NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2.

The transcription factor nuclear factor kappa B (NF-kappaB) is constitutively active in both cancer cells and stromal cells of breast cancer; however, the precise role of activated NF-kappaB in cancer progression is not known. Using parental MCF10A cells and a variant that expresses the myoepithelial marker p63 stably overexpressing the constitutively active p65 subunit of NF-kappaB (MCF10A/p65), we show that NF-kappaB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin. P65 also suppressed the expression of p63 and the putative breast epithelial progenitor marker cytokeratin 5/6. MCF10A/p65 cells were phenotypically similar to cells undergoing epithelial to mesenchymal transition (EMT). MCF10A/p65 cells failed to form characteristic acini in three-dimensional Matrigel. Analysis of parental and MCF10A/p65 cells for genes previously shown to be involved in EMT revealed elevated expression of ZEB-1 and ZEB-2 in MCF10A/p65 cells compared to parental cells. In transient transfection assays, p65 increased ZEB-1 promoter activity. Furthermore, MCF10A cells overexpressing ZEB-1 showed reduced E-cadherin and p63 expression and displayed an EMT phenotype. The siRNA against ZEB-1 or ZEB-2 reduced the number of viable MCF10A/p65 but not parental cells, suggesting the dependence of MCF10A/p65 cells to ZEB-1 and ZEB-2 for cell cycle progression or survival. MCF10A cells chronically exposed to tumor necrosis factor alpha (TNFalpha), a potent NF-kappaB inducer, also exhibited the EMT-like phenotype and ZEB-1/ZEB-2 induction, both of which were reversed following TNFalpha withdrawal.

Long term review of laparoscopic sacrocolpopexy.

OBJECTIVE: Assessment of long term outcome following laparoscopic sacrocolpopexy. DESIGN: Retrospective follow up study using standardised examination with pelvic organ prolapse quantification system (POP-Q) and questionnaires. SETTING: A tertiary urogynaecology unit in the North West of England. POPULATION: One hundred and forty consecutive cases who had a laparoscopic sacrocolpopexy at St Mary's Hospital, Manchester, between 1993 and 1999. METHODS: Women completed questionnaires and were examined in gynaecology clinic or sent postal questionnaires if unable to attend the clinic. MAIN OUTCOME MEASURES: Adequacy of vault support and recurrent vaginal prolapse assessed by POP-Q score. Assessment of prolapse, urinary and bowel symptoms and sexual function using questionnaires. RESULTS: One hundred and three women were contacted after a median of 66 months. Sixty-six women were examined and a further 37 women filled in questionnaires only. Recurrent vault prolapse occurred in 4 of the 66 women who were examined. Prolapse had recurred or persisted in 21 of 66 women, with equal numbers of anterior and posterior vaginal wall prolapse. Overall, 81/102 (79%) said that their symptoms of prolapse were 'cured' or 'improved'; 39/103 (38%) still had symptoms of prolapse. For every two women who were cured of their urinary or bowel symptoms, one woman developed worse symptoms. CONCLUSIONS: Among the 66 women available for examination laparoscopic sacrocolpopexy provided good long term support of the vault in 92%. Forty-two percent of these women had recurrent vaginal wall prolapse. Despite this, 79% of women felt that their symptoms of prolapse were cured or improved following surgery.

Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.

The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affinity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.

Morphologic transformation of the thymus in developing zebrafish.

The morphologic transformation of the developing zebrafish thymus from 1 week postfertilization (wpf) to 15 wpf is described. The thymus overall morphology changed from a small pouch-like shape at 1 wpf to a conical shape between 2 and 3 wpf before acquiring a more complex shape from 4 wpf onward. Rapid growth rate along the lateral axis at the region near the pharyngeal epithelium occurred between 1 and 2 wpf, whereas rapid growth rate along the dorsal-ventral axis occurred between 3 and 6 wpf. Expansion of thymocyte population beginning from 1 wpf became more evident by 2 to 3 wpf, as indicated by the apparent increase of different sizes of lymphocytes, recombination activating gene-1 (rag-1), and T-cell antigen receptor alpha chain constant region (TCRAC) -positive cells. Tissue section in situ hybridization (ISH) analysis with rag-1 probe reveals that cortex-medullary regionalization has begun between 1 and 2 wpf as rag-1 expression clearly demarcated the cortex, whereas the medulla was rag-1 negative. The presence of TCRAC-positive cells in the medulla by 2-3 wpf, suggests that the thymic selection processes had begun. The zebrafish thymus is morphologically mature by 3 wpf. Early signs of thymic involution were observed in zebrafish aged 15 wpf.

The course of pregnancy in a patient with nail-patella syndrome.

INTRODUCTION: The nail-patella syndrome is a rare autosomal dominant condition with high penetrance. Pregnancy in such a patient is rare and we believe this to be the first report of a live birth occurring in a patient with nail-patella syndrome. CLINICAL PICTURE: A 25-year-old patient presented in her first pregnancy with nephrotic syndrome associated with characteristic bone abnormalities and nail dysplasia and was later diagnosed to have nail-patella syndrome. In her second pregnancy, the course of her pregnancy was complicated by further deterioration of renal function with superimposed pre-eclampsia resulting in early delivery at 28 weeks. CONCLUSION: Such pregnancies should be regarded as high risk and managed jointly with the renal physician in a tertiary care centre to ensure an optimal outcome to the mother and baby.

The influence of age on aerosol deposition in children with cystic fibrosis.

Nebulized aerosols are commonly used to deliver drugs for the treatment of respiratory disease in children, but there are inadequate data on the dose of drug depositing in the lungs in this age group, and the effect of age on this dose. We therefore aimed to quantify total and regional deposition of nebulized aerosol in children of widely differing age. Twelve infants (median age 0.8 yrs, range 0.3-1.4 yrs) who were asleep, and eight older children (median age 10.8 yrs, range 6.3-18.0 yrs) with cystic fibrosis were studied. Radiolabelled normal saline aerosol was generated by a Turret nebulizer, with a driving flow of 9 l.min-1. All subjects inhaled aerosol via the nasal route, whilst the older children undertook a second study with inhalation via the oral route. Following aerosol inhalation, planar and single-photon emission computed tomography (SPECT) scans were obtained. For the nasal route, total lung deposition was lower in infants (median 1.3%, range 0.3-1.6%) than in older children (median 2.7%, range 1.6-4.4%). For the older children inhaling via the nasal or oral route, there was no influence of age on lung, upper respiratory tract, or the sum of upper respiratory tract and lung deposition. We conclude that the dose of a nasally inspired aerosol reaching the lungs of infants who are asleep is approximately half that for older children, when the nebulizer is operating at 9 l.min-1. Age does not affect deposition of nasally or orally inspired aerosols in older children.

Comparison of efficacy of salbutamol and sodium cromoglycate in the prevention of ticarcillin-induced bronchoconstriction.

Nebulized ticarcillin can cause bronchoconstriction in children with cystic fibrosis (CF). We assessed whether pretreatment with salbutamol or sodium cromoglycate (SCG) would prevent this side-effect using a randomized, double-blind, placebo-controlled design. Fifteen children with CF received pretreatments of saline, SCG, or salbutamol, in random order, one on each day. Baseline lung function was measured before and after pretreatment, and after ticarcillin nebulization. On the control day (saline pretreatment), ticarcillin caused a reduction in forced expiratory volume in one second (FEV1), which was maximal 10 minutes after receiving the aerosol and persisted for 120 minutes. The mean maximal fall in FEV1 was 9%. Pretreatment with salbutamol abolished the fall in FEV1 seen with ticarcillin at all time points. Pretreatment with SCG diminished the maximal fall in FEV1 at 10 minutes (mean, 4%) and resulted in the FEV1 returning to baseline within 120 minutes. These data suggest that pretreatment with salbutamol is more effective in preventing ticarcillin-induced bronchoconstriction in the doses used in routine clinical practice, than it is with SCG.

Bronchial responsiveness to histamine in infants and older children.

Normal infants have been shown to respond to a relatively low concentration of inhaled histamine. However, those studies used partial maximal expiratory flow volume (PMEFV) curves to assess lung function. In order to directly compare responsiveness between infants and older children, we compared bronchial responsiveness to histamine between a group of 45 normal infants, median age 4 wk (range 2 to 6 wk) and a group of 30 nonasthmatic older children, median age 10 yr (range 5 to 15 yr) using PMEFV curves in both groups to assess lung function. In the infant group, PMEFV curves were generated using the forced expiratory flow volume technique. For the older children, PMEFV curves were generated by voluntary effort. The provocative concentration of histamine that produced a 40% fall (PC40) in maximum flow at functional residual capacity (VmaxFRC) was calculated from the PMEFV curves. The geometric mean PC40 of the infants (1.02 g/L) was lower than the geometric mean of the older children (3.4 g/L) (p less than 0.001). However, these results were then corrected for dilution of the aerosol due to air entrainment (AE). Corrected values of PC40 were not significantly different between infants and older children. These results demonstrate the importance of accounting for AE in the evaluation of histamine responsiveness and suggest that bronchial responsiveness may be similar in normal infants and older children.

Bronchial response to nebulized antibiotics in children with cystic fibrosis.

Nebulized antibiotics are being used increasingly in children with cystic fibrosis. We assessed the effect of nebulized antibiotic solutions of varying tonicity on lung function in 12 children aged 5-15 yrs with cystic fibrosis. Baseline forced expiratory volume in one second and (FEV1) was measured, followed by a single nebulization of normal saline (272 mosmol.kg-1), tobramycin (248 mosmol.kg-1), or ticarcillin (3,080 mosmol.kg-1). All children received each of these, administered randomly, one per day. FEV1 was remeasured 5, 15 and 30 min after completion of the nebulization. Ticarcillin (mean fall 10.7% (SD 8.9)) caused a larger fall in FEV1 than normal saline (4.8% (4.3), p less than 0.05). The fall in FEV1 for ticarcillin was greater than for tobramycin (1.2% (2.0), p less than 0.05). Normal saline did not result in a significantly larger fall in FEV1 than tobramycin (p greater than 0.05). Bronchoconstriction to ticarcillin persisted at 30 min. We conclude that nebulized antibiotics can affect lung function in children with cystic fibrosis if the solutions are hypertonic.

Biochemical mechanisms of cardiac hypertrophy.

Rapid cardiac growth in adult rats and neonatal pigs involves more efficient use of existing components of the protein synthesis pathway and synthesis of new ribosomes and mRNA to increase the capacity for protein synthesis. Greater efficiency of synthesis can be induced by mechanical perturbations that stretch the ventricular wall, including increased cardiac work and increased ventricular pressure development in beating hearts, and increased aortic and intraventricular pressure in arrested-drained hearts. The biochemical signal linking stretch to more efficient protein synthesis has not been identified. Preferential synthesis of new ribosomes occurs in the first two hours of exposure of Langendorff preparations to high aortic pressure or within four hours after injection of thyroid hormone into normal rats. The rate of protein degradation is either accelerated or unchanged in hypertrophing hearts but is inhibited by induction of cardiac work or high aortic pressure in Langendorff preparations. Overall, increased capacity for, and efficiency of, protein synthesis are the major factors accounting for cardiac growth.

Congenital renal salt-losing syndrome and failure to thrive in infancy.

An infant with congenital renal salt-losing syndrome is reported. The importance of recognizing this rare disorder is emphasized because early detection, and treatment with sodium supplementation, results in marked growth improvement and may prevent volume depletion, electrolyte disturbance, and cardiovascular collapse. The clinical features and biochemical abnormalities seen in the congenital renal salt-losing syndrome are described. In this condition, natriuresis occurs in the face of normal renal and adrenal function and, as the child gets older, spontaneous resolution occurs. Other causes of hyponatraemia in infancy are discussed.