Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?

STUDY QUESTION: Is human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated? SUMMARY ANSWER: LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated. WHAT IS KNOWN ALREADY: The human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia. STUDY DESIGN, SIZE, DURATION: The study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women's hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC). MAIN RESULTS AND THE ROLE OF CHANCE: LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0.01), however, they did not correlate in luminal and stratum basalis epithelium (r = 0.5 and 0.13, respectively). Endometrial LGR5 demonstrates a dynamic spatiotemporal expression pattern, suggesting hormonal regulation. Oral and local progestogens significantly reduced endometrial LGR5 mRNA levels compared with women not on hormonal treatment (P < 0.01). Our data were in agreement with in silico analysis of published endometrial microarrays. LARGE SCALE DATA: We did not generate our own large scale data but interrogated publically available large scale data sets. LIMITATIONS, REASONS FOR CAUTION: In the absence of reliable antibodies for human LGR5 protein and validated lineage markers for the various epithelial populations that potentially exist within the endometrium, our study does not formally characterise or examine the functional ability of the resident LGR5+ cells as multipotent. WIDER IMPLICATIONS OF THE FINDINGS: These data will facilitate future lineage tracing studies in the human endometrial epithelium; to identify the location of stem cells and further complement the in vitro functional studies, to confirm if the LGR5 expressing epithelial cells indeed represent the epithelial stem cell population. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Wellbeing of Women project grant (RTF510) and Cancer Research UK (A14895). None of the authors have any conflicts of interest to disclose.

Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK.

The extracellular, matrix-modifying enzyme lysyl oxidase (LOX) has recently been linked to colorectal cancer (CRC) progression, in particular to the stages of invasion and metastasis. In this report, we use cell lines expressing a catalytically inactive mutant form of LOX to show that catalytic activity is required for LOX-mediated effects on proliferation and invasion in both in vitro and in vivo models of CRC. Furthermore, we use rheology to measure the relative stiffness of modified collagen matrices and subcutaneous tumors, and show that LOX-induced collagen cross-linking results in stiffening of the matrix both in vitro and in vivo. We observe a strong association between matrix stiffness and activation of the FAK (focal adhesion kinase)/SRC-signaling pathway, with a stiffer environment resulting in increased FAK/SRC phosphorylation and a more proliferative and invasive phenotype. We are the first to show a direct relationship between LOX enzymatic activity and tissue stiffness, and to demonstrate a role for stiffness in driving CRC progression. Our findings provide significant evidence to suggest that therapeutic inhibition of LOX activity may provide a novel effective treatment option for patients with metastatic CRC.

Molecular phylogeny supports the paraphyletic nature of the genus Trogoderma (Coleoptera: Dermestidae) collected in the Australasian ecozone.

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

A military-civilian resource-sharing agreement.

A first-of-its-kind resource-sharing agreement between the US Department of Defense and a civilian hospital was undertaken with resounding success. This article discusses how Sacred Heart Medical Center, Spokane, Wash, and the 92nd Medical Group at Fairchild Air Force Base, Spokane, Wash, developed the project and accomplished the integration, which continues to benefit each organization.

Expression of antioxidant enzymes in human prostatic adenocarcinoma.

BACKGROUND: Antioxidant enzymes (AEs), which catalyze the conversion of reactive oxygen species (ROS) to water, include catalase (CAT), manganese-containing superoxide dismutase (MnSOD), and copper and zinc-containing superoxide dismutase (CuZnSOD). Previous work has indicated that MnSOD, CAT, and CuZnSOD levels are nearly always low in cancer cells. METHODS: Formalin-fixed, paraffin-embedded tissue from 31 radical prostatectomy specimens was immunohistochemically stained with polyclonal antibodies to CAT, MnSOD, and CuZnSOD. RESULTS: Malignant glands are typically stained with less intensity than benign/ hyperplastic glands. Marked heterogeneity of staining intensity was seen in the malignant glands for each of the three enzymes. A similar, though less marked, spectrum of heterogeneity of staining intensity was observed in the benign/hyperplastic epithelium contained in the specimens. No statistically significant correlation was found between intensity of staining for any of the three antioxidant enzymes and Gleason score, tumor stage, or preoperative prostate-specific antigen (PSA). CONCLUSIONS: Cellular levels of CAT, MnSOD, and CuZnSOD in prostatic adenocarcinoma reveal that many tumors appear to have decreased levels of expression. The finding that malignant prostate epithelium may have lowered expression of AEs suggests that further study of the role of AEs in malignant transformation in the prostate is warranted.

Bronchoscopic evaluation of pulmonary infiltrates following bone marrow transplantation.

STUDY OBJECTIVE: To determine the impact of fiberoptic bronchoscopy (FOB), including quantitative bacterial cultures obtained by BAL and protected specimen brushing on therapeutic decisions and outcome in bone marrow transplant (BMT) patients. DESIGN: Retrospective review of all BMT patients undergoing FOB during a 4-year period. SETTING: A tertiary care university hospital. RESULTS: Three hundred five patients underwent BMT; 71 (23%) had FOB to assess pulmonary infiltrates. Allogeneic BMT recipients underwent FOB 3.37 times more often than autologous recipients (p < 0.001). Pathogens were identified in 31 (46%) patients undergoing FOB; bacteria were most commonly isolated although 86% of patients had received broad-spectrum empiric antibiotics. Therapy was changed in 20 (65%) patients when a microorganism was identified and in 9 (22%) with nondiagnostic results (p = 0.0026), but isolation of a presumed pathogen had no apparent effect on survival. There were 19 (27%) FOB complications, including bleeding in 8 (11%) patients and death in 2 (3%). Major complications were associated with prolonged prothrombin time (p = 0.006) and were more common (36% vs 14%; p < 0.05) in patients who had protected specimen brushing vs BAL alone. Mortality at 40 months in BMT patients not requiring FOB was 33% compared with 61% mortality in those undergoing FOB (p < 0.001); mortality was 96% in patients with respiratory failure requiring mechanical ventilation. CONCLUSION: FOB is diagnostically useful in the evaluation of some BMT patients with pulmonary complications and often influences therapy, although no impact on survival was clearly demonstrated. FOB should be performed only after benefits of the procedure are weighed carefully against its increased risk in this select population.

Pneumonia in intubated trauma patients. Microbiology and outcomes.

To describe the epidemiology of nosocomial pneumonia in trauma patients and its impact on outcome, we performed a retrospective case-control analysis. Quantitative bronchoscopic cultures were collected from 62 intubated patients with suspected pneumonia. Patients with proven pneumonia had higher abdominal injury scores. Those with bronchoscopy-negative pneumonitis were older. Age and injury severity were used to match two controls to each case. The incidence of pneumonia was 5.8% Streptococci and Hemophilus were common pathogens, but gram-negative rods were isolated more frequently after lengthier intubation. Polymicrobial infections were common. There were no serious complications of bronchoscopy, and culture results often led to antibiotic therapy. No excess mortality could be attributed to pneumonia. Patients with pneumonia and those with bronchoscopy-negative pneumonitis required prolonged care compared with others (p < 0.05). Patients with pneumonia did not receive excess ventilation or hospitalization but incurred hospital charges 1.5 times higher than controls (p = 0.04). Pneumonia was confirmed in less than half of those suspected of having it on the basis of clinical findings. When severity of injury was considered, pneumonia was associated with neither increased mortality nor increased hospital care, but the clinical features suggesting respiratory infection identified trauma patients requiring prolonged hospitalization and incurring higher costs.

Decision making in nosocomial pneumonia. An analytic approach to the interpretation of quantitative bronchoscopic cultures.

Quantitative cultures of specimens obtained at fiberoptic bronchoscopy have been used to diagnose nosocomial pneumonia in research settings, but their clinical role remains controversial. We reviewed the literature comparing these culture techniques with other accepted methods to diagnose pneumonia in mechanically ventilated patients and extracted data to describe the receiver operator characteristics (ROC) of quantitative cultures of protected specimen brush (PSB) and bronchoalveolar lavage (BAL) samples. Analysis of ROCs reveals that these tests have a discriminating power comparable or superior to that of many widely accepted routinely used tests. Current data do not suggest that either culture technique offers an advantage over the other. Since benefits of antibiotic therapy of pneumonia and risks of treatment of noninfected critically ill patients are not well quantified, universally applicable recommendations for appropriate values to define an abnormal test result cannot logically be made. Multiple decision analytic tools show that values lower than those previously recommended are more appropriate in patients suspected of having pneumonia unless the risk of antibiotic therapy is judged to be extreme. On the basis of these findings, we suggest guidelines for clinicians' interpretation of PSB and BAL quantitative culture results.

Cl- channels of the gastric parietal cell that are active at low pH.

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative evaluation of detection assays for Chlamydia trachomatis.

The performances of a commercial nucleic acid hybridization test (Gen-Probe Pace 2 Chlamydia trachomatis) and two commercial enzyme immunoassays (EIAs) (Abbott Chlamydiazyme and Pharmacia Chlamydia EIA) were evaluated against cell culture for the detection of C. trachomatis infection, with cervical swabs obtained from 1,037 women visiting a public sexual health center. The positivity rate by cell culture was 4.7%. Sensitivity and specificity for each test were as follows: Gen-Probe, 95.8 and 98.3%; Chlamydiazyme, 80.4 and 99.3%; Pharmacia EIA, 80.8 and 99.1%. Analysis of discrepant results with probe confirmation assay (Gen-Probe) and direct immunofluorescence (Syva Microtrak) revealed 12 cases of C. trachomatis infection for which culture was negative, resulting in the definition of a true-positive case as opposed to a culture positive. The positivity rate by true-positive definition was 5.9%, and sensitivity and specificity for each test were as follows: Gen-Probe, 96.7 and 99.6%; Chlamydiazyme, 77.5 and 100%; Pharmacia EIA, 77.0 and 100%; cell culture, 80.0 and 100%. We conclude that the Gen-Probe Pace 2 C. trachomatis test is a sensitive and specific alternative to cell culture for the detection of C. trachomatis.