Intramacrophage survival of uropathogenic Escherichia coli : differences between diverse clinical isolates and between mouse and human macrophages

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24 h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24 h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.

Oestrogen and progestin responses in human endometrium

Our understanding of the mechanisms of the actions of oestrogens and progestins have evolved from the simple concept of nuclear receptor-mediated regulation of transcription to a highly sophisticated, finely tuned interplay between various coregulators, other signaling cascades and transcription factors. The net result of these complex regulatory mechanisms is a steroid-, cell-, or tissue-specific action of oestrogens and progestins. their antagonists or selective modulators of their receptors. In this review, we have attempted to shed some light on the regulation of the actions of oestrogens and progestins on the human endometrium. (C) 2003 Elsevier Science Ltd. All rights reserved.

Triphasic pattern in the ex vivo response of human proliferative phase endometrium to oestrogens

The aim of this study was to evaluate the ex vivo oestrogen responsiveness of human proliferative phase endometrium using short-term explant cultures. The effects of oestrogen (17beta-E2) on proliferation and the expression of oestrogen-responsive genes known to be involved in regulating endometrial function were evaluated. Three distinct response patterns could be distinguished: (1) the menstrual (M) phase pattern (cycle days 2-5), which is characterised by a complete lack in the proliferative response to 17beta-E2, while an increased expression of AR (2.6-fold, P<0.01), PR (2.7-fold, P<0.01) and COX-2 (3.5-fold, P<0.01) at the mRNA level was observed and a similar upregulation was also found for AR, PR and COX-2 at the protein level; (2) the early proliferative (EP) phase pattern (cycle days 6-10) with 17beta-E2 enhanced proliferation in the stroma (1.7-fold, P<0.05), whereas the expression of AR, PR and COX-2 were not affected at the mRNA and protein levels and ER-a mRNA and protein levels were significantly reduced by 17beta-E2; (3) the late proliferative (LP) phase pattern (cycle days 11-14), which is characterised by a moderate stimulation of proliferation (1.4-fold, P<0.05) and PR mRNA expression (1.7-fold, P<0.01) by 17beta-E2. In conclusion, three distinct response patterns to 17beta-E2 could be identified with respect to proliferation and the expression of known oestrogen-responsive genes in human proliferative phase endometrium explant cultures.

Oestrogen-modulated gene expression in the human endometrium

To identify key regulatory mechanisms in the growth and development of the human endometrium, microarray analysis was performed on uncultured human endometrium collected during menstruation (M) and the late-proliferative (LATE-P)-phase of the menstrual cycle, as well as after 24 h incubation in the presence of oestradiol (17beta-E2). We demonstrate the expression of novel gene transcripts in the human endometrium. i.e. mucin-9, novel oestrogen-responsive gene transcripts, i.e. gelsolin and flotillin-1, and genes known to be expressed in human endometrium but not yet shown to be oestrogen responsive, i.e. connexin-37 and TFF1/pS2. Genes reported to be expressed during the implantation window and implicated in progesterone action, i.e. secretoglobin family 2A, member 2 (mammaglobin) and homeobox-containing proteins, were up-regulated in uncultured LATE-P-phase endometrium compared to M-phase endometrium. Some gene transcripts are regulated directly by 17beta-E2 alone, others are influenced by the in vivo environment as well. These observations emphasise that the regulation of endometrium maturation by oestrogen entails more then just stimulation of cell proliferation.

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1 alpha and CA-IX in the menstrual and early proliferative phases. HIF1 alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed toy estrogen during the late proliferative phase.

Estrogen metabolizing enzymes in endometrium and endometriosis

BACKGROUND Estradiol (E-2) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)l in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS Aromatase and 17 beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17 beta-HSD, EST and STS were readily detectable. Only 17 beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17 beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS In endometriosis lesions, the balance is tilted in favor of enzymes producing E2. This is due to a suppression of types 2 and 4 17 beta-HSD, and an increased expression of aromatase and type 1 17 beta-HSD in ectopic endometrium.

Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human

To date, research into the biological processes and molecular mechanisms associated with endometrial receptivity and embryo implantation has been a focus of attention, whereas the complex events that occur in the human endometrium during the menstrual and proliferative phase under the influence of estrogen have received little attention. The objective of this review is to provide an update of our current understanding of the actions of estrogen on both human and rodent endometrium, with special emphasis on the regulation of uterine growth and cell proliferation, and the value of global gene expression analysis, in increasing understanding of these processes.

Haemoglobin expression in human endometrium

BACKGROUND: The general concept that haemoglobin is only a carrier protein for oxygen and carbon dioxide is challenged since recent studies have shown haemoglobin expression in non-erythroid cells and the protection of haemoglobin against oxidative and nitrosative stress. Using microarrays, we previously showed expression of haemoglobins alpha, beta, delta and gamma and the haeme metabolizing enzyme, haeme oxygenase (HO)-1 in human endometrium. METHODS: Using real-time quantitative PCR, haemoglobin alpha, beta, delta and gamma, and HO-1 mRNA levels were assessed throughout the menstrual cycle (n = 30 women). Haemoglobin and HO-1 protein levels in the human endometrium were assessed with immunohistochemistry. For steroid responsiveness, menstrual and late proliferative-phase endometrial explants were cultured for 24 h in the presence of vehicle (0.1% ethanol), estradiol (17 beta-E-2, 1 nM), progestin (Org 2058, 1 nM) or 17 beta-E-2+Org 2058 (1 nM each). RESULTS: All haemoglobins and the HO-1 were expressed in normal human endometrium. Haemoglobin mRNA and protein expression did not vary significantly during the menstrual cycle. Explant culture with Org 2058 or 17 beta-E-2+Org 2058 increased haemoglobin gamma mRNA expression (P < 0.05). HO-1 mRNA levels, and not protein levels, were significantly higher during the menstrual (M)-phase of the cycle (P < 0.05), and were down-regulated by Org 2058 in M-phase explants and by 17 beta-E-2+Org 2058 in LP-phase explants, versus control (P < 0.05). CONCLUSIONS: The haemoglobin-HO-1 system may be required to ensure adequate regulation of the bioavailability of haeme, iron and oxygen in human endometrium.

Effects of selective oestrogen receptor modulators on proliferation in tissue cultures of pre- and postmenopausal human endometrium

We characterised the effects of selective oestrogen receptor modulators (SERM) in explant cultures of human endometrium tissue. Endometrium tissues were cultured for 24 h in Millicell-CM culture inserts in serum-free medium in the presence of vehicle,17 beta-estradiol (17 beta-E2,1 nM), oestrogen receptor (ER) antagonist ICI 164.384 (40 nM), and 4-OH-tamoxifen (40 nM), raloxifene (4 nM), lasofoxifene (4 nM)and acolbifene (4 nM). Protein expression of ER alpha, ER beta 1 and Ki-67 were evaluated by immunohistochemistry (IHC). The proliferative fraction was assessed by counting the number of Ki-67 positive cells. Nuclear staining of ER( and ER(1 was observed in the glandular epithelium and stroma of pre- and postmenopausal endometrium. ER(1 protein was also localized in the endothelial cells of blood vessels. Treating premenopausal endometrium tissue with 17 beta-E2 increased the fraction of Ki-67 positive cells (p < 0.001) by 55% in glands compared to the control. Raloxifene (4 nM) increased (p < 0.05) the Ki-67 positive fraction. All other SERMS did not affect proliferation in this model. Treating postmenopausal endometrium with 17(-E2 increased (p < 0.001) the fraction of Ki-67 positive cells by 250% in glands compared to the control. A similar effect was also seen for 4-OH-tamoxifen, whereas the rest of SERMs did not stimulate proliferation. We demonstrated that oestradiol increases the fraction of proliferating cells in short term explant cultures of postmenopausal endometrium. In addition, we were able to reveal the agonistic properties of 4-OH-tamoxifen and confirm that raloxifene and next-generation SERMs acolbifene and lasofoxifene were neutral on the human postmenopausal endometrium. (C) 2008 Elsevier Ltd. All rights reserved.

Olfactomedin-4 regulation by estrogen in the human endometrium requires epidermal growth factor signaling

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17 beta-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17 beta-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-alpha is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-alpha (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controts. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation. (Am J Pathol 2010, 177:2495-2508: DOI: 10.2353/ajpath.2010.100026

A high and low fear phenotype mouse model

Fear-related illnesses such as post-traumatic stress disorder (PTSD) impose a tremendous burden on individual quality of life, families, and the national economy. In the military population, 17-20% of services members returning from deployment are diagnosed with PTSD. While treatments have improved for PTSD and are helpful for some, many people continue to suffer despite therapy. The aim of this research is to examine fear memory behaviourally and at the cellular level in the amygdala by using a unique inter-cross strain of high and low fear phenotype mice. An extended outcross C57BL/6J x DBA/2J (F8) are selected for high or low Pavlovian fear memory to context and cue. On presentation of either the original learning context or the cue (tone) mice display high or low levels of freezing as a behavioural measure of fear. In order to identify key aspects of the cellular basis of this difference in fear memory behaviour we are making measurements of protein levels and neuron numbers of a known pathway involved in the consolidation of a long term fear memory (pMAPK). Ongoing studies aim to determine if high fear behaviour is associated with differential signalling in the lateral amygdala compared to low fear behaviour. Additionally, by blocking this pathway in the lateral amygdala (LA), we aim to reduce fear behaviour following Pavlovian fear conditioning. This research will help to unravel the cellular mechanisms underlying high fear behaviour and advance the field toward targeted treatment and improved outcomes, ultimately improving human quality of life.

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions

Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional program associated with intramacrophage survival, we performed host–pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated, and quantified the mammalian and bacterial transcriptomes. BMMs responded to the two UPEC strains with a broadly similar gene expression program. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes upregulated at 24 h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

Conservation genetics of the water mouse, Xeromys myoides Thomas, 1889

The water mouse, Xeromys myoides, is currently recognised as a vulnerable species in Australia, inhabiting a small number of distinct and isolated coastal regions of Queensland and the Northern Territory. An examination of the evolutionary history and contemporary influences shaping the genetic structure of this species is required to make informed conservation management decisions. Here, we report the first analysis undertaken on the phylogeography and population genetics of the water mouse across its mainland Australian distribution. Genetic diversity was assessed at two mitochondrial DNA (Cytochrome b, 1000 bp; D-loop, 400 bp) and eight microsatellite DNA loci. Very low genetic diversity was found, indicating that water mice underwent a recent expansion throughout their Australian range and constitute a single evolutionarily significant unit. Microsatellite analyses revealed that the highest genetic diversity was found in the Mackay region of central Queensland; population substructure was also identified, suggesting that local populations may be isolated in this region. Conversely, genetic diversity in the Coomera region of south-east Queensland was very low and the population in this region has experienced a significant genetic bottleneck. These results have significant implications for future management, particularly in terms of augmenting populations through translocations or reintroducing water mice in areas where they have gone extinct.

Comparative analysis of genome maintenance genes in naked mole rat, mouse, and human

Genome maintenance (GM) is an essential defense system against aging and cancer, as both are characterized by increased genome instability. Here, we compared the copy number variation and mutation rate of 518 GM-associated genes in the naked mole rat (NMR), mouse, and human genomes. GM genes appeared to be strongly conserved, with copy number variation in only four genes. Interestingly, we found NMR to have a higher copy number of CEBPG, a regulator of DNA repair, and TINF2, a protector of telomere integrity. NMR, as well as human, was also found to have a lower rate of germline nucleotide substitution than the mouse. Together, the data suggest that the long-lived NMR, as well as human, has more robust GM than mouse and identifies new targets for the analysis of the exceptional longevity of the NMR.