detected by barium study in 12 cases (92.3 %), and all cases were on the left side. Pathologic detection of the fistula was achieved in only 8 cases (69.2 %). Immunohistochemistry showed abnormal distribution of calcitonin-positive cells in 12 cases (92.3 %). No recurrence has occurred in any of the patients. Conclusions Surgical removal of the inflammatory tissue is worthy of consideration in suspected PSF cases as in patients who exhibit similar manifestations without preoperative detection of the tract.

Decrease of parafollicular thyroid C-cells in experimental esophageal atresia: further evidence of a neural crest pathogenic pathway

Adriamycin-induced experimental esophageal atresia (EA) is often associated with malformations of neural crest (NC) origin, such as abnormal pharyngeal pouch derivatives like the thymus and the parathyroids. The aim of the present study was to examine whether NC-derived thyroid C-cells were abnormal in a rat model. Pregnant rats received intraperitoneally either 2 mg/kg Adriamycin (EA) or vehicle (controls) on days 8 and 9 of gestation. Fetuses were recovered on day 21, and blocks including the trachea and thyroid were fixed in formalin, coronally sectioned at 3-mum widths, and stained with standard hematoxylin and eosin until the largest area of thyroid was reached. From this point on, the 1st, 10th, and 20th slices were immunohistochemically stained with anti-calcitonin antibody. Positively-stained cells in each section of the gland were counted using a computer-assisted image analysis method, and the results were averaged. The distribution of the cells within the gland was assessed as well. Comparisons between EA and control rats were made by nonparametric tests with a significance threshold of p<0.05. The number of C-cells was dramatically reduced in EA animals compared with controls (32.4+/-36 vs. 92.3+/-60.5, p<0.001). Histology of the thyroid was similar in both groups, but the distribution of positive C-cells within the gland followed an abnormal pattern in EA rats. Adriamycin causes a pattern of NC-derived malformations, including a severe decrease in thyroid C-cells accompanied by abnormal distribution or migration patterns. These results represent further evidence of the involvement of NC organogenic control dysregulation in the pathogenesis of EA and its associated malformations. The similarities between the rat model and the clinical picture strongly support investigating other subclinical NC-derived anomalies in patients with EA.

C-cells in colloid goiter.

The aim of this investigation was to quantitatively evaluate C-cells in colloid goiters, analyzing 36 thyroids that were obtained through thyroidectomy from 24 patients with goiter and 12 normal glands from adult patients without thyroid disease, which were used as the control group. On average, 6 different thyroid areas were sampled and labeled by immunohistochemistry with a monoclonal anticalcitonin antibody, utilizing the avidin-biotin-peroxidase complex. C-cells were counted in fields measuring 1 square centimeter, and the mean number of cells per field was then calculated. Data were statistically analyzed using the Mann-Whitney test. In the colloid goiter group, the number of C-cells ranged from 0 to 23 per field, while in normal controls they ranged from 20 to 148 per field. These results demonstrate a significant decrease of C-cell number in the colloid goiter group compared with control group, indicating that the hyperplastic process is restricted to follicular cells, to the detriment of C-cells, which probably cease to receive trophic stimuli.

Subcommissural organ–Reissner's fiber complex of the teleost Clarias batrachus responds to GABA treatment

Subcommissural organ (SCO) is a highly specialized ependymal gland located in the roof of the third ventricle. The secretory products of the SCO, which condense to form Reissner's fiber (RF), were recently found to cross-react with the anti-calcitonin antibody. To understand the mechanisms regulating the formation of the RF and the possible function of these discrete structures, we studied the response of the SCO–RF complex to intracranially administered GABA, using immunocytochemical labeling with anti-calcitonin antibody. Although the SCO–RF complex of control fish was intensely immunostained, 1 h after GABA treatment, the ependymal cells revealed partial loss of immunoreactivity; the RF showed occasional loss of immunoreactivity with its diameter increased by about 56% of the control value. Following 2 h of GABA treatment, the SCO revealed dramatic loss of calcitonin-like immunoreactivity from the ependymal cells. The RF showed a dual response in this group, while in some segments the RF appeared conspicuously thick, elsewhere it appeared thin. The mean diameter was, however, not significantly different from the normal. Following 4 h of GABA treatment, while calcitonin-like immunoreactive material made its reappearance in the SCO, the RF diameter was uniformly reduced to about 35% of the control value. The responses by the RF as well as the SCO to intracranially administered GABA were blocked by pretreatment with bicuculline, a GABA A receptor antagonist. The results suggest that GABA, acting via GABA A receptors, may trigger the release of secretory material from the SCO and induce histomorphological changes in the RF indicative of discharge of stored material.

Unpaired Ultimobranchial Glands of the African Lungfish, Protopterus dolloi

The ultimobranchial glands of juvenile African lungfish (Protopterus dolloi) (14 individuals; total body length 25-205 mm) were immunohistochemically examined. In individuals larger than 36 mm, one ultimobranchial gland was close to the left afferent branchial arteries. The topography of the ultimobranchial gland was similar to that of salamanders and sharks, but not to teleosts. With body growth, the ultimobranchial gland was vascularized and the parenchymal cells were gradually immunostained with anti-calcitonin antibody. In all individuals examined, the ultimobranchial gland existed only on the left side of the pharynx. These observations are discussed from a phylogenetic viewpoint.

Brain uptake of 11C-labelled stable prostacyclin receptor ligand studied by positron emission tomography

The present study proposed a simple sensitive and specific immunoassay for the quantification of calcitonin (CT) in human serum with water-soluble multi-walled carbon nanotubes (MWNTs). The COOH group of MWNTs could react with the NH group of rhodamine S (Rhod.S) molecules to form Rhod.S-MWNTs, which could emit room temperature phosphorescence (RTP) on acetate cellulose membrane (ACM) and react with Tween-80 to form micellar compound. Tween-80-Rhod.S-MWNTs (TRM), as a phosphorescent labelling reagent, could dramatically enhance the RTP signal of the system. The developed TRM phosphorescent reagent was used to label anti-calcitonin antibody (AbCT) to form the TRM-AbCT labelling product, which could take high specific immunoreaction with CT, and the ΔIp (= Ip2 − Ip1, Ip2 and Ip1 were the phosphorescence intensity of the test solution and the blank sample, respectively) of the system was linear to the content of CT. Hence, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) was established for the determination of CT in human serum. This sensitive (limit of quantification (LOQ) was 8.0 × 10−14 g mL−1), accurate, selective and precise method has been applied to determine CT in human serum and predict primary osteoporosis and fractures, with the results in good agreement with those obtained by chemiluminescence immunoassay (CLIA). Simultaneously, the structure of MWNTs was characterized with scanning electron microscopy (SEM) and infrared spectroscopy (IR), and the reaction mechanisms of both labelling AbCT with TRM and SSRTPIA for the determination of trace CT were discussed.

Co-expression of vimentin and 19S-thyroglobulin in follicular cells located in the C-cell complex of dog thyroid gland.

The localization of vimentin, an intermediate filament protein, in the dog thyroid cells was examined by immunohistochemistry with a monoclonal antibody against vimentin and polyclonal anti-19S-thyroglobulin and anti-calcitonin antibodies. In the dog thyroid gland, C-cells are usually distributed as large cell groups in an interfollicular location. In addition, C-cell complexes that are special C-cell clusters associated with other epithelial elements and cysts are frequently located around the parathyroid gland IV. The C-cell complexes are remnants of embryonic ultimobranchial bodies and retain fetal characteristics for long periods. Follicular cells in various stages of differentiation are observed in the C-cell complexes and large C-cell groups of postnatal dogs: (a) cell masses not yet organized into follicles, (b) primitive and small follicles storing colloid, and (c) small follicles without colloid storage. The immature follicular cells in the C-cell complexes and large C-cell groups express immunoreactivity for vimentin in addition to 19S-thyroglobulin. When the follicular cells stored considerable amounts of colloid in the follicular lumina, vimentin immunoreactivity disappeared from them. Typical thyroid follicles showed no immunoreactivity for vimentin. In dog fetuses in which follicular cells were arranged in cell clusters or were vigorously forming follicles, no immunoreactivity for vimentin was observed in the thyroid parenchyma. These results indicate that vimentin is specifically expressed in the immature follicular cells derived from the ultimobranchial anlage. The vimentin filaments may participate in increased cellular activities in the cells, i.e., thyroglobulin synthesis and folliculogenesis.

Distribution of C cells in the thyroid gland with pyriform sinus fistula

A pyriform sinus fistula can cause acute thyroiditis or recurrent infection in the neck. This fistula is believed to be a remnant of the branchial apparatus, although its origin has yet to be pinpointed. The spatial distribution of C cells in the thyroid gland was mapped by immunohistologic method in four patients with a pyriform sinus fistula. The C cells were identified immunohistologically with anticalcitonin antibody. The stained calcitonin-positive cells also crossreacted with the antibodies to carcinoembryonic antigen, chromogranin A, and neuron-specific enolase. The C cells were mainly distributed near the end of the fistula, and in three patients their concentration per unit volume of thyroid tissue was found to be inversely proportional to the distance from the end of the fistulas. Comparison of distant locations of the left-sided thyroid lobe in patients and the same region in control subjects showed a similar number of C cells. Thus this limited distribution of C cells suggested that the pyriform sinus fistula was either a remnant of the ultimobranchial body, the result of disturbed migration of the C cell in the fetus, or both.

Distribution of C cells in the thyroid gland with pyriform sinus fistula

A pyriform sinus fistula can cause acute thyroiditis or recurrent infection in the neck. This fistula is believed to be a remnant of the branchial apparatus, although its origin has yet to be pinpointed. The spatial distribution of C cells in the thyroid gland was mapped by immunohistologic method in four patients with a pyriform sinus fistula. The C cells were identified immunohistologically with anticalcitonin antibody. The stained calcitonin-positive cells also crossreacted with the antibodies to carcinoembryonic antigen, chromogranin A, and neuron-specific enolase. The C cells were mainly distributed near the end of the fistula, and in three patients their concentration per unit volume of thyroid tissue was found to be inversely proportional to the distance from the end of the fistulas. Comparison of distant locations of the left-sided thyroid lobe in patients and the same region in control subjects showed a similar number of C cells. Thus this limited distribution of C cells suggested that the pyriform sinus fistula was either a remnant of the ultimobranchial body, the result of disturbed migration of the C cell in the fetus, or both. (OTOLARYNGOL HEAD NECK SURG 1995;112:268-73.)

C-cell hyperplasia associated with chronic lymphocytic thyroiditis: A retrospective quantitative study of 112 cases

Since the first description by Wolfe et al of C-cell hyperplasia (CCH) in asymptomatic relatives of patients suffering from a medullary thyroid carcinoma (MTC), several investigators have described CCH associated with a chronic lymphocytic thyroiditis (CLT) not within the context of MTC or multiple endocrine neoplasia (MEN). We report the study of C-cell density in 112 cases of CLT on retrospective surgical material to determine the frequency of the association between CCH and CLT. The cases of CLT were compared with 19 normal thyroid glands obtained at necropsy. C cells, immunoreactive with a polyclonal anti-calcitonin (CT) antibody, were counted at high magnification (×400) and the number of low-power magnification (×100) microscopic fields (LPFs) containing at least 50 C cells per slide was assessed. Image analysis was performed to determine the C-cell density expressed in number of C cells/cm 2. C-cell hyperplasia was defined by the following criteria: C-cell density > 40 cells/cm 2 and the presence of at least three LPFs containing more than 50 C cells. Twenty percent of the cases of CLT showed a CCH thus defined, and four of them had an elevated serum CT level. Statistical analysis showed no clinical or biological correlation with the presence of CCH. However, the frequency of CCH was higher if a follicular cell carcinoma was associated with CLT. This study confirms a pathological association between CCH and CLT, provides new criteria for the definition of CCH on surgical pathology material, and reports four cases with an elevated serum CT level not within the context of MTC or MEN.

Small Cell Carcinoma of the Uterine Cervix Showing Cushing's Syndrome Caused by Ectopic Adrenocorticotropin Hormone Production

A uterine cervical cancer is reported in a woman who developed Cushing's syndrome. The tumor measured 1.3 x 0.7 cm, and was a pure small cell carcinoma, identical to that in the lung. The primary tumor cells showed argyrophilia with Grimelius staining and reacted positively to the anti-chromogranin antibody. Clinically, the neoplasm behaved in an aggressive manner in spite of adjuvant chemotherapy and radiotherapy, and the patient died of widespread metastasis. Cushing's syndrome was noted after the occurrence of liver metastasis with an elevation of the serum adrenocorticotropin hormone (ACTH) level. At autopsy, metastatic tumor cells from the liver reacted immunohistochemically positively not only to anti-ACTH but also to antichromogranin, anti-gastrin and anti-calcitonin antibodies. This is the first report of an immunohistochemical analysis of, and comparison of primary and metastatic sites in cervical carcinoma showing Cushing's syndrome.

An Immunohistochemical Study of Calcitonin–containing Cells in Benign and Malignant Thyroid Lesions

Parafollicular cells (C-cells) in benign and malignant thyroid lesions were studied immunohistochemically with a polyclonal anti-calcitonin (CT) antibody. The C-cells were seen most frequently in the middle third of the lateral lobes in the thyroid gland of normal individuals and patients with Graves' disease and chronic thyroiditis, although in the latter the number of such cells was significantly decreased (p less than 0.05). In adenomatous goiter, C-cells were present in nodular lesions from an early stage of nodule development (frequency about 19%), whereas in the later stage these cells were rarely observed inside type 1 nodules, which were generally characterized by an admixture of follicles with considerably different sizes. However, C-cells were not observed inside type 2 nodules, which were composed of similar-sized follicles, or in the parenchyma of 56 cases of benign and malignant thyroid tumors. These findings suggest that since C-cells are present in nodular lesions, the histogenesis of adenomatous goiter is quite different from that of follicular adenoma; thyroid neoplasms generally contain no C-cells in the parenchyma.

Voltage‐gated sodium and potassium currents and their variation in calcitonin‐secreting cells of the chick.

1. The electrical properties of dissociated ultimobranchial cells from chick embryos (18-20 days after fertilization) were studied using whole-cell patch electrodes. Antibodies for immunohistological identification of calcitonin-secreting cells in the preparation were obtained by immunizing rabbits with a conjugated analogue of eel calcitonin. 2. In a proportion of cells, spike-like action potentials were generated in response to depolarization when cells were immersed in standard saline containing 140 mM-Na+ but no Ca2+. When the membrane potential was shifted from a holding potential (-83 - -103 mV) to a test depolarization (-50 mV or more positive) under voltage-clamp conditions, a transient inward current was produced which was followed by a slowly developing outward current. 3. The inward current was identified as a Na+-carried current, since (1) the kinetics of the current seemed fast and the amplitude consistently depended on the holding potential, (2) replacement of external Na+ with choline ions reversibly abolished the current, and (3) external application of tetrodotoxin (1 microM) abolished the current completely. The cells in which inward currents were detected showed intense to intermediate degrees of staining with anti-calcitonin antibodies. 4. In some other cells, no regenerative potentials were evoked even with intense depolarization, but a delayed decrease in membrane depolarization during the current pulse was observed. Voltage-clamp experiments in these cells revealed the existence of slowly developing outward currents, and the cells showed an intermediate degree of antibody staining. 5. The outward currents in both types of cells were selectively diminished in the presence of K+ channel blockers such as tetraethylammonium (1-10 mM) or 4-aminopyridine (1 mM). When the pipette contained 120 mM-CsCl, none of the dissociated cells exhibited any appreciable outward currents. Thus, the outward currents were most likely to be membrane potential-dependent K+ currents. The potential dependency of activation and inactivation of the currents were consistent with those of delayed K+ rectifier. 6. In the remaining cells, only passive responses of membrane potentials were observed with current injection. No discernible voltage-dependent inward or outward currents were detected under voltage-clamp conditions. Although these cells had a similar appearance to the two types of cells previously mentioned under phase-contrast microscopy, none of them showed significant antibody staining. These cells were presumed to represent non-secretory or supporting cells within the gland.(ABSTRACT TRUNCATED AT 400 WORDS)

Homeostatic role of anticalcitonin antibodies in calcium metabolism in experimental diabetes mellitus

In experimental alloxan diabetes marked hypocalcemia and hyperglycemia are observed in the blood of rats during the acute period of the disease (from 1.5 to 2 months). After 2.5-3.5 months of diabetes calcium level reaches the upper normal limit. Simultaneously, antibodies to calcitonin appear. It is quite likely that excessive calcitonin secretion, leading to hypocalcemia, decreased insulin secretion and hyperglycemia take place in the acute stage of diabetes. When diabetes persists the antibodies to calcitonin which reveal a homeostatic reaction blocking the excess of calcitonin appear. This normalizes the level of calcium in the blood and prevents the further increase in the blood sugar level.

Immunocytochemical study of Wulzen's cone of the bovine pituitary.

The Wulzen's cone of the bovine adenohypophysis presents a variable development and general arrangement. It is joined to the pars intermedia with no intervening connective tissue. It is covered by a single layer of cubical cell epithelium on the side of the hypophysial cleft. Immunofluorescence reveals the presence of different glandular cell types. The most abundant cells are those demonstrated by an anti-oPRL antibody and are either isolated or clustered. Other cells react with anti-hGH, anti-bLH, anti-beta aLH or anti-beta hTSH antibodies. Some cells react simultaneously with anti-beta MSH, anti-1-24ACTH, anti-17-39ACTH, anti-beta LPH and anti-beta endorphin antibodies. Cell types other than the numerous prolactin cells appear only as isolated elements. We did not observe cells reacting with anti-leu-enkephalin, anti-met-enkephalin or anti-calcitonin antibodies either in the Wulzen's cone or in the pars distalis or pars intermedia.

Biological and immunological properties of tritiated salmon calcitonin

Tritiated salmon calcitonin was prepared by methylation of the free amino groups using tritiated sodium borohydride as precursor. Specific radioactivity was measured in competitive inhibition studies with specific anticalcitonin antibodies or tubular membranes as binding sites for calcitonin. The value observed, approx. 4 Ci/mmol, corresponded to methylation of one third of the available N-H bonds. Tritiated calcitonin prepared in this way retained full biological activity as assessed in vitro by stimulation of adenylate cyclase and in vivo by rat bioassay. Tritiated calcitonin specifically bound to isolated renal cells and nonspecific binding did not exceed 10% of total binding. Equilibrium was obtained after 15 min incubation. The hormonereceptor complex could be dissociated in the presence of an excess of unlabelled calcitonin. This data shows that tritiated calcitonin can be used in metabolic and receptor studies.