Human Fallopian Tube Organ Culture Research Articles

Interleukin-1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection

Chlamydia trachomatis infection is associated with severe Fallopian tube tissue damage leading to tubal infertility and ectopic pregnancy. To explore the molecular mechanisms behind infection an ex vivo model was established from human Fallopian tubes and examined by scanning electron microscopy and immunohistochemistry. Extensive tissue destruction affecting especially ciliated cells was observed in C. trachomatis infected human Fallopian tube organ culture. Interleukin-1 (IL-1) produced by epithelial cells was detected after infection. Addition of IL-1 receptor antagonist (IL-1RA) completely eliminated tissue destruction induced by C. trachomatis. The anti-inflammatory cytokine IL-10 reduced the damaging effect of C. trachomatis infection, however, to a lesser extent than IL-1RA. Furthermore, IL-1 was found to induce IL-8, a neutrophil attractant, using a signal transduction pathway involving p38 MAP kinase. Consequently, IL-1 has the potential to generate a cellular infiltrate at the site of infection in vivo. Blocking the IL-1 receptors by IL-1RA eliminated tissue destruction and cytokine production. Hence, these studies show the importance of IL-1 in initiating the tissue destruction observed in the Fallopian tube following C. trachomatis infection. Because leukocytes are absent in the ex vivo model, this study strongly indicates that IL-1 is the initial proinflammatory cytokine activated by C. trachomatis infection.

Chlamydia trachomatis enhances the expression of matrix metalloproteinases in an in vitro model of the human fallopian tube infection

Objective: The sequelae of sexually transmitted Chlamydia trachomatis infection include fallopian tube scarring, which implies modification of the extracellular matrix. Our objective was to describe the production of two matrix metalloproteinases in response to chlamydial infection in vitro. Study Design: Human fallopian tube organ cultures were infected with Chlamydia, and the production of matrix metalloproteinases was assessed by gelatin zymography, antigen capture enzyme-linked immunosorbent assay, immunohistochemistry, and in situ zymography. Results: Significantly elevated levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 were found in supernatants of infected segments. Immunohistochemistry and in situ zymography revealed that epithelial cells tended toward matrix metalloproteinase-2 production and that matrix metalloproteinase-9 exhibited a more diffuse stromal staining pattern. Conclusion: Matrix metalloproteinase-2 and matrix metalloproteinase-9 are expressed in vitro in response to chlamydial infection. Enhanced matrix metalloproteinase production in some individuals in response to infection may lead to tubal scarring through the increased turnover and subsequent repair of the extracellular matrix. (Am J Obstet Gynecol 2002;187:1377-83.)

Neisseria gonorrhoeae mutants altered in toxicity to human fallopian tubes and molecular characterization of the genetic locus involved.

In an effort to identify potential cytotoxins expressed by Neisseria gonorrhoeae, we have identified a locus that, when mutated in the gonococcus, results in a significant increase in toxicity of the strain to human fallopian tube organ cultures (HFTOC). This locus, gly1, contains two open reading frames (ORFs) which are likely cotranscribed. ORF1 encodes a polypeptide of 17.8 kDa with a signal sequence that is recognized and processed in Escherichia coli and N. gonorrhoeae. The 15.6-kDa processed polypeptide has been observed in membrane fractions and filtered spent media from cultures of E. coli expressing gly1 and in outer membrane preparations of wild-type N. gonorrhoeae. The gly1 locus is not essential for bacterial survival, and it does not play a detectable role in epithelial cell adhesion, invasion, or intracellular survival. However, a gly1 null mutant causes much more damage to fallopian tube tissues than its isogenic wild-type parent. A strain complemented in trans for the gly1 mutation showed a level of toxicity to HFTOC similar to the level elicited by the wild-type parent. Taken together, these results indicate an involvement of the gly1 locus in the toxicity of N. gonorrhoeae to human fallopian tubes.

Tumor necrosis factor-alpha response to infection with Chlamydia trachomatis in human fallopian tube organ culture : Ault K.A.; Tawfik O.W.; Smith-King M.M.; Gunter J.; Terranova P.F. USA Am J Obstet Gynecol 1996 175/5 (1242–1245)

Tumor necrosis factor-alpha response to infection with Chlamydia trachomatis in human fallopian tube organ culture : Ault K.A.; Tawfik O.W.; Smith-King M.M.; Gunter J.; Terranova P.F. USA Am J Obstet Gynecol 1996 175/5 (1242–1245)

Tumor necrosis factor-α response to infection with Chlamydia trachomatis in human fallopian tube organ culture

OBJECTIVE: Our purpose was to determine whether tumor necrosis factor-α is produced in response to infection with Chlamydia trachomatis in the fallopian tube. STUDY DESIGN: Fallopian tubes were harvested at the time of abdominal hysterectomy and processed by standard tissue culture techniques. Tubal segments were inoculated with Chlamydia trachomatis serotype E/UW-5/CX. At 48 hours of incubation supernatant fluid was assayed for tumor necrosis factor- α. Tubal segments were stained for chlamydial inclusions and tumor necrosis factor- α by use of immunohistochemical techniques. RESULTS: Mean tumor necrosis factor-α levels for infected segments were 92.1 ± 21.3 pg/ml (mean ± SEM) and for control segments were 61.9 ± 13.9 pg/ml ( p = 0.03 by paired t test). Tumor necrosis factor- α was predominantly localized in the tubal epithelium. CONCLUSIONS: Tumor necrosis factor-α is produced in response to chlamydial infection by the human fallopian tube. It is an important proinflammatory cytokine and may promote the production of other cytokines and immune-mediated damage of the fallopian tube. (Am J Obstet Gynecol 1996;175:1242-5.)

Digital confocal microscopy allows measurement and three-dimensional multiple spectral reconstruction of Neisseria gonorrhoeae/epithelial cell interactions in the human fallopian tube organ culture model.

A strategy for measuring Neisseria gonorrhoeae attachment and invasion in the human Fallopian tube organ culture (FTOC) model via computerized image analysis (CIA) combined with "digital" confocal microscopy (DCM) was tested. DCM on serial image stacks of fluorescent latex beads reduced out-of-focus light propagation in the Z-axis (p < 0.005) and improved the shape factor of lateral three-dimensional reconstructions of the beads (p < 0.001). Sections of tissue infected for 44 hr with piliated, Opa+ gonococci were stained with fluorescein-labeled monoclonal anti-gonococcal antibodies, rhodamine-labeled phalloidin, and Hoechst 33342. Serial images collected at identical focal planes for each fluorochrome were subjected to DCM. Epithelial cytoplasmic regions of interest defined by rhodamine-stained actin were superimposed on the corresponding fluorescein-stained and Hoechst-stained images. Fluorescent objects defined by gray-scale threshold were measured by computerized image analysis using different border treatments to differentiate attached from intracellular gonococci or count cell nuclei. Compared with raw images, measurement of DCM images was less dependent on threshold choice (p < 0.05). DCM augments conventional microscopy in removing out-of-focus light from fluorescent images, in reconstructing three-dimensional images, and in quantitatively differentiating extracellular from intracellular gonococci in a natural target tissue.

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.

Cytopathic effects of the pathogenic Neisseria. Studies using human fallopian tube organ cultures and human nasopharyngeal organ cultures.

Infection of mucosal surfaces by N. gonorrhoeae and N. meningitidis may result in inflammation indicating potential injury to host cells. We used human fallopian tube organ cultures (FTOC) and human nasopharyngeal organ cultures (NPOC) to study the mechanisms by which gonococci and meningococci damage human mucosal surfaces. Early in the course of FTOC infected with gonococci and NPOC infected with meningococci, damage was most apparent to ciliary activity. Loss of ciliary activity was accompanied by sloughing of ciliated cells. The damage to ciliated cells was not associated with attachment of gonococci or meningococci to these cells or the presence of organisms within ciliated cells. Infection with the commensal N. subflava did not result in significant damage to human FTOC or NPOC ciliary activity. LPS appears to be a major toxin of gonococci for human FTOC ciliated cells. Gonococcal peptidoglycan fragments also damage FTOC ciliary activity. Both piliated (P+) and nonpiliated (P-) gonococci and meningococci damage FTOC and NPOC ciliary activity, but P+ organisms damage ciliary activity more rapidly than P- organisms. Damage to FTOC ciliated cells was produced by less than 10 micrograms/ml of purified gonococcal and meningococcal LPS. By 1-2h after exposure to LPS, vesicles containing LPS were distributed throughout the cytoplasm of ciliated cells. Polymyxin B neutralized LPS-induced damage, suggesting that the lipid A portion of LPS was the toxic moiety. In contrast, purified gonococcal and meningococcal LPS at 100 micrograms/ml did not damage human NPOC or FTOC from rabbits, pigs and cows. These studies indicate that N. gonorrhoeae and possibly N. meningitidis damage ciliated epithelial cells indirectly by release of toxins from the organisms. The differences in susceptibility of FTOC and NPOC to LPS may suggest changes in density of receptors for LPS and may help explain variation in severity of gonococcal and meningococcal interactions at different human mucosal surfaces.

Analysis of damage to human ciliated nasopharyngeal epithelium by Neisseria meningitidis.

We used an in vitro model of human nasopharyngeal tissue in organ culture to evaluate the effects of Neisseria meningitidis on human cilia and ciliary function. Encapsulated, viable meningococci damaged ciliated epithelium of nasopharyngeal organ cultures, whereas Neisseria subflava, a commensal species, did not. Meningococcus-induced ciliary damage was due to loss of ciliated cells to which meningococci were not attached. Damage was seen with piliated and nonpiliated meningococci and did not appear to require the presence of other specific meningococcal surface proteins. Meningococcal viability was a requirement for both ciliary damage and interactions of meningococci with microvilli of nonciliated epithelial cells. That is, filter-sterilized supernatants from meningococcus-infected organ cultures, heat-killed meningococci at high inoculum, and purified meningococcal or gonococcal lipopolysaccharide at concentrations of 100 micrograms/ml did not damage ciliary activity of nasopharyngeal organ cultures. In contrast, meningococcal lipopolysaccharide at 10 micrograms/ml markedly damaged ciliary activity of human fallopian tube organ cultures, suggesting a selective toxicity of lipopolysaccharide for specific human ciliated cells. Damage to nasopharyngeal ciliated epithelium by N. meningitidis may be an important first step in meningococcal colonization of the human nasopharynx, but meningococcal lipopolysaccharide does not appear to be directly responsible for this toxicity.

In vitro modeling of acute salpingitis caused by Neisseria gonorrhoeae

Normal human fallopian tube organ culture was used as an in vitro model to study Neisseria gonorrhoeae infections. The effects of various gonococcal colony phenotypes on morphology of the epithelium were studied by scanning electron microscopy. After 30 minutes' incubation, there was striking attachment of piliated transparent phenotypes to the epithelium; however, there was no obvious pathology. After 24 hours' incubation, there were microcolony formation, slight swelling and hyperplasia of the mucosa, and occasional focal necrosis and sloughing of ciliated cells. Tissue from acute salpingitis showed widespread destruction of mucosa, hyperplasia, and crypt formation. Duplication of these findings in vitro may require longer incubation and the addition of other host factors.