[Immunocytochemical study of enterochromaffin cells in carcinoma of the large intestine].

In a series of 130 cases of adenocarcinoma of the large intestine, enterochromaffin (EC) cells were detected in 54 cases (41.5%) by immunocytochemistry with anti-chromogranin monoclonal antibody. Among the 54 cases, 30 were found positive for serotonin, 14 for somatostatin, 11 for glucagon, 5 for pancreatic polypeptide, and only one for gastrin. The cases with EC cells (++) or polypeptide positive cells exhibited higher grade of differentiation, earlier stage of tumour extension and higher survival rate than those without EC cells. A significant difference of the EC cell population pattern among different histological grades of the tumours and nonneoplastic mucosa was found. The proportion of hormone, especially polypeptide positive cells was the highest in the mucosa and lowest in the moderately poorly differentiated carcinomas. The incidence, methodology and clinicopathological significance of EC cells found in the tumours are discussed.

Secretin-cells of the mammalian intestine contain serotonin.

Various endocrine cells contain biogenic amines in addition to their peptide hormones. In the digestive tract, one of these amines is serotonin that is regularly present in enterochromaffin (EC-) cells. Previously, it has been assumed that other entero-endocrine cell types also contain this amine. Moreover, it was presumed that chromogranin A, an acidic glycoprotein, is involved in storage mechanisms for biogenic amines in endocrine cells. Using immunohistochemical techniques, we now exemplarily investigated cholecystokinin (CCK-) and secretin (S-) cells of five adult mammalian species for their content of serotonin and of chromogranin A. In all mammalian species, CCK-cells were devoid of serotonin but contained chromogranin A immunoreactivity of varying densities. In contrast, S-cells of all mammals were immunoreactive for serotonin; however, immunoreactivities for this biogenic monoamine were heterogeneous and varied from dense to faint or lacking immunostainings. Likewise, immunoreactivities for chromogranin A in S-cells showed inter-species and inter-cellular heterogeneities. S-cells containing serotonin were simultaneously immunoreactive for chromogranin A and the density of immunoreactivities for both were correlated in given S-cells. Based on mutual relationships of chromogranin A and serotonin immunoreactivities, we assume that chromograinin A is virtually a prerequisite for the S-cells' content of serotonin and that this protein participates in storage mechanisms for biogenic amines in endocrine cells. S-cells have now to be added to the family of amine-storing endocrine cells. Basically, serotonin-storing endocrine cells in the digestive tract cannot be simply regarded as enterochromaffin (EC-) cells any longer; the current nomenclature and classification of entero-endocrine cells should be reviewed in this respect.

Papillary adenoma of the stomach. Pathologic and immunohistochemical study.

A total of 13 gastric papillary adenomas composed of 8 papillary and 5 papillotubular adenomas were examined pathologically and immunohistochemically. They showed a dome-like or pedunculated appearance and were located at the antrum, except for one adenoma. Histologically, the adenoma cells showed atypia in varying degree and focal adenocarcinoma was noted in seven lesions. The number of goblet cells was apparently smaller in the papillary than in the tubular portion. Lysozyme was present at the supranuclear region in most papillary adenoma cells, whereas it was concentrated in Paneth's granules in tubular adenoma cells. No difference was found in the distribution and frequency of carcinoembryonic antigen, secretory component, and carbohydrate antigen CA 19-9 between papillary and tubular adenomas. Paucity of endocrine cells also characterized gastric papillary adenoma. Different phenotypic expressions might reflect the difference in histogenesis between papillary adenoma and tubular adenoma.

Time course of expression of neuropeptide Y, calcitonin gene-related peptide, and NADPH diaphorase activity in neurons of the developing murine bowel and the appearance of 5-hydroxytryptamine in mucosal enterochromaffin cells.

Serotoninergic and cholinergic neurons are known to appear earlier in the ontogeny (day E12) of the murine gut than those containing substance P or vasoactive intestinal peptide (day E14). It has also been demonstrated that proliferating neural precursors coexist with mature neurons in developing enteric ganglia. These observations have led to the hypotheses that peptidergic neurons develop later than those that utilize small molecule neurotransmitters and that the activity of early developing neurons may affect the phenotypic expression of coexisting neuroblasts. As a partial test of these hypotheses we studied the phenotypic expression of neurons recognized by antisera to neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP), and of those visualized by the histochemical demonstration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity. NADPH diaphorase activity, which is coexpressed with NPY immunoreactivity in all submucosal and many myenteric neurons, was first found on day E11 in clusters of cells in the dorsal mesogastrium. These cells also expressed neurofilament reactivity and thus were developing along a neuronal lineage. Enteric neurons that expressed NADPH diaphorase activity were visualized in the stomach one day later, on day E12. At this time, NADPH diaphorase-containing cells could no longer be demonstrated in the dorsal mesogastrium. NPY immunoreactivity first appeared in the wall of the bowel on day E12, when it was seen in cells in the presumptive stomach. By day E13, the entire length of the bowel contained NPY-immunoreactive neurons. Cells that displayed NADPH diaphorase activity were found at this time at both ends of the alimentary tract, but did not appear in the ileum until day E18. In contrast, CGRP immunoreactivity could not be detected anywhere in the gut until day E17, but by day E18 all regions of the bowel contained CGRP-immunoreactive neurons. Endogenous 5-HT was first detected at day E16 in mucosal epithelial cells in all segments of the gut except the stomach, where it appeared at day E18. The NPY/NADPH diaphorase set of neurons thus develop before the acquisition of a detectable level of endogenous 5-HT or enteric neural 5-HT receptors (which arise in the foregut at day E14). These observations demonstrate that enteric neurons that express small molecule neurotransmitters do not necessarily develop earlier than peptidergic neurons as a class; however, various types of enteric neurons do appear in a sequential order.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemistry of chromogranin A and B, and secretogranin II in the canine endocrine pancreas.

The chromogranins are large acidic proteins contained in secretion granules of adrenal medullary cells. These proteins are also presumed to be regular constituents of other endocrine cells, e.g., pancreatic endocrine cells. In our previous immunohistochemical studies performed in the endocrine pancreas of 10 mammalian species, only canine endocrine cells lacked any immunoreactivity for chromogranin A. Therefore, this problem was reinvestigated in the present study. Antisera against bovine and rat chromogranin A, B, and C (= secretogranin II) were applied under various conditions of the immunohistochemical protocol. Upon meeting certain requirements, chromogranin-immunoreactivities were found also in canine pancreatic B- (insulin), PP- (pancreatic polypeptide), and EC- (enterochromaffin) cells. The immunohistochemical data suggest that canine chromogranins partially differ structurally or immunologically from bovine chromogranins and completely from rat chromogranins. In addition, the present findings confirm our previous findings about both interspecies differences in the cellular localization of chromogranins in the endocrine pancreas and the peculiar localization of secretogranin II to pancreatic PP-cells. Finally, the present methodological studies have shown that even the buffer used in the immunohistochemical protocol may be decisive for a false-positive or false-negative immunostaining.

An immunohistochemical study of neuroendocrine cells in gynecologic tumors.

Various gynecologic tumors with argyrophilia were studied immunohistochemically for chromogranin using two antibodies, antichromogranin and antineuroendocrine. Of seven small cell carcinomas of the cervix, four were immunoreactive with antichromogranin and seven with antineuroendocrine. Argyrophil cells of six cervical adenocarcinomas were all immunoractive with both antibodies. Type I argyrophil cells of 20 endometrial carcinomas were likewise stained positively. However, of the 30 endometrial carcinomas with type II argyrophil cells, 19 showed positive immunoreactivity for chromogranin and 22 for neuroendocrine. Of the ovarian tumors tested, argyrophil cells of 11 mucinous tumors, three carcinoid tumors, and the pancreatic tissue of a malignant mixed germ cell tumor were all chromogranin- and neuroendocrine-immunoreactive. Type I argyrophil cells of five endometrioid carcinomas of the ovary were also immunoreactive with both antibodies. Of the 13 endometrioid carcinomas with type II argyrophil cells, only four showed positive immunoreactivity for chromogranin and only five for neuroendocrine. In conclusion, both antichromogranin and antineuroendocrine detect the specific neuroendocrine markers in close association with argyrophilia in gynecologic tumors, the latter being more sensitive for small cell carcinoma of the cervix, and for type II argyrophil cells in adenocarcinoma of the endometrium and endometrioid carcinoma of the ovary.

Solid teratomas and mixed müllerian tumors of the ovary: a clinical, histological, and immunocytochemical comparative study.

The differential diagnosis between solid teratomas and malignant mixed Müllerian tumors (MMTs) can be difficult, because both tumours have an epithelial as well as a mesenchymal component. Sometimes an MMT is incorrectly diagnosed as a solid teratoma. This has prognostic and therapeutic consequences, because MMTs have a worse prognosis. In this study 20 solid ovarian teratomas and 15 MMTs of the ovary have been compared according to their clinical, histomorphological, and immunocytochemical characteristics. Teratomas occur in the younger age group and are characterized by the presence of argyrophil cells which are immunoreactive to a wide range of neurohormonal peptides and are in addition characterized by the presence of GFAP immunoreactive tissues (glial fibrillar acidic protein). MMTs occur in the older age group and are characterized by the presence of sarcomatous elements and by the absence of an organoid arrangement, neuroectoderm, skin, argyrophilia, and immunoreactivity to GFAP and neurofilament.

Calbindin immunoreactivity is a characteristic of enterochromaffin-like cells (ECL cells) of the human stomach.

Immunoreactivity for the calcium binding protein, calbindin D28k has been localized in enterochromaffin-like (ECL) cells of the human stomach. The reactivity was observed with three different antisera, raised against bovine brain, primate brain, and chicken intestinal calbindin. The ECL cells were closed endocrine cells located at the bases of the oxyntic glands. They were not found in other regions of the stomach. No other gastric endocrine cells were reactive with these antisera.

Chromogranin A (CgA) in the gastro-entero-pancreatic (GEP) endocrine system. II. CgA in mammalian entero-endocrine cells.

Chromogranin A (CgA) and related acidic proteins are widely distributed in the organism. They are also present in entero-endocrine cells and in other members of the paraneuron family. Therefore, CgA has been claimed as an universal marker of this cellular community. To yield precise data about the distribution of CgA in entero-endocrine cells, all segments of the gastro-intestinal tract of five mammalian species (man, cattle, pig, cat, guinea-pig) were investigated immunohistochemically for CgA. In serial semithin plastic sections, all CgA-immunoreactive endocrine cells were identified for resident amines or peptides. CgA could be found in ten hormonally identified endocrine cell types and in two or three other endocrine cell types. Entero-endocrine cells containing amines (histamine, serotonin) regularly exhibited CgA-immunoreactivities. In contrast, peptide-containing endocrine cells were largely heterogeneous: Their CgA-immunoreactivities varies among the species, among the gastro-intestinal segments, and even among the members of the same cell population. Hence, seen histochemically, CgA is no universal marker for entero-endocrine cells. Seen biochemically, the observed heterogeneities of CgA-immunoreactivities theoretically can be attributed to various factors (species-specificities of CgA, subclasses of chromogranins, processing of CgA or its pro-protein). Most probably, these heterogeneities are caused by species- or cell-specific differences in the extent of processing of CgA. In addition, some findings point to certain interrelations between the processing or storage of CgA and resident peptides in the secretion granules of enteroendocrine cells.

Cytology and arrangement of enterochromaffin (EC) cells in the human stomach.

Serotonin containing enterochromaffin (EC) cells in the human gastric mucosa were observed using serial semithin sections immunostained by Sternberger's PAP method and reconstructed by computer-assisted methods. In oxyntic glands, EC cells displayed a marked pleomorphism which suggests their plasticity or active movement. They sometimes possessed multipolar cytoplasmic processes directly contacting the neighboring epithelial cells and/or gastric lumen. In the antropyloric glands, they are exclusively the "closed-type," which fails to contact the lumen, and are often arranged touching other EC cells (cluster formation), apparently exhibiting polynuclear enterochromaffin syncytia. This syncitium-like arrangement is interpreted as the morphological counterpart of a possibly synchronized function of these cells. The morphological differences of EC cells in their shape, luminal endings and arrangement between both regions may be indicative of regional differences in their functions. Furthermore, the present study provides the first three-dimensional visualization of EC cells in the human stomach.

Neuroendocrine cells in intestinal lamina propria. Detection with antibodies to chromogranin A.

This study details immunohistochemical experiments showing the location of chromogranin A-containing cells within the lamina propria of rat and human gut. The presence of this marker confirms the presence of neuroendocrine type cells in the lamina propria.

Intranuclear bundles of microfilaments and microtubules in chromaffin cells of the auricle of the heart of a lungfish, Protopterus aethiopicus.

Intranuclear microtubular-microfilamentous rod-like inclusions were investigated in chromaffin cells of the auricle of the heart of lungfishes. In conventional electron microscopy, these inclusions reveal a wide variety in appearance, depending on their orientation to the plane of sectioning. Whereas originally they were merely interpreted as a bundle of microfilaments, application of a goniometer stage showed the rod- or spindle-shaped intranuclear inclusions to have a basic substructure of parallel arranged microtubules among microfilaments, which are clearly connected to chromatin granules, occasionally penetrating dense areas of chromatin. The chemical nature and biological significance of these structures, which so far remain enigmatic, are discussed.

Distribution of serotonin-immunoreactive paraneurons in the lower urinary tract of dogs.

Morphological and quantitative studies were made on serotonin-containing paraneurons throughout the lower urinary tract in male and female dogs. Using an anti-serotonin antiserum, the cells were consistently demonstrated to be dispersed in the epithelium from the vesico-urethral junction to the external urethral ostium. They occurred most frequently in the urethra proximal to the urogenital diaphragm in both sexes. The total number of the serotonin-immunoreactive cells in the urethra was estimated to be 36.2 X 10(4) (SD 9.9 X 10(4] in the male (n = 3) and 15.6 X 10(4) (SD 2.1 X 10(4] in the female (n = 3). Besides the urethra, the prostate and vaginal vestibule contained several serotonin-immunoreactive cells. The urethral serotonin cells were basically bipolar basal-granulated cells that extended the basal cytoplasm to the basement membrane and reached the lumen with an apical process. Modified cell shapes were, however, also frequent, and included bifurcated apical and/or basal processes or a laterally directed basal process. Occasional serotonin cells possessed a threadlike basal process with varicosities and a terminal bouton, reminiscent of a neuronal process. Immunoreactivity for chromogranin A, a carrier protein common to endocrine paraneurons, was demonstrated in all of the urethral serotonin cells. The chromogranin A-immunoreactive granules accumulated more densely in the basal and perinuclear regions of the cell. It is hypothesized that the serotonin-immunopositive paraneurons may receive chemical and/or physical information from urine and, in response to it, secrete serotonin which presumably causes the contraction of the musculature of the urethra.

[Immunohistochemical study of 4 cases of mucoid and argyrophilic carcinoma of the breast]. / Etude immunohistochimique de 4 cas de carcinomes mucoïdes et argyrophiles du sein.

Three to 5% of breast carcinomas are argyrophilic, including some which are mucinous and thus "composite", whereas there are no argyrophilic cells in normal breast nor in benign breast pathology. This raises the problem of the origin and type of these argyrophilic cells. We carried out a histologic and immunohistochemical study in 4 such cases of mucoid tumors containing at least 50% argyrophilic cells. Two of these tumors presenting node involvement were also studied immunohistochemically. The histologic study showed colloid and intragalactophoric proliferation areas in cell cases and some endocrine areas in 2 out of 4 cases. Argyrophilic cells were present in all of these areas. True mucoargyrophilic amphicrine cells were found primarily in colloid areas. None of these tumors were argentaffin. Immunohistochemical study was performed by the PAP method using antibody directed against VIP, ACTH, PP, somatostatin, bombesin, calcitonin, gastrin, prolactin and GH. Three out of four tumors were positive with VIP. Moreover one of them contained ACTH cells and a metastasis of this tumor contained bombesin cells. No tumor was positive with the other anti-sera tested. This study is related to the rare series in the literature which report secretion of ACTH, catecholamins, bombesin, gastrin, VIP, PP, somatostatin, prolactin, etc. The number of cases reported to date remains too low to show a significant prognostic difference between amphicrine tumors and other mammary carcinomas.

Immunohistochemical demonstration of chromogranin in endometrial carcinomas with argyrophil cells.

Forty-five endometrial carcinomas, 36 of which contained argyrophil cells and nine of which were nonargyrophilic by the Grimelius method, were examined immunohistochemically for chromogranin. Chromogranin immunoreactivity was present in 19 of the 36 tumors with argyrophil cells (53 per cent) and in none of the nine tumors lacking these cells. All six of the tumors that contained argyrophil cells resembling enterochromaffin cells were chromogranin-positive, with the staining corresponding to the argyrophilia. In contrast, only 13 of the 30 tumors in which argyrophilia was present in the apical region or throughout the cytoplasm of the cells showed chromogranin immunoreactivity. In seven of these tumors, an excellent correlation existed between the distribution of argyrophilia and chromogranin positivity, but in the other six tumors argyrophilia was more pronounced than chromogranin immunoreactivity. Adjacent to one tumor, unusual cells in which argyrophil granules were packed predominantly in the basal portion of the cytoplasm were encountered in a focus of atypical hyperplasia; these cells were also chromogranin-positive. The present observations suggest that endometrial carcinoma cells with diffuse or apical chromogranin immunoreactivity may represent an early stage in the development of cells resembling those of the enterochromaffin type.

Historical introduction: the Italian contribution to the discovery of 5-hydroxytryptamine (enteramine, serotonin).

In 1949-1951 two independent lines of research led to the discovery and elucidation of the structure of serotonin and enteramine, and their identification with 5-hydroxytryptamine. This is briefly described, with emphasis on the Italian contribution to the solution of the problem.

Argyrophil cells in Brenner tumors: histochemical and immunohistochemical analysis.

Argyrophil cells were identified by the single-impregnation Grimelius technique in 11 of 28 (39%) Brenner tumors, accounting for less than 1% of the tumor cell population in all the cases. All tumors with argyrophil cells were stained to demonstrate calcitonin, somatostatin, gastrin, adrenocorticotropic hormone, neurotensin, insulin, glucagon, and serotonin; and four of them (three benign and one borderline) were also stained for chromogranins with the monoclonal antibody LK2H10. Serotonin was present in nine of the 11 cases with argyrophil cells. Neurotensin and somatostatin were found in one borderline tumor, which also contained serotonin. Chromogranin reactivity was demonstrated in all four cases in which it was examined. Ultrastructural examination of one tumor revealed that the argyrophil cells contained secretory granules, 80 nm in diameter, and had elongated cytoplasmic processes that extended between the more numerous nonargyrophil tumor cells. The argyrophil cells of Brenner tumors are similar to those of urothelium in the frequency with which they are immunoreactive for serotonin and the rarity with which they are reactive for peptide hormones. These cells differ from those of mucinous ovarian tumors, which often contain both serotonin and peptide hormones. The findings of this study lend additional support to the close similarity of the epithelial components of Brenner tumors and urothelium.

Chromogranin: a newly recognized marker for endocrine cells of the human gastrointestinal tract.

Existing methods for the histochemical demonstration of gastrointestinal cells are somewhat limited. Chromogranin represents a family of proteins that coexist with catecholamines in the secretory vesicles of adrenal medulla cells. In the present study, immunocytochemistry was used to test whether chromogranin is a marker for gut endocrine cells. Serial sections of each area of human gut were immunostained for chromogranin and for the amine and each of the peptides known to be present in mucosal endocrine cells. Chromogranin was immunostained in large numbers of endocrine cells in all tissues examined. All identified endocrine cell types were found, in serial sections or by sequential silver impregnations, to be chromogranin immunoreactive. However, the possibility exists that some chromogranin-immunoreactive cells contain a yet to be discovered endocrine substance. Immunostaining of chromogranin thus appears to provide a means for demonstrating all gastrointestinal mucosal endocrine cells identifiable by the methods described in this study.