Immunohistochemical demonstration of age-related deposition of vitronectin (S-protein of complement) and terminal complement complex on dermal elastic fibers.

Immunoreactivity of vitronectin was investigated in 100 skin specimens from different body regions in 87 individuals of different ages using monoclonal and polyclonal anti-vitronectin antibodies in an avidin-biotin-peroxidase complex technique. Vitronectin immunoreactivity was found in conjunction with dermal elastic fibers in all subjects older than 13 years. No vitronectin immunostaining was detected in subjects younger than six years, suggesting deposition of vitronectin during late childhood or early adolescence. Using an immunogold staining procedure, vitronectin immunoreactivity was ultrastructurally localized to the periphery of elastic fibers. The blood level of vitronectin in 20 healthy newborns was 67% of the adult level, suggesting active biosynthesis already in the fetus. To investigate whether vitronectin is deposited as part of the SC5b-9 complex or as uncomplexed protein, the immunoreactivity of vitronectin was compared with that of C9, using monoclonal and polyclonal antibodies against the C9 neoantigen. Distinct C9 neoantigen immunoreactivity was demonstrated in association with dermal elastic fibers in human skin in adults but only in subjects older than 30 years. The intensity of C9 neoantigen immunoreactivity appeared to increase with age and was found to be stronger in sun-exposed skin than in sun-protected skin. These findings indicate that uncomplexed vitronectin is deposited during childhood or early adolescence and that terminal complement complexes (C5b-9 and/or SC5b-9) are deposited on elastic fibers later on in life. Hypothetically, the tissue form of vitronectin may be involved in the prevention of tissue damage in proximity to local complement activation. In addition, it may be physiologically important as substratum for cells, stimulating cell migration and anchorage.

Pituitary adenylate cyclase activating polypeptide expression in sensory neurons.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide isolated from ovine hypothalami. The presence of PACAP-like immunoreactivity was recently demonstrated in nerve cell bodies of sensory ganglia in the rat. Since PACAP belongs to a large family of chemically related neuropeptides, we have, in the present study, tried to establish the synthesis of PACAP in neurons of sensory ganglia, using in situ hybridization with a 35S-labelled oligonucleotide probe complementary to PACAP mRNA. The expression of PACAP was compared to that of calcitonin gene-related peptide (CGRP) using a radiolabelled CGRP oligonucleotide probe. The PACAP probe labelled small to medium-sized neurons in the trigeminal ganglion and dorsal root ganglia at different levels, indicating the presence of PACAP mRNA. The CGRP probe labelled nerve cell bodies of varying size, outnumbering those labelled by the PACAP probe. In dorsal root ganglia, cells expressing PACAP constituted c. 10% and those expressing CGRP 46% of the total number of nerve cell bodies. Expression of PACAP was seen in a small subpopulation of cells expressing CGRP. We conclude that PACAP is synthesized in a subpopulation of neurons of sensory ganglia in the rat. Therefore, the recently described effects of PACAP--cutaneous vasodilation, potentiation of oedema formation and depression of nociceptive spinal reflexes--may be physiological and related to neurogenic inflammation and modulation of pain transmission.

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique.

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.

Kallikrein in rat pancreatic tissue after beta cell destruction or acinar cell atrophy.

The purpose of this study was to determine whether glandular kallikrein in rat pancreas is located in the beta cells of the endocrine pancreas or in the acinar cells of the exocrine pancreas. Kallikrein was measured by radial immunodiffusion and a direct radioimmunoassay in homogenates of pancreas obtained from 1) control rats, 2) rats with pancreatic beta cells selectively destroyed by streptozotocin, and 3) rats with acinar cell atrophy induced by pancreatic duct occlusion. Beta cell destruction was confirmed by the presence of hyperglycemia and by an almost total depletion of insulin-producing cells as demonstrated immunohistochemically. Acinar cell atrophy was confirmed histologically and by an almost total depletion of trypsin-like enzymes in pancreatic homogenates. The concentration of kallikrein in pancreatic homogenates was unchanged after beta cell destruction, whereas it was greatly decreased following acinar cell atrophy. Kallikrein was, by immunohistochemistry, demonstrated in the acinar cell only. The immunohistochemical localization of kallikrein agrees with the above results. These studies strongly indicate that kallikrein is predominantly located in the acinar cells of the exocrine pancreas.

Vasoactive intestinal peptide nerves in ocular and orbital structures of the cat.

Vasoactive intestinal polypeptide (VIP), a neuronal peptide of ubiquitous occurrence in the body, is known to have strong vasodilatory effects and to promote secretion from many exocrine glands. Nerves displaying VIP immunoreactivity (VIP nerves) were detected in several orbital structures of the cat. Such nerves were numerous in the lacrimal glands and somewhat less numerous in the Harderian glands and the tarsal glands. The nerves surrounded glandular acini and small blood vessels. Intraocularly, VIP nerves were seen in the ciliary processes, in the posterior third of the ciliary muscle, and around small to medium-sized blood vessels in the posterior uvea. VIP nerve fibers were absent from vessels in the anterior uvea. This distribution may explain why intracranial stimulation in the oculomotor nerve exit region dilates the vessels of the choroid but not those of the iris. A large number of VIP-immunoreactive nerve cell bodies were observed in the pterygopalatine ganglion. Extirpation of this ganglion resulted in the disappearance of VIP nerves from the intraocular structures and from the lacrimal and Harderian glands. Removal of the superior cervical ganglion and the ciliary ganglion did not affect the VIP nerve supply. The results suggest that the VIP nerves originate in the pterygopalatine ganglion.

Peptide-containing nerve fibres in normal human parathyroid glands and in human parathyroid adenomas.

There are only a few studies on the innervation of the human parathyroid glands and the content of neurotransmitters. We therefore studied the occurrence and distribution of peptide-containing and adrenergic nerve fibres and the coexistence pattern of neuromessengers by immunocytochemistry in normal (unaffected) and adenomatous parathyroid glands from patients undergoing surgery for parathyroid adenoma. The unaffected parathyroid glands had a moderate-to-rich supply of nerve fibres and terminals containing two general neuronal markers, protein gene product 9.5 (PGP 9.5) and synaptophysin, neuropeptide Y (NPY) and tyrosine hydroxylase (TH). They were seen close to blood vessels and, occasionally, among the endocrine cells. Only a few nerves contained calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP). The general density of innervation, using PGP 9.5 and synaptophysin as markers, varied greatly among the different adenomas examined. This applied also to the density of fibres and terminals containing specific types of messengers. Some of the tumours had a rich supply of TH- and NPY-containing nerve fibres, while others contained only few scattered fibres. The CGRP-containing fibres varied from moderate in number to no detectable fibres. The PACAP-, SP- and VIP-containing fibres were always very few or not detectable. It is not inconceivable that the wide variation in general density of the innervation and frequency of peptide-containing nerves among individual parathyroid adenomas is of significance for their hormone secretory behaviour.

Neuroendocrine differentiation in male breast carcinomas.

The presence of neuroendocrine differentiation, as expressed by cellular chromogranin immunoreactivity, was investigated in paraffin-embedded tissue material from 51 consecutive cases of male breast carcinoma. From six of these cases electron microscopic studies were included. Chromogranin-immunoreactive cells were present in solid cords and delineated tubular structures. Ultrastructurally, dense core secretory granules could be detected. The expression of neuroendocrine differentiation was 45%, which is between two and eight times higher than reported for female breast carcinomas by other investigators. The present findings suggest that male breast carcinoma is an exclusive tumour disease showing both similarities and discrepancies when compared to its female counterpart.

The influence of a histamine2-receptor antagonist on the healing of an experimentally induced gastric mucosal lesion.

The effect of an H2-receptor antagonist (ranitidine) on the healing of gastric mucosal lesions was studied. Mucosal lesions were induced by a standardized thermo-mechanical technique. The healing process was assessed by macro- and light microscopical examination. It was further evaluated by measurements of the tissue contents of hydroxyproline, a chemical compound reflecting collagen, and of DNA and RNA, reflecting cell frequency and protein synthesis respectively, in the gastric wall from both injured and wound-free areas. The healing process was more rapid in ranitidine-treated animals than in controls. After four weeks, however, the lesion in nine out of ten animals had healed in the ranitidine-treated group and seven of nine rats in the control group. At that time the amounts of hydroxyproline, DNA and RNA did not differ between the two groups. These findings may be taken as an indication that the tissue components of the healed lesions were similar in ranitidine-treated rats and in the saline controls, i.e. the different speeds of the healing process did not seem to influence the components of the scar tissue.

Two distinct VIP precursors in cartilaginous fish?

In the ray gut immunoreactive VIP has a dual localization in endocrine cells and in nerve fibers. Immunoreactive VIP and PHI were found to co-exist in the same nerve fibers. This is predictable, since VIP and PHI derive from the same precursor. However, PHI could not be demonstrated in the VIP immunoreactive endocrine cells. Chromatographic analysis (high performance liquid chromatography) of extracts of the gut revealed two different molecular forms of VIP, one large peak with an elution position similar to that of authentic porcine VIP and a minor peak with a different elution position. The results suggest either the existence of different precursors for VIP in endocrine cells and in neurons, or the different processing of the same precursor in neurons and endocrine cells.

Co-existence of glicentin and peptide YY in colorectal L-cells in cat and man. An electron microscopic study.

Electron microscopic immunocytochemistry using protein A-gold labelling of ultrathin sections revealed immunoreactive glicentin (gut-type glucagon) and peptide YY (PYY) in virtually all secretory granules in a population of L-type endocrine cells in feline colon and human rectum. The granules of the human glicentin/PYY cells were considerably smaller in size than those in the cat. In both species the results indicate co-existence of glicentin and PYY in the same secretory granules, despite the probable derivation of the two peptides from two different precursors.

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.

Trisomy 3 as the sole karyotypic change in a pediatric immature teratoma.

Cytogenetic analysis of short-term cultured cells from an immature ovarian teratoma of a 9-year-old girl disclosed an extra copy of chromosome 3 as the sole clonal abnormality. The fact that trisomy 3 was previously reported as the only karyotypic change in two ovarian germ-cell tumors--one teratoma NOS and one immature teratoma--suggests that gain of chromosome 3 constitutes an early and pathogenetically important change in a subset of female germ-cell tumors.

Complex karyotype in a childhood adrenocortical carcinoma.

Cytogenetic analysis of short-term cultured cells from an 11-cm adrenocortical carcinoma in a 3.5-year-old girl revealed the karyotype 46,XX,inv(9)(p11q12)c/[2]/56-57,XX,+2,+4,+5,+7,+8,inv(9)c,+10,+add (13)(p11), +14,+15,+19,+20,+20,+mar[cp19]. To our knowledge, this is the first description of an abnormal karyotype in a pediatric adrenocortical tumor. Inasmuch as the distinction between benign and malignant adrenocortical tumors is often difficult to make from clinical and histopathologic data alone, the present findings suggest that cytogenetic analysis may be a valuable adjunct in the differential diagnosis.

Parathyroid adenoma with t(1;5)(p22;q32) as the sole clonal chromosome abnormality.

Cytogenetic investigation of short-term cultures from a parathyroid adenoma revealed a t(1;5)(p22;q32) as the sole clonal chromosomal aberration. Karyotypic abnormalities have not previously been described in this tumor type.

Neuronal localization of pancreatic polypeptide (PP) and vasoactive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris).

Nerve fibres and cell bodies displaying vasoactive intestinal polypeptide (VIP) or pancreatic polypeptide (PP) immunoreactivity were demonstrated in ganglia of the earthworm (Lumbricus terrestris). VIP cell bodies were found in the most anterior ganglion of the ventral nerve cord, the subpharyngeal ganglion. Immunoreactive nerves were seen running in the center of the cord until about the 10th segment. PP cell bodies were found in the cerebral ganglion where VIP was lacking, in the subpharyngeal ganglion and in more posteriorly located ganglia of the ventral nerve cord. PP nerve fibres could be followed below the 10th segment of the cord.

Peptide-hormone- and serotonin-immunoreactive cells in normal and hyperplastic prostate glands.

Peptide-hormone- and serotonin-immunoreactive cells of endocrine type are present both in the normal prostatic gland and in the nodules of benign prostatic hyperplasia of man. They are located in the epithelium of the acini and the ducts of all the different parts of the gland, as well as in the urothelium of the prostatic part of the mucosa of the urethra. The endocrine cells are usually argyrophil, sometimes even argentaffin, and immunoreactive with neuron-specific enolase; they can be either of open or of closed type and usually occur widely scattered as single cells. Three kinds of endocrine cells were observed both in the normal gland and in the hyperplastic parenchyma. In the by far most prevalent type serotonin was found to co-exist with a peptide immunohistochemically related to the thyroid stimulating hormone (TSH). In a more rare type serotonin co-existed immunohistochemically with calcitonin. The third kind of endocrine cells was somatostatin-immunoreactive cells; they were also rather rare. The only difference observed between the normal and hyperplastic parenchyma was an increase in the number of all the three kinds of endocrine cells in the hyperplastic nodules. The endocrine cells could easily be visualized by means of silver-staining techniques, even using conventionally formalin-fixed, paraffin-embedded specimens.

Peptide-hormone- and serotonin-immunoreactive tumour cells in carcinoma of the prostate.

In order to establish the extent of neuroendocrine differentiation and the occurrence of neurohormonal peptides in the neoplastic cells of prostatic carcinomas, silver-staining and immunocytochemical techniques were used. All gave satisfactory results. The incidence of the neuroendocrine cells seemed to be higher in the fresh "Bouin-fixed" biopsy specimens than in the conventionally "formalin-fixed" specimens from archival paraffin blocks. All carcinomas demonstrated argyrophil cells as an integral element of the tumour. In highly differentiated carcinomas (grade I) these cells were scattered focally, intermingled with non-argyrophil cells in typical adenocarcinomas; their incidence was estimated to be about the same as in benign prostatic hyperplasia. Most of them were immunoreactive with antisera raised against serotonin and/or TSH (thyroid stimulating hormone). In moderately and poorly differentiated (grades II-III) carcinomas, however, the argyrophil cells were more numerous and showed greater variation in growth pattern; only occasionally they displayed a typical carcinoid-like structure. Moderately and poorly differentiated carcinomas also showed a greater variation in the number and kinds of peptide immunoreactivities than the highly differentiated carcinomas. In addition to serotonin- and TSH-immunoreactive cells as the most prevalent type, now also human chorionic gonadotrophin (HCG-alpha), adrenocorticotropic hormone (ACTH), leu-enkephalin, beta-endorphin, somatostatin, glucagon and calcitonin immunoreactive cells could be found within certain tumour areas and often with a distinctly patchy distribution. In two cases, where the tumour cells in the metastases were also investigated, they were found to be both argyrophil and immunoreactive with the same antisera as those of the primary tumour. Our findings emphasise the fact that prostatic carcinomas are more complex and heterogenous than previously thought, exhibiting endocrine differentiation as an integral element of virtually all prostatic adenocarcinomas.

Innervation of the feline eustachian tube.

The distribution of adrenergic, cholinergic and peptidergic nerves in the feline eustachian tube was studied using histochemical techniques. Adrenergic, acetylcholinesterase-positive and vasoactive intestinal polypeptide immunoreactive nerves were numerous in the tubal wall. All three types of nerve fibers occurred in the subepithelial layer, around small blood vessels and around the acini of seromucous glands. No nerves displaying substance P or enkephalin immunoreactivity were observed.

Occurrence and distribution of VIP nerves in the nasal mucosa and tracheobronchial wall.

Nerves displaying vasoactive intestinal peptide (VIP) immunoreactivity were detected in the upper respiratory tract of guinea pigs, rabbits and cats. VIP nerves were numerous in the cat, less numerous in the rabbit and rare in the guinea pig. In the nasal mucosa, fine varicose VIP nerves were found to surround nasal glands and small blood vessels. In the tracheobronchial wall VIP nerves were observed around seromucous glands, blood vessels and smooth muscle. Ganglia located in the walls of the trachea and main bronchi contained clusters of VIP immunoreactive nerve cell bodies, conceivably representing the origin of the VIP fibres found in this region.

Spontaneous argyrophil cell carcinoid in the glandular stomach: immunohistochemical study of gastric endocrine cells in normal and tumour-bearing mastomys.

Endocrine tumours (argyrophil cell carcinoids) are frequent in the oxyntic mucosa of mastomys. The tumour is notable for its high histamine content and for its high histidine decarboxylase activity. The tumour is thought to arise from the histamine-storing, enterochromaffin-like cells of the oxyntic mucosa. They are of two ultrastructurally distinguishable types, ECL cells and A-like cells, both of which have been demonstrated in the tumour. Identical cells have been demonstrated in the oxyntic mucosa of the rat; there is much evidence that in this species the functional activity and the number of these cells are determined by the serum gastrin concentration. However, tumours have never been found to arise from these cells in the rat. As an initial step in an attempt to explain the formation of the gastric endocrine tumour in the mastomys we examined the distribution and frequency of occurrence of endocrine cells in the mastomys stomach. Gastrin cells in the antrum of mastomys seemed to occur in about the same frequency as in the antrum of rat and mouse. 5-HT-storing enterochromaffin cells, however, were considerably more numerous in the mastomys, whereas the somatostatin cells in the antrum were fewer. The number of enterochromaffin-like cells and somatostratin cells in the oxyntic mucosa of mastomys was much lower than in the rat and mouse. Once developed, the gastric endocrine tumour seems to reduce the antral gastrin cell number; the larger the tumour the greater the reduction.