Permeability studies of the guinea pig placental labyrinth

Summary The permeability of the materno-fetal barrier in the chorioallantoic placenta was studied in guinea pigs with gestation at term applying a variety of electron-opaque tracers via maternal circulation. None of the tracers tested was seen permeating the syncytiotrophoblast in the materno-fetal direction up to a 20 min interval when the fine structure of the placenta was satisfactorily preserved. The lanthanum chloride, cationized ferritin and horseradish peroxidase bound to the trophoblast surface, apparently due to electrostatic forces more than to specific receptors, and no uptake of these probes was detected in the cytoplasm. Albumin-colloidal gold complex, also used as a tracer, yielded similar results. As reported in other species with more complex syncytiotrophoblastic organization, this layer investing maternal lacunae is a highly selective permability barrier.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Alexander von Humboldt StiftungCareer Member of CONICET     

Permeability studies of the guinea pig placental labyrinth

Summary Permeability of the fetal endothelium within the guinea pig placental labyrinth is studied by means of horse-radish peroxidase (HRP) and ionic lanthanum as diffusion tracers. The paracellular transport of HRP is restricted by the occluding junctions of the fetal endothelium. In contrast, ionic lanthanum readily permeates most of the intercellular junctions and rapidly infiltrates the basal lamina. Freeze-fracture replicas reveal zonulae occludentes connecting the fetal endothelial cells. The network of the zonulae occludentes is variable, exhibiting highly complex areas as well as single strand interconnections. A correlation between the permeability studies and freeze-fracture findings is discussed.Supported by grants from the Consejo Nactional de Investigaciones Cientificas y Técnicas (CONICET), the Alexander-von-Hemboldt-Stiftung and the Deutsche ForschungsgemeinschaftResearch Professor of the Alexander-von-Humboldt-Stiftung and Career Investigator of CONICET     

Neuropeptides in the human dorsal vagal complex: An immunohistochemical study

The distribution of twelve biologically active neuropeptides, i.e., thyrotropin-releasing hormone, corticotropin-releasing factor, pro-opiomelanocortin-derived peptides (adrenocorticotropic hormone, β-endorphin, α-melanocyte-stimulating hormone), leucine-enkephalin, dynorphin A, dynorphin B, cholecystokinin, substance P, galanin and calcitonin gene-related peptide, was examined by immunohistochemistry in the human dorsal vagal complex including the nucleus of the solitary tract, the dorsal motor nucleus of the vagus and the area postrema. Immunoreactivity of all the twelve neuropeptides was found widely distributed in the various subdivisions of the nucleus of the solitary tract, showing a unique distribution for every peptide. Neuronal cell bodies immunostained with leucine-enkephalin, galanin and dynorphin B were found in this region. There were no immunopositive perikarya for any of the peptides in the other structures studied. Fibers containing galanin, corticotropin-releasing factor, substance P, dynorphin B, thyrotropin-releasing hormone and calcitonin gene-related peptide were observed at a relatively high density in the nucleus of the solitary tract. In the same structure, a moderately dense network of fibers immunostained with dynorphin A, cholecystokinin and leucine-enkephalin, but only solitary pro-opiomelanocortin-derived peptides-containing fiber fragments were observed. In the dorsal motor nucleus of the vagus the most prominent network of fibers was found to contain thyrotropin-releasing hormone, galanin and substance P. In contrast to these, no β-endorphin immunoreactivity was detected. The area postrema contained only moderate to low densities of galanin-, substance P-, calcitonin gene-related peptide-, dynorphin B- and cholecystokinin-immunoreactive fibers.     

Permeability studies of the labyrinth in the guinea pig placenta

Summary A surgical procedure was developed for cannulation of umbilical vessels in the guinea pig placenta. This approach allows an administration of electron opaque tracers, perfusion of fixatives and injection of corrosion casting compounds with minimal disturbance to the fetus and the placenta.Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET) and the Alexander von Humboldt Stiftung     

Contraction of guinea pig trachea with antibodies to guinea pig IgE. An in vitro model for asthma

Rabbit antiserum to guinea pig IgE was prepared. This antiserum absorbed IgE antibodies to dinitrophenyl determinants when examined by passive cutaneous anaphylaxis. This antiserum also provoked contraction of tracheal rings from normal guinea pigs in vitro. This system is a new model for asthma in which only IgE among immunoglobulins reacts.     

Absence of adrenaline neurons in the guinea pig brain: a combined immunohistochemical and high-performance liquid chromatography study

The distributions of catecholamine (CA) neurons in the medulla oblongata of the guinea pig and rat were compared using immunohistochemistry with rabbit antisera against the CA synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (D beta H) and phenylethanolamine-N-methyltransferase (PNMT). TH and D beta H distributions were similar in the two species. In contrast, the central nervous systems of both normal and colchicine-treated guinea pigs failed to demonstrate immunoreactivity for PNMT, the synthetic enzyme for adrenaline. The concentrations of biogenic amines in the hypothalamus and medulla were determined in guinea pig and rat tissues by high-performance liquid chromatography with electrochemical detection. No adrenaline was detected in the guinea pig brain. Thus it appears that guinea pigs lack central neurons capable of synthesizing adrenaline.     

The innervation of the synovium of the knee joint in the guinea pig: an immunohistochemical and ultrastructural study

 The innervation of the knee joint synovial membrane of the guinea pig, i.e., the synoviocyte layer, the subjacent connective tissue and the connective tissue region beneath, was analyzed with immunohistofluorescence and electron microscopy. A screening of the innervation with antibodies against the general axon marker – protein gene product (PGP) 9,5 – revealed the presence of nerve fibers distributed in various regions of the knee joint synovial membrane. Confirmating previous studies, some of these nerve fibers stained with antibodies to tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP). In addition, dynorphin (DYN)-containing fibers were detected, which have not been reported previously in normal joints. In general, the immunoreactive fibers were observed close to the synoviocytes and at blood vessels. Fibers with colocalization of NPY- and TH-like immunoreactivities (LIs), as well as of DYN- and TH-LIs were demonstrated. In the electron microscope, bundles of unmyelinated fibers as well as single fibers were found in the connective tissue region below the synoviocytes. Varicose parts of the nerve fibers contained mainly small, clear vesicles. Small and large dense-cored vesicles were also seen, but less frequently. Denser portions of the plasma membranes of some axons were observed in these regions, facing the extracellular space. Myelinated fibers were also observed in some nerve bundles. These findings emphasize the complex innervation of the synovial membrane, with nerve fibers containing a host of neuroactive substances. Altogether, these fibers are probably involved in many functions such as vasoregulation and control of synovial secretion in addition to being a source of mediators in joint inflammation. Accepted: 22 November 1997     

Epithelioid sarcoma. An immunohistochemical study

The immunohistochemical findings in 14 epithelioid sarcomas, neoplasms of uncertain histogenesis, indicate that they react with antibodies against cytokeratin, epithelial membrane antigen, and vimentin. All cases were nonreactive for leukocyte common antigen, myoglobin, and Factor VIII-related antigen. These results point to the fact that epithelioid sarcoma expresses phenotypic characteristics more often associated with epithelioid neoplasms, rather than the mesenchymal profile of most soft tissue sarcomas. One explanation for this observation is that epithelioid sarcoma is in fact a carcinoma originating in the deep soft tissues. On the other hand, the pluripotential mesoderm has a known embryonic capacity to differentiate into epithelium and, therefore, it is plausible that epithelioid sarcoma is a mesenchymally derived neoplasm. Aside from histogenetic considerations, epithelioid sarcoma may be confused with a number of other neoplastic and granulomatous processes. Differential immunohistochemical stains are useful in selected instances wherein light and electron microscopic findings are diagnostically equivocal.     

Mesenchymal chondrosarcoma. An immunohistochemical study

Mesenchymal chondrosarcoma is an uncommon small-cell neoplasm of bone and soft tissue, the chondrogenic nature of which has been generally accepted. However, the phenotypic attributes of the small-cell population in this neoplasm have not been well characterized, and its relationship to "precartilage mesenchyme" remains unclear. In an attempt to address this issue, we performed an immunohistochemical analysis of nine cases, using antibodies to vimentin, S100 protein, Leu-7 antigen, neuron-specific enolase, synaptophysin, desmin, muscle-specific actin, cytokeratin, and epithelial membrane antigen, and the avidin-biotin-peroxidase complex (ABC) method. The small cells of mesenchymal chondrosarcoma failed to express S100 protein, whereas all components of the tumors (small cells, lacunar chondroblasts, and chondroid matrix) stained for Leu-7 antigen in six cases. Neuron-specific enolase was identified in the small cells of four cases and in the lacunar cells of seven. None contained desmin, actin, cytokeratin, epithelial membrane antigen, or synaptophysin. The immunophenotype of mesenchymal chondrosarcoma resembled that of embryonic cartilage and thus did not contradict the premise that this tumor was the neoplastic counterpart of fetal chondroid tissues. However, immunohistologic studies are not overly helpful in the differential diagnosis between mesenchymal chondrosarcoma and other small round cell lesions.     

Glomus tumours. An immunohistochemical study

The histological and immunohistochemical features of 20 glomus tumours (glomangiomas) were studied retrospectively in routinely processed material. The tumours came from 18 patients (9 women and 9 men, aged 25 to 80 years); two were recurring lesions. Twelve were classified as solid glomus tumours, eight as glomangiomas. Small nerve fibres were present in all except one. A variable number of mast cells were found in the stroma. The glomus tumour cells were negative when stained for Neuron-Specific Enolase, Glial Fibrillic Acidic Protein, S-100 Protein, Chromogranin, or with the Ulex Europaeus Lectin type 1. Conversely, all were found to be positive for Actin, Myosin, and Vimentin. Four exhibited an equivocal reaction for Desmin, the rest were negative. This immunohistochemical profile is in accordance with the findings of other investigators and can be helpful in differential diagnosis. It also shows the glomus cell to be related to smooth muscle cells and pericytes. The majority of these lesions are probably hamartomas, but a few may be true neoplasms.     

Ultrastructural development of guinea pig cytomegalovirus in cultured guinea pig embryo cells.

The ultrastructural development of guinea pig cytomegalovirus (GPCMV) in guinea pig embryo cells was studied using electron microscopy. Tubular structures were found in nuclei of virus infected cells, followed by the appearance of intranuclear inclusions containing virus nucleocapsids. While some nucleocapsids were enveloped at the inner nuclear membrane, others were released into the cytoplasm where they were associated with, or within, dense matrix which was subsequently enveloped by cytoplasmic membranes to form enveloped dense virions. Dense bodies without virus capsids were formed in the cytoplasm and enveloped in a similar manner. An involvement of the nuclear pores in the release of unenveloped virus capsids from the nucleus to the cytoplasm was postulated. Evidence that the enveloped dense virions and dense bodies shared common envelope antigen(s) was obtained by immunoelectron microscopy. The similarities and differences in the ultrastructural development of GPCMV and other cytomegaloviruses are discussed.     

Capillary hemangioblastoma. An immunohistochemical study

Previous studies using immunohistochemical methods to determine the presence of glial fibrillary acidic protein (GFAP) and Factor VIII-related antigen (FVIIIR:Ag) in stromal cells of capillary hemangioblastomas have yielded conflicting results with respect to the possible astrocytic and endothelial origins of these cells. This study included Ulex europaeus I lectin (UEAI), a more sensitive marker of endothelial cells. Antibodies were also used as markers of pericytes and a variety of markers were employed to identify different populations of histiocytes. Results of this investigation indicate that FVIIIR:Ag and UEAI are limited only to endothelial cells, and that GFAP is present in entrapped astrocytes only. Positivity of stromal cells was found with some of the histiocytic markers, but the authors were unable to conclude that these cells have a histiocytic origin. It was concluded that currently there is no evidence that stromal cells are derived from endothelial, pericytic, or astrocytic cells-their origin remains uncertain.     

An immunohistochemical study of endopeptidase-24.11 ("enkephalinase") in the pig nervous system

Endopeptidase-24.11, a plasma membrane ectoenzyme with the ability to hydrolyse a variety of neuropeptides, has been localized in the pig nervous system by an immunoperoxidase technique. The endopeptidase was mapped in cryostat sections of the fore and mid-brain to the following structures: caudate-putamen, globus pallidus, olfactory tubercle, nucleus interpeduncularis and substantia nigra. Endopeptidase-24.11-like immunoreactivity was also found in the pia mater, choroid plexus and ependymal lining of the central canal. In the spinal cord, weak staining was observed in the dorsal horn, but strong staining was found in the dorsal root ganglia and nerve roots. Within the central nervous system, endopeptidase immunoreactivity was confined to gray matter and within the positive areas of the striatum densely staining areas, corresponding to striosomes, were discernible. These well-defined structures were exploited in serial sections to examine the alignment of the enzyme-rich patches of neuropil with correspondingly strong staining for other antigens. A consistent match was observed with a monoclonal antibody to neurofilament protein, but there was a poor correlation with a polyclonal antibody to glial fibrillary acidic protein. Substance P-like and [Leu]enkephalin-like immunoreactivity were also studied in sections adjacent to those stained for the endopeptidase. Good matching between enzyme-rich and peptide-rich areas was observed, but some enkephalin-rich areas did not align with enzyme staining and indeed endopeptidase-rich areas were not necessarily matched with areas rich in either peptide. These findings suggest a neuronal rather than an astrocytic location for endopeptidase-24.11 in the CNS and lend support to the view that it plays a central role in neuropeptide metabolism at membrane surfaces. In the peripheral nervous system, the endopeptidase was located in Schwann cell membranes surrounding dorsal root ganglion cells and nerve fibres, while in the pituitary the main concentration was in the adenohypophysis, where only a proportion of the endocrine cells were found to be immunoreactive.     

An immunohistochemical study of branchial cysts.

Twenty five specimens of branchial cyst from the same number of patients have been examined. On staining with haematoxylin and eosin a consistent finding was that the mural lymphoid follicles were always aligned with their mantle zones towards the luminal epithelium. With conventional staining lymphatic sinuses were noted in 17 of the specimens, but with immunohistochemical staining these structures were apparent in 23 cysts. Their frequent occurrence in branchial cysts supports the theory that these lesions are derived from epithelial inclusions in lymph nodes. Immunohistochemical techniques for a range of other markers, using polyclonal and monoclonal antisera, showed a distribution of lymphoid and non-lymphoid tissue elements, as seen in lymph nodes and, for example, palatine tonsils. The lining luminal epithelium also shared many features in common with the crypt epithelium of tonsils.     

Prostatic malacoplakia. An ultrastructural and immunohistochemical study

A case of malacoplakia of the prostatic gland associated with prostatic nodular hyperplasia from a 69 years old man was presented, and its light and electron microscopic and immunohistochemical features were discussed along with its pathogenesis. This lesion was incidentally found in a transurethral prostatectomy specimen, and consisted of large number of epithelioid cells in which were typical cytoplasmic inclusions known as Michaelis-Gutmann bodies. Ultrastructurally, these inclusions showed a dense, central calcified bodies of various developmental stages. Immunohistochemical study using antilysosomal antibody revealed no lysosomal activity. Based on these findings, we could suspect that main problem for this development of malacoplakia is altered intracellular digestion process of foreign biologic materials.     

Ampullary somatostatinoma. An immunohistochemical and ultrastructural study

Two new cases of ampullary somatostatinoma are reported. In one case the tumor is associated with an increase in somatostatin-positive cells in the adjacent duodenal mucosa. Both tumors show a predominant glandular pattern with many psammomatous calcified bodies. Such bodies seem to arise by calcium phosphate encrustation of intraluminal cellular debris. The neoplastic cells contain two distinct types of intermediate filaments: the first is located along the plasma membrane and reacts to keratin antiserum; the other, appearing as paranuclear aggregates, reacts to neurofilament antiserum. The neoplastic cells show signs of intestinal differentiation (microvilli, glycocalyceal bodies, filamentous core rootlets) as well as of neuroendocrine differentiation (secretory granules, whorls of neurofilaments).     

Epithelial and melanoma antigens in gliosarcoma. An immunohistochemical study.

Gliosarcomas are mixed tumors with malignant glial and mesenchymal elements. The number of GFAP-positive tumor cells decreases with the increase of sarcomatous components, until whole areas may be GFAP negative. These distinct differentiations may, however, lead to false interpretations in small tissue samples. In this connection, it is of interest that, according to other reports, glial tumors may be positive for different anti-keratin antibodies and this prompted us to undertake a systematic investigation of the immunoreactivity of gliosarcomas using a panel of well-characterized monoclonal antibodies against cytokeratins (KL1, AE 1/3, Lu-5, CK-19, CK MNF 116 and Ma-903). These cases were further studied with the anti-epithelial non-cytokeratin antibodies EMA, HEA 125, Ber-EP4, CEA as well as the melanoma-antibody HMB-45, Leu-M1, GFAP and vimentin. As screening study we examined 20 cerebral metastatic carcinomas, 21 malignant gliomas (including 6 gliosarcomas) and 3 metastatic melanomas with the monoclonal antibodies KL1 and HMB-45. All cerebral metastatic carcinomas and 4/6 gliosarcomas were positive for KL1, whereas all melanomas, 2 metastatic carcinomas and 3 gliosarcomas showed an immunostaining with HMB-45. All gliosarcomas were positive with at least one of the tested anti-cytokeratin antibodies. The gliosarcomas did not show an immunoreaction in any of the cases when CEA, HEA 125, Ber-EP4, EMA or Leu M1 were applied. In our opinion, the monoclonal antibodies HEA 125 and Ber-EP4 could obviously be helpful in differentiating gliosarcomas from metastatic carcinomas.     

Intracellular study of tonic-type enteric neurons in guinea pig small intestine.

1. Intracellular recording revealed a population of myenteric neurons with electrical behavior that appeared to be equivalent to the tonic-type mechanosensitive neurons found in earlier studies that utilized extracellular recording of single units. 2. Electrical stimulation of the interganglionic fiber tracts evoked a slowly rising excitatory postsynaptic potential (slow EPSP) that was prolonged for several seconds after termination of the stimulus in these cells. The slow EPSP was associated with increased input resistance and augmented excitability of the somal membrane. The somal membranes had relatively low excitability in the absence of fiber tract stimulation. This was indicated by: 1) failure of depolarizing current pulses to elicit spike discharge in some cells; 2) when spikes were elicited by depolarizing current pulses, one to three spikes occurred, and these were seen only at the onset of the pulse; 3) passive invasion of the soma by current from spikes in the cell's processes did not trigger spikes; 4) the spikes were followed by prolonged hyperpolarizing afterpotentials associated with decreased input resistance. 3. Characteristics of the augmented excitability during the slow EPSP were: 1) endogenous discharge of trains of spikes, 2) spike discharge throughout 200 ms duration depolarizing current pulses, 3) electrotonic spike potentials from the processes triggered somal spikes, 4) postspike afterhyperpolarization was reduced or abolished. 4. The slow EPSP was reduced or abolished in Krebs solution with 16 mM Mg+2 and in HEPES-buffered Krebs with 1 mM Mn2+. It was enhanced in Krebs solution with 3 times normal Ca2+. Spike discharge in elevated Ca2+ eliminated the increase in input resistance associated with the slow EPSP. 5. Calcium availability was an important factor in regulation of membrane conductance and excitability in the perikaryon. The slow EPSP provides a mechanism whereby the soma of a multipolar neuron gates the spread of excitation between its dendrites and axon.