Establishment of a ciliated epithelial cell line from human Fallopian tube

Human tubal epithelial cells in primary culture were transfected with simian virus 40 (SV40) large T antigen plasmid, and an immortalized ciliated cell line, named as NT/T-S, was established without crisis. Transmission electron microscopy proved that NT/T-S cells had cilia, microvilli, junctional complexes, rough endoplasmic reticula, free ribosomes and microtubules. NT/T-S cells were evaluated preliminarily on the basis of co-culture study using surplus embryos at the 4- to 8-cell stage in our IVF and embryo transfer programme. All of the 133 embryos had >/=10% fragments (based on the surface area) and were unworthy of cryopreservation. Up to 57% (16/28) of the embryos with 10-30% fragments reached the blastocyst stage by co-culture. In contrast, blastocyst formation was observed in <10% of the control embryos, some of which were co-cultured with NFL/T cells (the immortalized human fetal liver epithelial cells) (1/16), and the others were incubated with the co-culture medium alone (1/18). Various cytokines/growth factors such as leukaemia inhibitory factor (LIF), interleukin (IL)-6, IL-8 and basic fibroblast growth factor were secreted by NT/T-S cells as well as by the tubal epithelial cells in primary culture. The establishment of a ciliated cell line will provide a valuable resource for the further studies of the Fallopian tube in the early events of pregnancy.     

A new virus cultivated only in organ cultures of human ciliated epithelium

Summary A new human respiratory virus has been isolated from a human adult with a common cold. The virus was cultivated and recognized only in organ cultures of human ciliated respiratory epithelium. The virus is ether-stable, acid-labile and it grows better at 33° C than at 37° C and in near neutral rather than alkaline medium. It is believed to be a rhinovirus with fastidious growth requirements.This investigation was supported by grants from: Alfred Österlunds Stiftelse, Malmö, Sweden, J. V. och Charlotta Lundgrens Stiftelse and the Medical Faculty, University of Lund, Sweden.     

Cytogenetic aberrations in primary cell cultures of the ovarian surface epithelium

Our objective was to determine the presence of chromosomal abnormalities in primary cultures of ovarian surface epithelial cells in women of different ages with no history of cancer. Throughout conventional cytogenetic techniques, we analyzed chromosome spreads of cultured ovarian epithelial cells from 10 donors who were 50 or more years old (B) and 16 controls between 20 and 49 years old (A), belonging to the mestizo population in Bogota DC, Colombia. Of the 26 cultures that were analyzed in passage 1, 61.5% had an abnormal chromosome complement (62.5% in A, and 60% in B). Abnormalities included polyploidies, endoduplications and monosomies. Deletions in chromosomes 3 and 11 were found in just one metaphase. None of the samples showed weaknesses or breakpoints. After transforming and applying the exact student's t-test for variance heterogeneity, we found significant differences in the frequency of metaphases, that were higher in A than in B (p=0.05), and in the frequency of polyploidies, which were higher in B than in A (p=0.044). Through the application of the Mann-Whitney test, we determined that the frequency of endoduplications was higher in A than in B (p=0.126), without reaching significant differences. There were no significant differences in the frequency of monosomies. The level of significance was set at p < or = 0.05. Taking into account that polyploidization is a marker of chromosomal instability and that the risk of cancer arising from the ovarian surface epithelium augments substantially after menopause, the increase in the frequency of age-associated polyploidies could be used as a predictor of ovarian cancer in women from an ethnically homogeneous population as the mestizo one in Bogota DC.     

白介素-2,白介素-13与输卵管粘膜上皮炎的相关性研究

目的探讨血清炎症因子白细胞介素-2（IL-2）,白细胞介素-13（IL-13）与输卵管粘膜上皮炎的关系。方法运用免疫组化结合病理图像半定量分析方法,检测30例输卵管粘膜上皮炎壶腹部标本（研究组）,30例正常输卵管壶腹部标本（对照组）的表达情况。结果IL-2在研究组输卵管粘膜中阳性表达与对照组比较,有升高趋势,但两组比较无统计学意义（P〉0.05）;IL-13在研究组输卵管粘膜中阳性表达高于对照组,两组比较有统计学差异（P〈0.05）;研究组输卵管炎患者粘膜上皮局部IL-2/IL-13显著下降（P〈0.05）。结论输卵管慢性炎会引起IL-2,IL-13表达增强,IL-2/IL-13比值下降提示可能出现TH1/TH2细胞的免疫功能失衡。     

Influenza A virus infection of primary differentiated airway epithelial cell cultures derived from Syrian golden hamsters

The ability of several different influenza A virus strains to infect and replicate in primary, differentiated airway epithelial cell cultures from Syrian golden hamsters was investigated. All virus strains tested replicated equivalently in the cultures and displayed a preference for infecting nonciliated cells. This tropism correlated with the expression of both alpha2,3- and alpha2,6-linked sialic acid on the nonciliated cells. In contrast, the ciliated cells did not have detectable alpha2,6-linked sialic acid and expressed only low amounts of alpha2,3-linked sialic acid. In contrast to clinical isolates, laboratory strains of influenza A virus infected a limited number of ciliated cells at late times post-infection. The presence of alpha2,3- and alpha2,6-linked sialic acid residues on the same cell type suggests that Syrian golden hamsters and differentiated airway epithelial cell cultures derived from hamsters may provide a system for studying the reassortment of influenza A virus strains which utilize different forms of sialic acid as a primary virus receptor.     

Fallopian tube obstruction as a sequela toChlamydia trachomatis infection

The association of tubal infertility and ectopic pregnancy with Chlamydia trachomatis infection was investigated using a case-control study design. Although culture methods failed to document active chlamydial infection in the majority of cases, serology revealed a significant association of Chlamydia trachomatis antibody with tubal infertility and ectopic pregnancy. Thirteen of 18 (72 %) women with tubal factor infertility and 18 of 32 (56 %) women with ectopic pregnancy had antibodies to Chlamydia trachomatis as compared to 11 of 49 (22 %) normal pregnant controls. Interestingly, only 7 of 18 (39 %) infertile women and 5 of 36 (14 %) women with ectopic pregnancy recalled a history of pelvic inflammatory disease. These results suggest that chlamydia-associated salpingitis, whether clinically evident or subclinical, is a major contributor to diseases of tubal dysfunction.     

Beta-galactosidase activity as a marker of senescence in primary cultures of the ovarian surface epithelium

Beta-galactosidase activity reflects the rate of cellular aging in vitro. Such activity was quantified at pH 6 in ovarian epithelial cells from 28 donors without a history of cancer, by the chemoluminiscent method. The cells were serially cultured until they achieved the state of permanent growth arrest. During the exponential growth phase, all cultures showed a similar pattern of growth and low beta-galactosidase activity. However, both in the onset of decrease replicative activity, as well as in the onset of the stationary phase, there was a significant rise in the enzyme activity. Our results showed that beta-galactosidase activity can be considered as a replicative senescence marker of the ovarian surface epithelium at pH 6.     

Secretory cell outgrowth,PAX2 and serous carcinogenesis in the Fallopian tube

The ‘p53 signature’ is a benign secretory cell outgrowth in the distal Fallopian tube that shares properties with ovarian serous cancer—including p53 mutations—and is a putative serous cancer precursor. We expanded the precursor definition to all secretory cell outgrowths (SCOUTs) of 30 or more cells and scored normal (N) and altered (A) expression of both p53 and PAX2, a gene down‐regulated in ovarian and endometrial cancer. SCOUTs were identified by BCL2/p73 staining in tubes from women with serous carcinoma, inherited mutations in BRCA1 or BRCA2 and controls. SCOUTs were prevalent in both proximal and distal tube and significantly associated with serous carcinoma versus the others (p < 0.001); 89% were PAX2 (A) and 26% were PAX2 (A)/p53 (A) (p53 signatures). PAX2 (A)/p53 (N) SCOUTs were free of p53 mutations; however, 12 of 13 p53 signatures were PAX2 (A). A tubal carcinoma and contiguous SCOUT were p53 (A)/PAX2 (A) and shared the same p53 mutation. SCOUTs are discretely localized alterations commonly containing altered expression of multiple genes within histologically benign tubal epithelium. Geographic distribution in the tube varies by genotype and immunophenotype, from regionally unrestricted (PAX2) to greater likelihood specific area (fimbria) of shared prevalence (PAX2 and p53). This study reveals, for the first time, an entity (SCOUT) that is associated with serous cancer, expands the topography of altered PAX2 expression in the female genital tract mucosa and highlights another potential pathway disturbance involved in early serous carcinogenesis in the Fallopian tube. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

An ultrastructural study of normal human thymic epithelium in primary cultures.

The ultrastructural study of a large series of samples of normal human thymic epithelial cells (TECs) in primary cultures is here presented. Cultured TECs are heterogeneous due to marked differences in their cytoplasmic organization. In particular, a spectrum of ultrastructural features have been observed, ranging from poorly-differentiated cells to secretory and epidermoid-like ones, as well as intermediate transitional forms and cells co-expressing both epidermoid-like and secretory features. Cells with peculiar secretory (neuroendocrine-like) characteristics have also been identified in the culture obtained from one of the donors. These data suggest the existence of a thymic epithelial cell type which may act as a dynamic unit, changing its cytoplasmic organization in response to microenvironmental signals. The types and interrelationships of cytoplasmic organelles observed in the secretory cells suggest that the production of thymic peptides involves the classical pathway of protein synthesis and secretion.     

Effects of cigarette smoke and alcohol on ciliated tracheal epithelium and inflammatory cell recruitment

Ciliated epithelium represents the first line of host defense against lung infection. Most alcoholics smoke and are at high risk for developing lung infections. We reported that cigarette smoke activates protein kinase C (PKC) and alcohol desensitizes ciliary beat frequency (CBF) to beta-agonists in bovine bronchial epithelial cells in vitro. The combined effect of smoke and alcohol exposure on mouse ciliated tracheal epithelium has not been studied in vivo. We hypothesized that previously observed in vitro effects of smoke and alcohol exposure could be replicated in vivo. Female C57BL/6 mice were exposed to whole body cigarette smoke only, 20% alcohol ad libitum in drinking water only, or the combination of cigarette smoke plus alcohol for 6 wk. Bronchoalveolar lavage (BAL) cell populations, CBF, and airway kinase activity were assessed. Total BAL cells were decreased in animals exposed to alcohol alone and increased in animals exposed to smoke alone. Mice receiving smoke and alcohol had cell levels similar to smoke alone. Baseline CBF was not affected in any group; however, isoproterenol stimulation of CBF was blunted by alcohol exposure and actually slowed below baseline in the smoke plus alcohol group. Isoproterenol-induced PKA activity was inhibited in mice receiving alcohol independent of smoke exposure. Smoke activated PKC independent of alcohol. The isoproterenol-induced slowing below baseline of CBF after combined smoke and alcohol exposure demonstrates a novel ciliary impairment likely related to the combination of alcohol-mediated PKA desensitization and smoke-stimulated PKC activation, possibly through acetaldehyde present in the vapor phase of cigarette smoke.     

Fallopian tube sperm perfusion used in a donor insemination programme.

Ninety-six couples were enrolled in a programme of Fallopian tube sperm perfusion (FSP). FSP is a method combining ovarian stimulation, ovulation induction and intra-uterine insemination with a 4 ml volume of sperm suspension. Frozen/thawed donor semen was used in all cycles. A total of 196 treatment cycles were started and of these 172 were completed. Twenty cycles were cancelled because of maturation of too many follicles, a low ovarian response, or formation of ovarian cysts. Five of the women enrolled in the programme did not have any inseminations. Forty-eight pregnancies occurred (27.9% per cycle) among 45 women (49.5%). There were five spontaneous abortions and one ectopic pregnancy. Sixteen pregnancies are on-going (greater than 12 weeks) and 26 women have delivered (including two sets of triplets and one set of twins). The pregnancy rate declined from the first attempt (34.1%) to the fourth attempt (14.3%). The pregnancy rate was significantly higher in women with three to five mature follicles compared to women with two or fewer mature follicles (P less than 0.05). Cycles in which human chorionic gonadotrophin (HCG) was administered on day 11 or later had a higher pregnancy rate than cycles where HCG was administered on day 10 (P less than 0.01). There was no difference in pregnancy rates between insemination with 8-10 x 10(6) motile spermatozoa and insemination with greater than 30 x 10(6) motile spermatozoa. FSP used in a programme of artificial insemination by donor using frozen/thawed donor semen is relatively simple to perform and gives a high pregnancy rate per cycle.     

Na-dependent Li-transport in primary nerve cell cultures

Primary cultures from chick embryonic brain were used to study the steady state distribution of lithium. The intra/extracellular Li+ ratio decreased by enhancing the external Na+ concentration. Ouabain did not influence this unequality. A phloretin-sensitive component was revealed in the Li uptake at low Na+ concentration. The findings might suggest the existence of a Na+-dependent Li+ countertransport system in these brain cell cultures.     

Physiology: Morphology and ultrastructure of Fallopian tube epithelium at different stages of the menstrual cycle and menopause

The Fallopian tube has been reported to undergo cyclical changes.However, many studies of tubal ultrastructure have either examinedone segment of the tube only or studied animal oviducts. Theaim of this study was to document in detail the combined morphologicaland ultrastructural features of the epithelial lining alongthe length of the tube in women at different stages of the menstrualcycle. We report an increase in the proportion of ciliated cellsalong the tube, being highest in the fimbriae, but no substantialdifference between the follicular and luteal phases of the menstrualcycle. In the late follicular phase, fragments of cytoplasmicand cellular material were seen in the isthmic lumen but notin the outer tubal segments. Similarly, surface domes and secretorygranules were more prominent in the mid-tube and ampullary sectionsthan in the fimbriae. This surface activity was followed byrelative quiescence in the early/mid luteal phase with reversionto a more active surface but with little secretory activityin the late luteal phase. These findings along the Fallopiantube substantiate the concept of functional differentiationbetween the different segments and necessitate further studiesto determine its clinical relevance.     

Successful conception following Fallopian tube recanalization in infertile patients with a unilateral proximally occluded tube and a contralateral patent tube

BACKGROUND: There are many published case reports of successful conception following transcervical Fallopian tube recanalization (T-FTR) in patients with bilateral proximally occluded Fallopian tubes. However, no serial trials have been published with respect to successful conception following unilateral tubal recanalization in infertile patients with a unilateral proximally occluded tube and a contralateral patent tube. This study was designated to analyse the success rate of T-FTR and the pregnancy rate due to natural fertilization in the lumen of the recanalized tube in these patients. METHODS: We have encountered only 11 patients with this abnormality in our department in the past 10 years. T-FTR with fluoroscopic guidance was performed in these patients, confirmed by at least two hysterosalpingographies to exclude tubal spasm. The uterine catheter devised by us was used during the procedure. RESULTS: All 11 Fallopian tubes were successfully opened by T-FTR. In the six patients who conceived, a preovulatory follicle was demonstrated on the side of the cannulated tube during the conception. The success rate of recanalization, the pregnancy rate due to fertilization in the lumen of the recanalized tube and the successful delivery rate were 100, 55 and 36% respectively. CONCLUSIONS: Our findings suggest that a functional and/or organic disorder in the patent tube resulted in infertility in patients with unilateral proximal tubal obstruction. Our results further show that recanalization of occluded tubes is an effective treatment. Thus, recognition of successful conception following T-FTR in these patients will be beneficial to our clinical approach to this infertile condition.     

Specialized pacemaking cells in the human Fallopian tube

It is postulated that the propagated electric activity of the gut is generated by the interstitial cells of Cajal (ICCs). We investigated the hypothesis of the presence of ICCs in the Fallopian tube (FT) as initiators of the tubal electric activity. Specimens from various parts of the FT were obtained from 21 female cadavers (mean age 38.2 +/-10.9 years) from the PostMortem Department of the Cairo University, Faculty of Medicine. Fixed sections were prepared and stained for c-kit. Controls for antisera specificity consisted of tissue incubated with normal rabbit serum substituted for the primary antiserum. Dendritic, c-kit-positive, ICC-like cells were detected in the tubal musculature of the studied specimens. They were distinguishable from the c-kit-positive nonbranching mast cells and from the c-kit-negative smooth muscle cells (SMC). Immunoreactivity was absent in the negative controls. We have for the first time identified cells in the FT with morphologic and immunologic phenotypes similar to the ICCs of the gut. These cells may be responsible for initiating the slow waves (SWs) recorded from the SMC of the FT. This is a preliminary study, and further studies are needed to investigate the functional role of these cells.