Cleaved caspase-3 expression is a potential prognostic factor for endometrial cancer with positive peritoneal cytology.

INTRODUCTION: Positive peritoneal cytology (PPC) in endometrial cancer remains a controversial topic. Cleaved caspase-3 (CC3) and Ki-67 are excellent markers of apoptotic and proliferating cells, respectively. The objective of this study was to determine the significance of CC3 and Ki-67 expression in peritoneal cytology samples as prognostic factors for endometrial cancer with PPC. METHODS: Sixty endometrial cancer specimens with PPC alone were divided into 51 endometrioid tumours (43 endometrioid carcinomas and eight carcinomas with squamous differentiation) and nine non-endometrioid tumours (two serous carcinomas, three clear cell carcinomas and four carcinosarcomas). CC3 and Ki-67 expression in peritoneal cytology samples were immunocytochemically assessed and correlated with disease-free survival (DFS) and overall survival (OS). RESULTS: Expression levels of CC3 and Ki-67 were not associated with any clinicopathological parameter. Patients with non-endometrioid tumours had significantly shorter DFS (P = .001) and OS (P = .001). Low CC3 expression (CC3Low ) was significantly associated with shorter OS (P = .02), but not DFS (P = .13). Multivariate analysis showed that non-endometrioid histology and CC3Low were independent prognostic factors. However, Ki-67 expression was not associated with survival. When endometrioid and non-endometrioid tumours were assessed separately, CC3Low was significantly associated with shorter DFS (P = .002) and OS (P = .002) in patients with non-endometrioid tumours. CONCLUSIONS: Our results suggest that CC3Low in peritoneal cytology samples is a poor prognostic factor in patients with endometrial cancers, especially non-endometrioid tumours. Immunocytochemical analysis of CC3 expression could potentially facilitate identification of patients with high-risk endometrial cancer with PPC.

Efficacy and safety of osteoporosis medications in a rat model of late-stage chronic kidney disease accompanied by secondary hyperparathyroidism and hyperphosphatemia.

This study showed that bisphosphonate was safe and effective for the treatment of bone disorders in stage 4 chronic kidney disease (CKD) rats. Intermittent teriparatide therapy showed an anabolic action on bone even under secondary hyperparathyroidism conditions without having an adverse effect on mineral metabolism in late-stage CKD. INTRODUCTION: Patients with late-stage CKD are at high risk for fragility fractures. However, there are no consensus on the efficacy and safety of osteoporosis medications for patients with late-stage CKD. In the present study, we aimed to examine the efficacy and safety of alendronate (ALN) and teriparatide (TPD) for treating bone disorder in late-stage CKD with pre-existing secondary hyperparathyroidism using a rat model of CKD. METHODS: Male 10-week-old Sprague-Dawley rats were subjected to a 5/6 nephrectomy or sham surgery and randomized into the following four groups: sham, vehicle (saline subcutaneous (sc) daily), ALN (50 µg/kg sc daily), and TPD (40 µg/kg sc daily). Medications commenced at 24 weeks of age and continued for 4 weeks. Micro-computed tomography, histological analysis, infrared spectroscopic imaging, and serum assays were performed. RESULTS: Nephrectomized rats developed hyperphosphatemia, secondary hyperparathyroidism (SHPT), and high creatinine, equivalent to CKD stage 4 in humans. ALN suppressed the bone turnover and increased the degree of mineralization in cortical bone, resulting in an improvement in the mechanical properties. TPD further increased the bone turnover and significantly increased the degree of mineralization, micro-geometry, and bone volume, resulting in a significant improvement in the mechanical properties. Both ALN and TPD had no adverse effect on renal function and mineral metabolism. CONCLUSIONS: BP is safe and effective for the treatment of bone disorders in stage 4 CKD rats. Intermittent TPD therapy showed an anabolic action on bone even under SHPT conditions without having an adverse effect on mineral metabolism in late-stage CKD.

Autoimmune arthritis deteriorates bone quantity and quality of periarticular bone in a mouse model of rheumatoid arthritis.

This study showed that autoimmune arthritis induces especially severe osteoporosis in the periarticular region adjacent to inflamed joints, suggesting that arthritis increases the fragility fracture risk near inflamed joints, which is frequently observed in patients with RA. INTRODUCTION: Periarticular osteoporosis near inflamed joints is a hallmark of early rheumatoid arthritis (RA). Here we show that rheumatic inflammation deteriorates the bone quality and bone quantity of periarticular bone, thereby decreasing bone strength and toughness in a mouse model of RA. METHODS: Female BALB/c mice and SKG mice, a mutant mouse model of autoimmune arthritis on the BALB/c background, were used. At 12 weeks of age, BALB/c mice underwent either Sham surgery or bilateral ovariectomy (OVX), and SKG mice underwent intraperitoneal injection of mannan to induce arthritis. Eight weeks later, the mice were killed and the femurs and tibias were subjected to micro-computed tomography, Fourier transform infrared (FTIR) spectroscopic imaging, X-ray diffraction, histology, and mechanical testing. RESULTS: SKG mice developed significant trabecular bone loss in both the distal metaphysis of the femur and the lumbar vertebral body, but the extent of the bone loss was more severe in the distal metaphysis. Neither SKG nor OVX mice exhibited changes in the geometry and matrix properties of the diaphysis of the femur, whereas SKG mice, but not OVX mice, did exhibit changes in these properties in the distal metaphysis of the femur. Bone strength and fracture toughness of the distal metaphysis of the tibia adjacent to the inflamed ankle joint were significantly decreased in SKG mice. CONCLUSIONS: Autoimmune arthritis induces periarticular osteoporosis, characterized by deterioration of cortical bone geometry and quality as well as by trabecular bone loss, leading to severe bone fragility in periarticular bone adjacent to inflamed joints.

Interaction of hydrophobic components in female urine before and after childbirth with P-glycoprotein in vitro.

The first urine in the morning (total 15 samples) and whole day urine (total 4 days, 17 samples) were collected from a young healthy woman during the pregnancy and lactation period, to examine the possible interactions of urine components (methanol extracts) with P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs). The interaction was evaluated by measuring the intracellular accumulation of rhodamine123, a P-gp substrate, in LLC-GA5-COL150 cells, or calcein, an MRP substrate, in Caco-2 cells in the absence and presence of urine components. Four first urine samples out of 12 collected before childbirth and one sample out of three collected after childbirth suppressed P-gp function significantly. The effect of pregnancy and lactation on P-gp inhibitory potencies of urine components was not observed. The whole day urine samples showed a clear circadian rhythm, in which three first urine samples in the morning out of four showed greater P-gp inhibitory potencies than other daytime samples. Interaction of urine components with MRPs was not detected. In conclusion, the concentration of endogenous P-gp inhibitor(s) was higher in the first urine in the morning, showing a clear circadian rhythm. Normal pregnancy and lactation appeared not to significantly affect the P-gp inhibitory potencies of urine components.

KRAS and BRAF mutations are rare and related to DNA mismatch repair deficiency in gastric cancer from the East and the West: results from a large international multicentre study.

BACKGROUND: Inhibitors of the epidermal growth factor (EGFR) signaling pathway have a major role in the treatment of KRAS wild-type colorectal cancer patients. The EGFR pathway has been shown to be activated in gastric cancer (GC). However, published data on KRAS and BRAF mutation status is limited in GC and has not been compared between GC from different geographic regions. METHODS: The prevalence of KRAS and BRAF mutations was established in 712 GC: 278 GC from the United Kingdom, 230 GC from Japan and 204 GC from Singapore. The relationship between KRAS/BRAF mutation status, DNA mismatch repair (MMR) status, clinicopathological variables and overall survival was analysed. RESULTS: Overall, 30 (4.2%) GC carried a KRAS mutation. In total, 5.8% of the UK GC, 4% of Japan GC and 1.5% of Singapore GC were KRAS mutant. KRAS mutant GC had fewer lymph node metastases in the UK cohort (P=0.005) and were more frequent in elderly patients in the Japan cohort (P=0.034). KRAS mutations were more frequent in MMR-deficient GC in the UK and the Japanese cohort (P<0.05). A BRAF mutation was only detected in a single Japanese GC. CONCLUSIONS: This large multicentre study demonstrated that KRAS mutations and DNA MMR deficiency have a role in a small subgroup of GC irrespective of country of origin, suggesting that this subgroup of GC may have developed along a common pathway. Further studies need to establish whether concomitant mutations or amplifications of other EGFR signalling pathway genes may contribute to the activation of this pathway in GC.

Radiographic and pathological analysis of small lung adenocarcinoma using the new IASLC classification.

AIM: To analyse the correlation between computed tomography (CT) findings of small lung adenocarcinomas and the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification of Lung Adenocarcinoma. MATERIALS AND METHODS: A retrospective review of 300 lung adenocarcinoma lesions (size ≤20 mm) after surgical resection in 295 consecutive patients was performed. Tumours were defined as air-containing type if the ratio of the maximum dimension of the tumour on mediastinal windows to the maximum dimension of the tumour on lung windows was ≤50%, and as solid-density type if the ratio was >50%. The incidence between CT findings (air bronchogram, vascular involvement, pleural tags, notches, and spiculation) and pathological findings were investigated. RESULTS: Of the 142 air-containing lesions, 114 were adenocarcinoma in situ (AIS), 28 were minimally invasive adenocarcinoma (MIA), and none of the lesions were invasive adenocarcinoma. Of the 158 solid-density lesions, 30 were AIS, 24 were MIA, and 104 were invasive adenocarcinoma. Notches and pleural tags were commonly observed in cases of invasive adenocarcinoma (p < 0.05). CONCLUSIONS: In the air-containing type of small lung adenocarcinomas, AIS and MIA were observed but no cases of invasive adenocarcinoma were found. The presence of notches and pleural tags were a significant factor in invasive adenocarcinoma.

Clinical outcome of endoscopic mucosal resection for esophageal squamous cell cancer invading muscularis mucosa and submucosal layer.

When a tumor invades the muscularis mucosa and submucosal layer (T1a-MM and T1b in Japan), esophageal squamous cell cancer poses 10-50% risk of lymph node metastasis. By this stage of esophageal cancer, surgery, although very invasive, is the standard radical therapy for the patients. Endoscopic mucosal resection (EMR) is the absolutely curable treatment for cancer in the superficial mucosal layer. Because of its minimal invasiveness, the indications of EMR may be expanded to include the treatment of T1a-MM and T1b esophageal carcinoma. To date, the clinical outcomes of EMR for T1a-MM and T1b patients have not been fully elucidated. Here, the retrospective analysis of the clinical outcomes is reported. Between January 1994 and December 2007, 247 patients underwent EMR at Kanagawa Cancer Center. Of these individuals, 44 patients with 44 lesions fulfilled the following criteria: (i) extended EMR treatment for clinical T1a-MM and T1b tumor; (ii) diagnosis of clinical N0M0; and (iii) follow up for at least 1 year, and negative vertical margin. These patients were reviewed for their clinical features and outcomes. Statistical analyses were performed by the Kaplan-Meier methods, the Chi-square test, and the Cox proportional hazard model. P-value of <0.05 was considered statistically significant. The data were analyzed in February 2009. Based on the informed consent and their general health conditions, 44 patients decided the following treatments immediately after the EMR: 2 underwent surgery, 1 underwent adjuvant chemotherapy, and 41 selected follow up without any additional therapy. Of the 41 patients, 20 selected this course by choice, 12 because of severe concurrent diseases, 2 because of poor performance status, and 7 because of other multiple primary cancers. Twelve patients died; two were cause specific (4.5%), eight from multiple primary cancers, one from severe concurrent diseases, and one from unknown causes. No critical complications were noted. Median follow-up time was 51 months (12-126). Five patients ultimately developed lymph node metastasis. One patient with adjuvant chemotherapy required surgery, and another was treated with chemotherapy whose subsequent death was cause specific. The other three patients received chemoradiotherapy and have not shown cause-specific death. Overall and cause-specific survival rates at 5 years were 67.3% and 91.8%, respectively. Among 41 patients treated by EMR alone, only one died from primary esophageal cancer (2.4%), and overall and cause-specific survival rates at 5 years were 75.6% and 97.6%, respectively. Multivariate analysis revealed that severe concurrent diseases including multiple primary cancers and the administration of 5-fluorouracil-based chemotherapy for multiple primary cancers significantly influenced survival (P= 0.025, hazard ratio [HR] 13.1 [95% confidence interval 1.5-114]) and (P= 0.037, HR 0.213 [95% confidence interval 0.05-0.914]), respectively. Eight and six patients developed metachronous esophageal squamous cell cancer and local recurrence, respectively. With the exception of one patient, they could be retreated endoscopically. EMR is a reasonable option for the patients with T1a-MM and T1b esophageal carcinoma without clinical metastasis, especially for the individuals with severe concurrent diseases. The prognostic factors for the benefit of EMR in such cases should be further examined.

Secretion of intelectin-1 from malignant pleural mesothelioma into pleural effusion.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare but fatal tumour. Although most MPM patients show pleural effusion at even the early stage, it is hard to diagnose as MPM at the early stage because a sensitive and reliable diagnostic marker for MPM has not been found in plasma or pleural effusion. METHODS: In this study, we investigated whether intelectin-1 was specifically contained in MPM cells and the pleural effusion of MPM patient by immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay. RESULTS: Malignant pleural mesothelioma cell lines, but not lung adenocarcinoma cell lines, secreted intelectin-1. In immunohistochemistry, epithelioid-type MPMs, but neither pleura-invading lung adenocarcinomas nor reactive mesothelial cells near the lung adenocarcinomas, were stained with anti-intelectin antibodies. Pleural effusion of MPM patients contained a higher concentration of intelectin-1 than that of lung cancer patients. CONCLUSION: These results suggest that detection of intelectin-1 may be useful for a differential diagnosis of epithelioid-type MPM in immunohistochemistry and that a high concentration of intelectin-1 in pleural effusion can be used as a new marker for clinical diagnosis of MPM.

Prognosis and morphometrical features of non-bronchioloalveolar cell adenocarcinoma: an assessment of the non-alveolar replacing area and high grade atypical area.

AIM: It has become obvious that the prognosis of bronchioloalveolar cell carcinoma (BAC) in small peripheral adenocarcinoma of the lung is good, but most cases actually treated as pulmonary adenocarcinoma in hospitals tend to be non-bronchioloalveolar cell carcinoma (non-BAC). The prognoses of non-BAC are greatly varied. We studied the relationships between the morphometrical features and the prognoses of non-BAC. METHODS: In total, 69 cases of non-BAC measuring

Immunochemical and immunohistochemical studies on the 27 S iodoprotein of dog thyroid with reference to thyroglobulin-like reaction of the parafollicular cells.

Our earlier finding that the thyroglobulin-like material responsible for the immunoreaction of parafollicular cells obtained in peak I fraction of Bio-Gel A-5m was followed up in the present study by an investigation of the immunochemical and immunohistochemical reactions of 27 S iodoprotein which was the most prominent material in the peak I fraction. The antibody was raised against completely purified 27 S iodoprotein which was obtained as follows: Thyroglobulin was extracted from dog thyroids and chromatographed initially on Bio-Gel A-5m and then on Bio-Gel A-50m. The area of 27 S migrated as a single bank on polyacrylamide gel slab electrophoresis. This was cut and eluted. Anti-27 S antiserum showed the same immunochemical patterns to 27 S and 19 S as anti-19 S antiserum with three different immunochemical methods: double diffusion test, one dimensional and two dimensional immunoelectrophoresis. The immunoperoxidase reactions of the anti-27 S antiserum and anti-19 S antiserum were restricted to follicular cells and luminal colloids. No reaction of the parafollicular cells was obtained by these antisera. Thus, 27 S iodoprotein shared common immunochemical and immunohistochemical properties with 19 S thyroglobulin. It was concluded that 27 S iodoprotein was not responsible for the thyroglobulin-like reaction of the parafollicular cells.

Molecular cloning and characterization of a novel human STE20-like kinase, hSLK.

We have cloned a human counterpart to a guinea pig STE20-like kinase cDNA, designated human SLK (hSLK), from a human lung carcinomatous cell line A549 cDNA library. hSLK cDNA encodes a novel 1204 amino acid serine/threonine kinase for which the kinase domain located at the N-terminus shares considerable homology to that of the STE20-like kinase family. The C-terminal domain of hSLK includes both the coiled-coil structure and four Pro/Glu/Ser/Thr-rich (PEST) sequences, but not the GTPase-binding domain (GBD) that is characteristic of the p21-activated kinase (PAK) family, polyproline consensus binding sites, or the Leu-rich domain seen in the group I germinal center kinases (GCKs). Northern blot analysis indicated that hSLK was ubiquitously expressed. hSLK overexpressed in COS-7 cells phosphorylates itself as well as myelin basic protein used as a substrate. On the other hand, hSLK cannot activate any of the three well-characterized mitogen-activated protein kinase MAPK (ERK, JNK/SAPK and p38) pathways. Moreover, hSLK kinase activity is not upregulated by constitutive active forms of GTPases (RasV12, RacV12 and Cdc42V12). These structural and functional properties indicate that hSLK should be considered to be a new member of group II GCKs.

Pilomatrix carcinoma of the axilla: CT and MRI features.

Pilomatrix carcinoma, a rare malignant soft tissue tumour, is the malignant variant of pilomatricoma. We report a case of pilomatrix carcinoma of the axilla. CT demonstrated a well-circumscribed, sand-like calcified mass. MRI showed diffusely inhomogeneous, mixed signal intensities with inhomogeneous enhancements. The MRI findings were different from those previously reported for pilomatricoma.

Neurotoxicity of advanced glycation end-products for cultured cortical neurons.

The Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in aging. AGEs are believed also to contribute to the pathology of Alzheimer disease (AD) and other neurodegenerative processes. Incubation of cortical neurons with 5 immunochemically distinct AGEs, designated AGEs-1 to -5, produced a dose-dependent increase in neuronal cell-death, as assessed by MTT assay, Trypan blue and Hoechst 33258 staining. The structural epitope designated AGE-2 was found to have the greatest cytopathic effect and the neurotoxicity of AGE-2 was neutralized by the addition of an anti-AGE-2-specific antibody, but not by other types of anti-AGE antibodies. Distinct classes of AGE structures also have been established to circulate in the blood of individuals with diabetes mellitus and end-stage renal disease treated by hemodialysis (DM-HD). We fractionated serum from normal control and DM-HD patients by gel filtration and identified 2 fractions that contained AGE epitopes-1 to -5 and as well as the defined AGE structure carboxymethyllysine (CML). The addition of these 2 fractions led to the death of cultured neuronal cells and this cytotoxic effect was completely prevented by the addition of the anti-AGE-2-specific antibody. We propose that the structural epitope AGE-2 is an important toxic moiety for neuronal cells.

Increased level of immunoreactive neuron-specific enolase in thyroid C cells from dogs and guinea pigs after chronic hypercalcemia.

Immunocytochemical localization of neuron-specific enolase (NSE) in thyroid C cells was investigated in various mammalian species. In bovine thyroid glands most of the C cells were weakly immunoreactive to anti-NSE antiserum. In other mammalian species, including dogs, guinea pigs, rabbits, cats, pigs, rats, hamsters, mice, and monkeys, some C cells or only a few C cells were weakly immunoreactive to the antiserum. It seems that normal C cells contain NSE in small amounts only or are devoid of NSE. After chronically induced hypercalcemia, C cells revealed hypertrophic and hyperplastic features. Whereas immunoreactive calcitonin was markedly decreased, marked increase of immunoreactive NSE was observed in C cells of both dogs and guinea pigs; almost all C cells were filled with reaction product for NSE. After administration of ethylenethiourea for a period of 3-8 months, C cells revealed a marked decrease of secretory granules and appearance of vesicular inclusions of various sizes, which were immunoreactive to the calcitonin antiserum, indicating a disturbance of calcitonin synthesis. No immunoreactivity for NSE was observed in C cells from dogs and guinea pigs so treated. In rabbits showing hypocalcemic tetany, calcitonin immunoreactivity was very intense and NSE immunoreactivity was faint to negative in the C cells. Thus, the level of NSE in C cells was clearly connected with the functional activity of the cells.

Somatostatin-like immunoreactivity in mammalian thyroid glands: contents and partial characterization.

Somatostatin (SRIF)-like immunoreactivity (SLI) in the thyroid glands of human and several animal species were compared, and the SLI peptides were characterized chromatographically and immunologically. All specimens were extracted with 2 M acetic acid, and the SLI content determined by RIA. The SLI concentrations in guinea pigs [34.3 +/- (SE) 4.8 ng/mg protein] and rabbits (9.4 +/- 0.8 ng/mg protein) were much greater than those in other mammals: dogs, rats, mice, and humans. On gel filtration of extracts of the guinea pig, rabbit and dog thyroids, the major peak of SLI (1.6 K SLI) coeluted with synthetic SRIF-14 (S-14). Two other forms of SLI ("big" SLI and 3 K SLI) were also detected, although their relative proportions to total SLI were small (2.3 to 8.2%). The 3 K SLI and 1.6 K SLI from guinea pig and rabbit thyroids contained peptides coeluting with synthetic SRIF-28 (S-28) and S-14, respectively, on reverse-phase high performance liquid chromatography. The dilution curves of the two molecular forms of SLI, i.e. 3 K SLI and 1.6 K SLI, were parallel to the displacement curves of S-28 and S-14 in the SRIF RIA. It is concluded 1) that the thyroid contents of SLI varied greatly from species to species, with the highest content being found in guinea pig thyroids; 2) that in guinea pigs, rabbits, and dogs, the predominant form of thyroid SLI is 1.6 K SLI; and 3) that the 3 K SLI and 1.6 K SLI peptides from guinea pig and rabbit thyroids are immunologically and chromatographically indistinguishable from S-28 and S-14, respectively.

Electron microscopic study on the development of the carotid body and glomus cell groups distributed in the wall of the common carotid artery and its branches in the chicken.

Development of the carotid body and the glomus cell groups in the wall of the common carotid artery and its branches was studied in chickens at various developmental stages by electron microscopy. At 8 days of incubation, the carotid body anlage consisted of mesenchyme-like cells, whereas the clusters of epithelial cells, which occasionally contained a few dense-cored vesicles and were accompanied by unmyelinated nerve fibers, were located in the region surrounding the carotid body anlage and in the wall of the common carotid artery. Subsequently, the granule-containing cells together with nerve fibers were detected in the periphery of the carotid body anlage. At 12 days of incubation, a large number of granule-containing cells (glomus cells) were dispersed throughout the carotid body parenchyma and were also widely distributed along the common carotid artery and its branches. The cells frequently extended long cytoplasmic processes that made contact with other glomus cells and nerve fibers. Synaptic junctions which showed desmosome-like thickening of pre- and postsynaptic membranes and accumulations of small clear vesicles (around 50 nm in diameter) were first detected along the contact between the long axons and glomus cells at 12 days of incubation. In the wall of the common carotid artery, interdigitations between the cytoplasmic processes of glomus cells and smooth muscle cells began to form. Sustentacular cells investing partly the glomus cells were also discerned both in the carotid body and around the arteries at this stage. At 14 days of incubation, the glomus cells expressed most of the characteristics of the mature cells, and the synaptic junctions displaying afferent morphology appeared; the secretory granules of glomus cells were accumulated near and attached to the desmosome-like thickening of apposed membranes.

Evidence to support the distal vagal ganglion as the origin of C cells of the ultimobranchial gland in the chick.

Formation and development of the ultimobranchial anlage were studied in chicken embryos by immunohistochemistry with the antibodies to class III beta-tubulin, TuJ1, human leukemic cell-line (HNK-1), and protein gene product (PGP) 9.5, all of which recognized neurons. Medial to the fourth aortic arch, the ultimobranchial anlage was formed by the extension of the ventral portion of the fourth pharyngeal pouch at 4.5 days of incubation. At 5 days of incubation, TuJ1-immunoreactive cells with long cell processes began to enter the ultimobranchial anlage, which displayed a follicle structure. At 6 days of incubation, numerous neuronal cells that were continuous with the distal vagal ganglion (nodose ganglion) and expressed immunoreactivity for TuJ1, HNK-1, and PGP 9.5 were found to be in direct contact with the peripheral portion of ultimobranchial anlage. The TuJ1 antibody reacted only with the neuronal cells, whereas the HNK-1 and PGP 9.5 antibodies reacted with both endodermal epithelial cells and the neuronal cells in the ultimobranchial anlage. Subsequently, the ultimobranchial anlage rapidly increased in size; the follicle wall was thickened and a central cavity disappeared. The TuJ1-immunoreactive cells were distributed throughout the ultimobranchial parenchyma in 7-day-old embryos. The neuronal cell streams from the distal vagal ganglion to the ultimobranchial anlage were still prominent at 8 days of incubation. Almost all parenchymal cells of the ultimobranchial glands were intensely immunoreactive for TuJ1, HNK-1, and PGP 9.5 between 10 and 16 days of incubation. These results indicate that the neuronal cells from the distal vagal ganglion enter into the ultimobranchial anlage and give rise to C cells, i.e., C cells differentiate along the neuronal lineage. During embryonic development, C cells of the chick ultimobranchial glands transiently express a number of neuronal properties.

Innervation of the serotonin-immunoreactive cells distributed in the wall of the common carotid artery and its branches in the chicken.

In the chicken, serotonin-immunoreactive cells were widely distributed not only in the carotid body but also in the wall of the common carotid artery and around each artery arising from the common carotid artery. Almost all of the serotonin cells in the wall of the common carotid artery were intensely immunoreactive to the neuropeptide Y, met- and leu-enkephalin antisera, whereas in the carotid body only a few cells were immunoreactive to these antisera. Innervation of the serotonin cells in and around arteries of chickens was investigated by immunohistochemistry and electron microscopy, in comparison with that of the carotid body. The serotonin cell groups in and around arteries, as well as the carotid body, received numerous peptidergic nerve fibers. Calcitonin gene-related peptide (CGRP)- and substance P-immunoreactive varicose nerve fibers were densely distributed, and somatostatin-immunoreactive fibers were moderately distributed in the serotonin cell groups. Galanin- and vasoactive intestinal peptide (VIP)-immunoreactive fibers were sparsely distributed in the cell groups. By electron microscopy, the serotonin cells in and around arteries were characterized by the presence of numerous dense-cored vesicles, 70-220 nm in diameter. The granule-containing cells were in close association with numerous axons. Naked axons regarded axon terminals were frequently apposed on the granular cells. The axon terminals were usually long and often partly invested the granular cells. Numerous synaptic junctions were detected along the contact between the granular cells and axon terminals. Most of the synaptic junctions showed afferent morphology; the secretory granules were accumulated near and attached to the asymmetrical membrane thickenings. Thus, the serotonin cells in and around arteries, like the carotid body, constitute chemoreceptive tissue.

Immunocytochemical localization and development of multiple kinds of neuropeptides and neuroendocrine proteins in the chick ultimobranchial gland.

The ultimobranchial gland is an endocrine organ consisting of C cell groups. In chickens, the glands are richly supplied by nerve fibers immunoreactive for neurofilaments. It was found by immunocytochemical staining that C cells of chick ultimobranchial glands showed immunoreactivities for multiple kinds of neuropeptides and neuroendocrine proteins in addition to calcitonin, i.e., calcitonin gene-related peptide (CGRP), somatostatin, neurotensin, chromogranin A, and tyrosine hydroxylase. Furthermore, enkephalin-immunoreactive cells that showed long cytoplasmic processes and large cell bodies, being distinct from the C cell feature, were detected. The densities of these cells per unit area of ultimobranchial gland were assessed using computer-assisted image analysis system; calcitonin cells were 42.9 +/- 10.0%; CGRP cells 26.9 +/- 5.6%; neurotensin cells 8.6 +/- 6.9%; somatostatin cells 3.1 +/- 1.4%; chromogranin A cells 11.8 +/- 1.8%; tyrosine hydroxylase cells 10.0 +/- 5.2%; enkephalin cells 2.9 +/- 1.3%. Dense distributions of peptidergic nerve fibers were also detected in chick ultimobranchial glands. Numerous varicose fibers immunoreactive for substance P were distributed in the close vicinity to C cell clusters and blood vessels. Enkephalin-immunoreactive fibers were also prominent around C cell clusters. Galanin-, vasoactive intestinal peptide (VIP)-, and tyrosine hydroxylase-immunoreactive fibers were distributed around blood vessels only. Subsequently, the ontogeny of these neuropeptides, neuroendocrine proteins, and peptidergic innervations was examined in chickens at various developmental stages. In 10-day-old embryos, weak to moderately intense immunoreactivity for calcitonin was already present in almost all C cells. Immunoreactivities for somatostatin, CGRP, and tyrosine hydroxylase began to appear at this age. At 12 days of incubation, substance P-immunoreactive fibers were first detected in the parenchyma of ultimobranchial glands. Considerable numbers of enkephalin-immunoreactive fibers and cells were also observed. At 14 days of incubation, the largest populations of somatostatin- and enkephalin-immunoreactive cells were attained; the densities of somatostatin- and enkephalin-immunoreactive cells per unit area were 21.2 +/- 3.2% and 12.9 +/- 3.1%, respectively. Substance P-immunoreactive fibers became numerous throughout the gland at this age. Thereafter, calcitonin-, CGRP-, tyrosine hydroxylase-immunoreactive cells progressively increased in number with embryonic age, whereas somatostatin- and enkephalin-immunoreactive cells started to decrease. Chromogranin A- and neurotensin-immunoreactive cells began to appear at 16 days and 18 days of incubation, respectively. Galanin-, VIP-, and tyrosine hydroxylase-immunoreactive fibers were inconspicuous during embryonic life.

Ultrastructural localization of neuropeptide Y and expression of its mRNA in the glomus cells distributed in the wall of the common carotid artery of the chicken.

In the chicken, glomus cells are widely distributed in the carotid body and in the wall of the common carotid artery and around its branches. The cells located in the wall of the common carotid artery express intense immunoreactivity for neuropeptide Y (NPY). They contain abundant dense-cored vesicles ranging from 70 to 220 nm in diameter. In this study, we examined ultrastructural localization of NPY in the glomus cells by using the postembedding immunogold method. Gold particles representing immunoreactivity for NPY were specifically localized on the dense-cored vesicles of the glomus cells. In addition, the localization of NPY mRNA in the glomus cells was examined by in situ hybridization with digoxigenin-labeled chicken NPY cRNA probe. A strong hybridization signal for NPY mRNA was detected in the glomus cells located in the wall of the common carotid artery. Few glomus cells of the carotid body, however, displayed labeling for NPY mRNA. Northern blot analysis with the chicken NPY exon 4 probe demonstrated that a single band for NPY mRNA was present in the poly (A) + RNA isolated from the common carotid artery where the glomus cells were distributed. Furthermore, the expression of NPY mRNA in the common carotid artery was confirmed by the reverse transcription-polymerase chain reaction. These results indicate that the chicken glomus cells are able to produce NPY but that the level of its translation varies according to the location of the cells.