A simple technique for the long-term non-polarised and polarised culture of human fallopian tube epithelial cells

Fallopian tubes were obtained from 25 women undergoing abdominal hysterectomy. Pieces of fallopian tube mucosa were placed in culture flasks containing minimum essential medium in Earle's salts supplemented with fetal bovine serum. First passage was carried out after 7-10 days and subcultures in 4-5 days. For polarised cell culture, epithelial cells were seeded onto an extracellular matrix system. New epithelial cells were seen on day 2-3 of the primary culture and epithelial patches on day 7-10. Cells reached confluence in 4-5 days in subcultures. The cells could be subcultured for 7-11 passages with a life span of 42-60 days. Epithelial origins of the cells were confirmed by immunofluorescence staining with anti-cytokeratin antibody. Polarised cells showed a columnar pattern, microvilli on their apical surface and basally located nucleus whereas non-polarised cells were flat. It was concluded that the human fallopian tube epithelial cells can be cultured in vitro to create non-polarised and polarised cell layers by using a simple and reproducible technique and this system can be a potential model to study function of the fallopian tube.     

Gap junctions in hemodichorial and hemotrichorial placentae

Gap junctions were found to be a constant feature of chorioallantoic placentae with two or three trophoblastic layers. The gap junctions connect layers I and II in hemodichorial and layers II and III in hemotrichorial placentae. Although the gap junctions vary in form and in the packing density of membrane-associated particles, they cover an extensive surface area in all species examined. The gap junctions always connect adjacent membranes of two trophoblastic layers, which show no evidence of micropinocytotic activity; at least one of these trophoblastic layers is syncytial. It is therefore concluded that the gap junctions play an important role in diaplacental transport. We consider that gap junctions act as molecular sieves, resulting in limitations in the transport of large molecules. The passage of small molecules, on the contrary, would be facilitated by the gap junctions.     

Angiotensin II receptors and angiotensin II stimulation of ciliary activity in human fallopian tube

Using an antibody (6313/G2) directed against a specific sequence in the extracellular domain of the type 1 angiotensin II receptor (AT1), we demonstrated the presence of angiotensin II (AII) receptors in human fallopian tube. Immunoperoxidase staining for AT1 receptor showed positive staining in the epithelium of the tubal mucosa. The intensity of staining varied depending upon the hormonal status at the time of salpingectomy, being strongest in the proliferative phase of the ovarian cycle and weakest after menopause. Ligand binding assay confirmed that the AII receptor concentration was highest in the mucosa of fallopian tubes from premenopausal women. Mucosa from the ampullary segment had higher concentrations of AII receptor than the fimbrial and isthmic segments in both premenopausal and postmenopausal women. Displacement studies using specific AII receptor subtype antagonists showed that approximately 60% of the total activity could be displaced by CGP42112B (type 2 specific) and 40% by losartan (AT1 specific). Immunoblotting confirmed that the antibody detected a protein of approximately 60 kDa. Functional studies showed that AII had a stimulatory action on tubal ciliary beat frequency, but had no significant effect on myosalpingseal activity. This effect was achieved at nanomolar concentrations of AII; further increases in the AII concentration were without additional effect. The stimulatory effect of AII was inhibited by the specific AT1 antagonist losartan, whereas the type 2 antagonist, CGP42112B, had no effect. The data demonstrate that AII may play an important role in ovum transport and fertility.     

Lectin histochemistry of fallopian tube epithelial cells. Relation to ovum transport and ovum pickup

BACKGROUND: This study was designed to investigate whether collagen type I degradation is altered in patients with essential hypertension and whether this alteration could be related to disturbances in the serum matrix metalloproteinase pathway of collagen degradation. A second aim of the study was to assess whether some relation exists between serum markers of collagen type I degradation and left ventricular hypertrophy in hypertensive patients. METHODS AND RESULTS: We measured serum concentrations of carboxy-terminal telopeptide of collagen type I (CITP) as a marker of extracellular collagen type I degradation, of total matrix metalloproteinase-1 (MMP-1), or collagenase, of total tissue inhibitor of metalloproteinases 1 (TIMP-1), and of MMP-1/TIMP-1 complex in 37 patients with never-treated essential hypertension and in 23 normotensive control subjects. Serum concentrations of free MMP-1 and free TIMP-1 were calculated by subtracting the values of MMP-1/TIMP-1 complex from the values of total MMP-1 and total TIMP-1, respectively. Measurements were repeated in 26 hypertensive patients after 1 year of treatment with the ACE inhibitor lisinopril. Baseline free MMP-1 was decreased (P<0.001) and baseline free TIMP-1 was increased (P<0.001) in hypertensives compared with normotensives. No significant differences were observed in the baseline values of CITP between the 2 groups of subjects. Hypertensive patients with baseline left ventricular hypertrophy exhibited lower values of free MMP-1 (P<0.01) and CITP (P<0.05) and higher (P<0.001) values of free TIMP-1 than hypertensive patients without baseline left ventricular hypertrophy. Treated patients attained an increase (P<0.001) in free MMP-1 and a decrease (P<0.05) in free TIMP-1. In addition, serum CITP was increased (P<0.05) in treated hypertensives compared with normotensive subjects. CONCLUSIONS: These findings suggest that systemic extracellular degradation of collagen type I is depressed in patients with essential hypertension and can be normalized by treatment with lisinopril. A depressed degradation of collagen type I may facilitate organ fibrosis in hypertensive patients, namely, in those with left ventricular hypertrophy.     

Interfollicular fibroblasts in the human thyroid gland: recognition of a CD34 positive stromal cell network communicated by gap junctions and terminated by autonomic nerve endings

While epithelial structure and functions have been substantially investigated in many organs, the mesenchymal elements have received less attention. Compared with follicular epithelial cells, there are a few morphological studies on the stroma of human thyroid gland. In order to characterize more fully and assess its possible functions, 15 samples of surgical and autopsy human thyroid tissue were studied by classical histology, immunohistochemistry, transmission electron microscopy, electron microscopic immunohistochemistry, and scanning electron microscopy. In human thyroid gland, the interfollicular connective tissue surrounding the follicles contained collagenous matrix, fibroblasts, unmyelinated nerve fibers with Schwann cells, small blood vessels, lymphatics, lymphocytes, plasma cells, macrophages, and mast cells. At the ultrastructural level, gap junctions between the cytoplasmic processes of interfollicular fibroblasts constituted a novel observation. Immunohistochemistry using a monoclonal antibody against Cx43 confirmed the distribution of gap junctions between stromal fibroblastic cells, which was compatible with the ultrastructural findings. The frequent and intimate association of fibroblastic processes with nerve terminals was also shown. Interfollicular stromal fibroblasts also stained with CD34. The main constituent of the human thyroid stromal tissue was a CD34 positive reticular network involving fibroblasts, mononuclear cells and nerve terminals. It represents a highly ordered stroma, with potential structural and functional similarities to the stroma of bone marrow (Yamazaki and Allen, 1990).     

Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes

The hepatocytes in the mature normal liver are tightly coupled through gap junctions, except during compensatory hyperplasia (regeneration) after partial hepatectomy when the gap junctions become down-regulated. The significance of this down-regulation has been a long-standing enigma. The present study of hepatocytes in primary culture and in the regenerating liver aimed at defining the relationship, if any, between hepatocyte gap junctional communication and proliferation. Gap junctional down-regulation in the regenerating liver appeared to be a specific phenomenon because desmosomes and the surface contact area between neighboring hepatocytes remained constant. All agents and conditions (dexamethasone in vivo; dexamethasone, cyclic adenosine monophosphate, serum, and high cell density in vitro) delaying gap junctional down-regulation also increased the lag before the cells reached competence to enter S phase. This raised the possibility that hepatocyte DNA replication was inhibited through preservation of gap junctions. However, we disproved this assumption by showing that the DNA replication (more specifically the G1/S transition rate constant) was inhibited even in hepatocytes completely devoid of gap junctional communication. The teleological advantage of linking gap junctional down-regulation to hepatocyte G1 progression therefore may not be to trigger DNA replication but to ensure that proliferating hepatocytes and hepatocytes responsible for liver-specific metabolic functions maintain separate pools of metabolites and signaling molecules.     

Gap junctions and particle aggregates in the tegumentary syncytium of a trematode

The tegumentary syncytium of a Trematode is studied by transmission EM and freeze-fracture with the following results. (1) Infoldings of the basal plasma membrane suggest that transport of water and solutes occur through the tegument. (2) Heterocellular gap junctions are found between the tegumentary cell bodies and the parenchymal cells. Gap junctional particles, 8 nm in diameter, are visible on the P face of membrane and form an irregular pattern. (3) Orthogonal arrays of small particles (6 nm in diameter) are abundant on the P face of the tegument basal plasma membrane and on the cell necks connecting tegumentary cell bodies to the tegument. (4) Hemidesmosomal particles are found on the E face of the tegument basal plasma membrane. The significance of these structures with respect to tegumentery permeability and exchanges with parenchyma are discussed.     

Neurogenic relaxation mediated by vasoactive intestinal polypeptide (VIP) in the isthmus of the human fallopian tube

The smooth musculature of the Fallopian tube is important for normal ovum transport, fertilization and implantation. Little is known about the factors controlling the motor activity of the isthmic sphincter. Studies were performed on smooth muscle preparations from the human tube in vitro. Electrical field stimulation of the nerves in the isthmic region reduced the motor activity, particularly in the circular muscle. The response was unaffected by adrenergic and cholinergic antagonists, but blocked by tetrodotoxin, suggesting a neural involvement. Vasoactive intestinal polypeptide (VIP) was considered a likely candidate for the neural mediation of this response in view of the high density of VIP-containing nerve fibres in this region, and in view of the fact that exogenous VIP causes a marked reduction of the tubal motor activity. To test whether VIP might be the endogenous mediator of this effect, nerve stimulation was carried out in the presence of large amounts of exogenous VIP in order to occupy all VIP receptors; the motor inhibitory action of VIP was counteracted by vasopressin. Under these conditions, nerve stimulation failed to reduce isthmic motor activity. This was not due to vasopressin since reduction occurred in the presence of this peptide alone. The results suggest that VIP is responsible for the neurogenic inhibition of motor activity in the isthmus region of the human Fallopian tube.     

Receptor tyrosine kinase expression in human bone marrow stromal cells

A case of crossed aphasia is presented in a strongly right-handed 77-year-old white female without history of familial sinistrality or prior neurological illness. She developed a right middle cerebral artery infarction documented by CT and accompanied by obvious clinical signs of a conduction aphasia with some resolution but continuing obvious language defect after 9 weeks in rehabilitation. Comprehensive neuropsychological and aphasia testing suggested anomalous lateralization of phonologic-output aspects of language, emotional prosody, motor planning and body schema modules with usual lateralization of lexical-semantic aspects of language and visuo-spatial functions. Experimental validation of the uncrossed lexical-semantic aspects of language using tachistoscope methods found support for the Alexander-Annett theory that different aspects of language can be dissociated in their lateralization. The subject had difficulty identifying a semantic associate of a picture presented to the left visual field (7 errors out of 10) relative to right visual field presentation (2 errors out of 10). Bilateral free naming errors (6 and 5 errors in the left and right visual fields, respectively) occurred consistent with the aphasic presentation, suggesting phonologic-output dysfunction from the right cerebral vascular accident. Implications of the results for aphasia classification are discussed.     

Ovarian steroid-modulated stromelysin-1 expression in human endometrial stromal and decidual cells

This study examined steroid-regulated expression of the metalloproteinase stromelysin-1 in primary human endometrial stromal and decidual cells. Immunoblot analysis using a specific polyclonal antibody against stromelysin-1 revealed that the progestin medroxyprogesterone acetate (MPA) produced a time-dependent reduction in a band at 50,000 mol wt. Although the cells were refractory to estradiol (E2) alone, E2 plus MPA further reduced the intensity of this stromelysin-1 zone. By 6 days of incubation, MPA inhibited levels of secreted stromelysin-1 by one third, and E2 plus MPA inhibited stromelysin-1 levels by two thirds compared with the control values. This differential responsiveness of the stromal cells to the two steroids is reported for several biochemical end points of decidualization. Northern analysis indicated pronounced inhibition of stromelysin-1 messenger ribonucleic acid (mRNA) by E2 plus MPA over a concentration range that simulated circulating progesterone levels of the luteal phase (10(-8) mol/L) through pregnancy (10(-6) mol/L). After suppression of stromelysin-1 expression in the stromal cell monolayers by E2 plus MPA, steroid withdrawal led to a several-fold enhancement of stromelysin-1 mRNA by 4 days and of the stromelysin-1 protein by 7 days. Given its actions in degrading several extracellular matrix components and activating other MMP zymogens, steroid withdrawal-enhanced stromelysin-1 activity could mediate a proteolytic cascade that promotes the rapid tissue destruction and vascular disruption associated with menstruation. Stromelysin-1 expression by cultured decidual cells isolated from first trimester endometrium was also reduced by MPA and synergistically reduced by E2 plus MPA. As activation of the 92-kilodalton gelatinase/type IV collagenase, a crucial mediator of trophoblast invasiveness, is stromelysin-1 dependent, reduced decidual stromelysin-1 production could help to limit trophoblast invasion.     

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.     

Associations between TGF-beta1 receptors in human bone marrow stromal cells

Bone marrow stromal cells are required for sustained haemopoiesis. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine present in the bone marrow microenvironment which regulates the expression of several cytokines, cytokine receptors and cell adhesion elements. The TGF-beta receptors type I and II, and endoglin, mediate TGF-beta1 binding to the membrane of human bone marrow stromal cells. [125I]TGF-beta1-affinity labelling experiments showed that three different anti-endoglin monoclonal antibodies co-immunoprecipitated a 68 kD TGF-beta1-labelled polypeptide together with TGF-beta1/endoglin complexes. Here, we have shown that the 68 kD receptor corresponds to the type I receptor, indicating that endoglin and the type I receptor associate on the membrane of these cells upon ligand binding. The expression of endoglin by stromal cells was found to be up-regulated by TGF-beta1, but not by IL-1beta. The association of endoglin with signalling components of the TGF-beta receptor system on the membrane of bone marrow stromal cells might modulate TGF-beta1 access to the signalling pathways, and therefore it could regulate TGF-beta1-mediated stromal cellular responses.     

Estrone formation from dehydroepiandrosterone in cultured human breast adipose stromal cells

OBJECTIVE: To describe the phenotype in a family with dominantly inherited cone-rod dystrophy with chromosome assignment to a 19q locus, and to correlate this with current classifications of this retinal dystrophy. DESIGN: A detailed clinical examination including Goldmann perimetry was undertaken in all family members. Six members under the age of 30 years underwent dark-adapted electroretinography, color contrast-sensitivity measurement, dark-adapted static perimetry, and dark adaptometry. PATIENTS: The study included 34 affected and 22 unaffected patients in four generations of a pedigree that manifested autosomal dominant cone-rod retinal dystrophy linked to a chromosome 19q locus by genetic linkage analysis. RESULTS: Loss of visual acuity occurred in the first decade of life, onset of night blindness occurred after 20 years of age, and little visual function remained after the age of 50 years. Central and, later, peripheral retinal fundus changes were associated with central scotoma, pseudoaltitudinal field defects, and finally global loss of function. Psychophysical and electrophysiologic testing before the age of 26 years showed more marked loss of cone than rod function. CONCLUSIONS: The phenotype associated with this mutation does not fit well into previous subtypes of cone-rod dystrophy. Further studies will be needed to correlate specific genetic mutations in this group of conditions with the various clinical phenotypes.     

Requirement for transglutaminase in progesterone-induced decidualization of human endometrial stromal cells

Differentiation of endometrial stromal cells (decidualization) is essential for embryo implantation and maintenance of pregnancy. By sequential complementary DNA subtractive hybridization, one of the messenger RNAs (mRNA) induced by progesterone in human endometrial stromal cells decidualized in vitro was identified as that of a tissue transglutaminase type II (TGase). TGase mRNA was induced within 6 h after the addition of progesterone to the culture, and the effect was dose dependent. Both the TGase inhibitor monodansylcadaverine and oligodeoxynucleotide complementary to the TGase mRNA inhibited the decidualization, as assessed by PRL production and morphological transformation. Expression of TGase mRNA in human decidua and endometria exposed to high levels of progesterone in vivo was demonstrated by Northern blotting and in situ hybridization. These data suggest that TGase is necessary for the decidualization of human endometrial stromal cells and that clarification of the mechanism of action of TGase will facilitate further insight into the diagnosis and treatment of infertility.     

Gap junctional intercellular communication of primary and asbestos-associated malignant human mesothelial cells

We examined gap junctional intercellular communication (GJIC) of primary human mesothelial cells and cell lines of asbestos-associated human pleural mesotheliomas, and the effect of asbestos and other mineral fibres on these cells. In homologous cultures, the GJIC capacity of six out of seven tumour cell lines was markedly less than for primary mesothelial cells. This defect in GJIC appeared not to be at the expression level of mRNA and protein of the gene encoding the 43 kDa gap junction protein. In heterologous cocultures of tumour cells and primary mesothelial cells, however, 80-90% of the tumour cell/normal cell contacts were functional. Exposure of primary mesothelial cells to TPA, a phorbol ester tumour promoter, resulted in marked inhibition of GJIC, being an action common to numerous tumour promoters. Such an effect though was not observed with the carcinogenic mesothelioma-inducing mineral fibres chrysotile and amosite, neither with glass wool. These results suggest that a permanent defect in GJIC capacity is a common feature of human mesothelioma cells, but how mineral fibres are involved in the process of mesotheliomagenesis is still unclear.     

Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized

We have investigated the synthesis, axonal transport, and processing of the beta-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a t1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]-methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is approximately 110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

Rapid sprouting of filopodia in nerve terminals of chromaffin cells, PC12 cells, and dorsal root neurons induced by electrical stimulation

Rapid morphological changes induced by direct electrical stimulation of nerve terminals were studied by using video-enhanced differential interference contrast microscopy at a very high magnification (12,000x). We used mainly cultured bovine chromaffin cells, which developed neurite-like processes, and PC12 cells, which showed neuronal differentiation upon NGF treatment. In a few cases, primary neurons of the rat dorsal root ganglion were also examined. Brief pulse stimulation of the terminals and varicosities induced exocytosis accompanied by rapid formation of filopodia. These filopodia, 0.1-0.2 micron in diameter and up to 10 microns in length, formed within a few hundreds of milliseconds and then retracted within tens of seconds. They could also be induced by K depolarization. This rapid filopodial sprouting strongly depended on the presence of extracellular Ca2+ and could be abolished in a medium containing a Ca chelator (EGTA) or La2+. Anti-cytoskeletal agents colchicine and cytochalasin B failed to block this response completely but lidocaine fully suppressed it. Quantitative analysis of exocytosis and filopodial sprouting showed that they were independent events, not directly linked to each other, having different thresholds usually higher for filopodial formation. In PC12 cells, the extent of filopodial sprouting varied with the state of differentiation of the cells, suggesting a functional role of rapid sprouting during a particular phase of their differentiation. Filopodia could be induced with greater ease by repetitive stimulation. The same responses may occur at growth cones approaching the target cells or even at mature synapses particularly after repetitive electrical activity, possibly playing a role in use-dependent synapse formation or plasticity.