Chlamydia trachomatis infection of human fallopian tube organ cultures

The pathogenic events that precede Chlamydia trachomatis salpingitis in the human fallopian tube have not been fully described. We used a model of human fallopian tubes in organ culture (HFTOC) infected with strain E/UW-5/CX of C. trachomatis to study these events. The model supported sustained C. trachomatis infection as demonstrated by recovery of viable C. trachomatis from medium and tissue over 5-7 d. However, the level of infectivity was low. Maximal infection occurred at 72 h after initial inoculation. In contrast to gonococcal infection of the HFTOC, C. trachomatis did not damage overall ciliary function of HFTOC. However, a local direct cytotoxic effect characterized by loss of microvilli and disruption of cell junctions was noted when multiple chlamydial elementary bodies attached to mucosal cells. Beginning at 24 h, and continuing throughout the course of C. trachomatis infection of HFTOC, ruptured epithelial cells releasing elementary bodies were noted. Chlamydial inclusions were seen in the mucosa by 72 h in approximately 6% of both ciliated and nonciliated epithelial cells. Mucosal inclusions contained all forms of the C. trachomatis developmental cycle. These data suggest that factors present in the human fallopian tube may limit susceptibility to chlamydial infection but support the use of the HFTOC model in the study of the pathogenesis of C. trachomatis salpingitis.     

Energy metabolism of the human fallopian tube.

The consumption of oxygen (QO2), the production of lactate and the profile of four key metabolic enzymes were measured in small samples of human oviductal mucosa (endosalpinx) removed at surgery. The QO2 in the absence of substrate was 3.4 microliters O2 (mg dry wt)-1 h-1, a value typical of quiescent tissue. The QO2 was stimulated by glucose, but diminished by glutamine and acetoacetate. Tissue lactate production was low and not increased by glucose. Hexokinase had the highest activity of the enzymes measured, followed by 2-oxoglutarate dehydrogenase; 6-phosphofructokinase and glycogen phosphorylase had low activities. The data are consistent with the proposition that glucose is a major metabolic fuel for human endosalpinx.     

Cytopathic effects induced by Epstein-Barr virus replication in epithelial nasopharyngeal carcinoma hybrid cells.

NPC-KT cl.S61, a subclone derived from an epithelial-nasopharyngeal carcinoma hybrid cell line (NPC-KT), showed cytopathic changes characteristic of herpesvirus replication, including formation of multinucleated giant cells and inclusion bodies, when Epstein-Barr virus replicative cycle was induced by 5-iodo-2'-deoxyuridine. Acyclovir (an inhibitor of herpesvirus DNA polymerase), Epstein-Barr virus-immune human serum, or 2-deoxyglucose (an inhibitor of the glycosylation) interfered with syncytium formation, indicating that a virus-specified glycoprotein belonging to the late group is responsible for cell fusion induced by Epstein-Barr virus replication in cl.S61 cells.     

Isolation and monolayer culture of human fallopian tube epithelial cells

Summary In the present study we have established a pure monolayer culture system of human fallopian tube epithelial cells. The cells were isolated using collagenase digestion, and were cultured in Medium 199 supplemented with 15% fetal bovine serum. The epithelial cells derived from primary and secondary culture were characterized using immunocytochemical staining and electron microscopy. The cells continued to grow for 2 to 3 wk once the monolayer culture of the cells was established. It is currently possible to maintain the cultures until the third generation. Proliferation of these cells was enhanced by epidermal growth factor but not by basic-fibroblast growth factor, insulin, transferrin, estradiol-17β, or progesterone. This culture system offers a good model for determining characteristics of the tubal epithelium and would permit effective study of co-culture with embryos.     

Experimental pathogenicity of mollicutes from bovines with reproductive disorders in rabbit fallopian tube organ culture.

Mollicutes (10) belonging to Mycoplasma and Acholeplasma isolated from various reproductive disorders were tested in rabbit fallopian tube (FT) organ culture. Parameter for describing pathogenic status of Mollicutes in rabbit FT organ culture included multiplication of organisms, and its effect on ciliary activity along with histopathological changes in FT explants. M. mycoides (LC, Y-Goat), M. bovoculi, M. bovigenitalium, Mycoplasma sp. and A. oculi were categorized as pathogenic; A. axanthum and A. laidlawii as mildly pathogenic; and M. bovis, M. arginini. and A. granularum, as nonpathogenic to rabbit FT organ culture. Thus, rabbit FT organ culture is recommended for use as a suitable and economical in vitro model to assess the pathogenicity of Mollicutes of reproductive tract origin.     

Immunohistochemical localization of galactosyltransferase in the normal human ovary and fallopian tube

Galactosyltransferase (UDP-galactose: 2-acetamido-2-deoxy beta-D-glucopyranose beta-(1-4) transferase) in human tissue specimens from ovaries and the corresponding fallopian tubes was localized immunohistochemically for light microscopy. An affinity-purified rabbit anti-human milk galactosyltransferase antibody was used. Intracellular galactosyltransferase was found to be localized to the juxtanuclear (Golgi) region of the secretory cells of the fallopian-tube epithelium and to the ovarian stromal cells involved in steroid-hormone production. Cell-surface galactosyltransferase was localized to ciliated cells of the fallopian-tube epithelium. During the follicular phase of the menstrual cycle, galactosyltransferase was found only in the Golgi regions of theca interna cells of the ovarian graafian follicle, and in the fallopian tube was found predominantly on the cilia of epithelial cells. During the luteal phase, galactosyltransferase was abundant in the Golgi regions of granulosa lutein cells of the corpus luteum, and was predominant in the secretory cells of the tubal epithelium. Galactosyltransferase was not detected on the mesothelial ovarian surface. The results demonstrate that the cellular distribution and location of galactosyltransferase correlates with phenotypic differentiation and varies during the human female hormonal cycle.     

Primary culture and immortalization of human fallopian tube secretory epithelial cells

Primary human fallopian tube secretory epithelial cell (FTSEC) cultures are useful for studying normal fallopian tube epithelial biology, as well as for developing models of fallopian tube disease, such as cancer. Because of the limited ability of primary human FTSECs to proliferate in vitro, it is necessary to immortalize them in order to establish a cell line that is suitable for long-term culture and large-scale in vitro experimentation. This protocol describes the isolation of FTSECs from human fallopian tube tissue, conditions for primary FTSEC culture and techniques for establishing immortal FTSEC lines. The entire process, from primary cell isolation to establishment of an immortal cell line, may take up to 2 months. Once established, immortal FTSECs can typically be maintained for at least 30 passages.     

Studies on Infectious Mononucleosis: I. Cytopathic Agents and Immunofluorescence Studies

Viral studies were carried out on throat swabs, rectal swabs and washed white blood cells from 27 cases of infectious mononucleosis (positive Paul-Bunnell-David-sohn test), and from 22 controls.Four cytopathic agents were isolated in the test group, two of which were readily subcultured for at least three successive passages.Three cytopathic agents were recovered in the control group, two of which have been identified as adenovirus type 5 and adenovirus type 3. The unidentified agents tested so far are sensitive to ether and to pH 3. The results of acridine-orange staining and the immunofluorescence technique, using a conjugated control serum and two conjugated convalescent infectious mononucleosis sera, indicate that the isolated agent or agents in the test group are RNA-type agents with a cytoplasmic cycle of development. The overall results of this study lead the authors to suspect a respiratory syncytial-like myxovirus as the as yet unidentified agent which they recovered.     

Primary culture of human fallopian tube epithelial cells and co-culture of early mouse pre-embryos.

We have established a monolayer culture system for human fallopian tube epithelial cells. The cells were isolated from tubes using collagenase digestion, and were cultured in Ham's F-10 supplemented with 15% fetal calf serum. The epithelial cells derived from culture were characterized using immunocytochemical staining and electron microscopy. These cells were stained with antikeratin and anti-epithelial membrane antigen, but showed no staining after treatment with antivimentin. Electron microscopy showed many microvilli on the cell surface and tight junctions or desmosomes at areas of cell-cell contact. Cell proliferation was enhanced by epidermal growth factor, but not by fibroblast growth factor, insulin, transferrin, estradiol-17 beta, or progesterone. The 2-cell ICR mouse pre-embryos were co-cultured for 4 days with tubal epithelial cells (A) (n = 98), in cell-conditioned medium (B) (n = 83), or in medium alone (C) (n = 72). During the first 24 h in culture, for groups A and B, the rates of cleavage to the 4-cell stage were 90.9% and 81.9%, respectively. Cleavage rates in these two groups were significantly higher (P = 0.0012, P less than 0.00001) than in group C (56.9%). After 72 h in culture, the rates of development to the blastocyst stage were significantly higher for groups A and B compared to group C (89.6% and 73.5% vs. 54.5%, P less than 0.00001, P = 0.0002). These results suggest that factor(s) from tubal epithelial cells may facilitate the development of mouse pre-embryos throughout the pre-implantation stages.     

Characterization and localization of estrogen and progesterone receptors of human fallopian tube

The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopian tube was investigated by immunohistochemical localization with specific monoclonal antibodies. Nuclear immunostaining was observed. Intense PR immunostaining was seen in tissues obtained at mid cycle and luteal stages of the normal menstrual cycle. On the other hand, enhanced staining for ER was seen in early follicular phase and mid cycle. Menopausal tissues showed negligible staining for both ER and PR. The ER and PR were characterized for their molecular size, anatomical distribution and levels during the menstrual cycle and in menopause. ER protein was present throughout the cycle and also during menopause. Western blot analysis revealed two forms of ER approximately 66 kDa and a truncated from approximately 49 kDa in hFT. Presence of A [approximately 90 kDa] and B [approximately 120 kDa] isoforms of human PR was detected. Follicular and early luteal tissue possessed relatively high concentration of immunoreactive PR whereas it was almost undetectable in menopausal tissues. These results suggests that ER and PR are regulated by the changing ovarian steroid hormones.     

Hairy-root organ cultures for the production of human acetylcholinesterase

Background  

Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.     

Aggregated proteoglycan synthesis in organ cultures of human nucleus pulposus.

Proteoglycans isolated under associative conditions in the presence of protease inhibitors from human nucleus pulposus contained 17% aggregate and 83% non-aggregating monomer (Kav = 0.5 on Sepharose CL-2B). Isolated aggregate after reduction and alkylation was resolved into two components (Kav = 0.15 and 0.43) on Sepharose CL-2B. Labeled proteoglycans isolated from parallel samples pulsed with [35S]sulfate and chased for up to 18 h were present largely as aggregated material (up to 78%). Reduction and alkylation of the labeled samples gave a labeled proteoglycan monomer with Kav = 0.15. Both the labeled and unlabeled chondroitin sulfate chains had the same distribution on Sepharose CL-6B and equivalent molecular weights (Mr = 2.0 x 10(3)). After chondroitinase ABC digestion, the unlabeled keratan sulfate-protein core was polydisperse with a Kav = 0.38 on Sepharose CL-4B while the labeled keratan sulfate-protein core had a Kav = 0.05. This indicates that the newly synthesized proteoglycan had a large core protein and suggests that the proteoglycans present in nucleus pulposus are originally synthesized as large molecular weight, aggregating proteoglycans.     

Biomorphosis of the human fallopian tube mucosa; a contribution to the problem of morphology and aging of the uterine tube. II. Histochemical findings

The present histochemical findings of investigations on the biomorphosis of the human tubal mucosa indicate that mucosubstances and glycogen localized in the epithelium exhibit age-dependent changes with regard to occurrence and localization. In the embryo-fetal time PAS-positive, diastase-resistant substances are localized in the epithelium, at first basally, and later perinuclearly. In the neonatal phase the distal tubal epithelium has only a weak PAS reaction, and the proximal epithelium has a detectable supranuclear activity. In the end of the 1st decade of the life the epithelium possesses a periodate reactive diastase-sensitive material densely deposited in the preampullar and ampullar parts of the uterine tube, preferably. Afterwards PAS-positive diastase-sensitive and diastase-resistant substances, respectively, are regularly present, in which in the fertile age of the women a regular pattern of the PAS activity can be demonstrated. In the period of the regressive age it is possible to establish a increasing disturbance of the usual cellular picture of the tubal epithelium. In connection with the structural changes a increase of histochemical different reacting cell groups is evident. As a result, a dissociated cellular picture has developed. Epithelial glycoproteins and glycogen can be detected in the mucosa up to the phase of the senium.