Transforming growth factor-alpha (TGFalpha) is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. EGFR signaling activates several intracellular signaling pathways including phosphatidylinositol 3'-kinase (PI3K). We previously showed that induction of lung-specific TGFalpha expression in transgenic mice caused progressive pulmonary fibrosis over a 4-week period. The increase in levels of phosphorylated Akt, detected after 1 day of doxycycline-induced TGFalpha expression, was blocked by treatment with the PI3K inhibitor, PX-866. Daily administration of PX-866 during TGFalpha induction prevented increases in lung collagen and airway resistance as well as decreases in lung compliance. Treatment of mice with oral PX-866 4 weeks after the induction of TGFalpha prevented additional weight loss and further increases in total collagen, and attenuated changes in pulmonary mechanics. These data show that PI3K is activated in TGFalpha/EGFR-mediated pulmonary fibrosis and support further studies to determine the role of PI3K activation in human lung fibrotic disease, which could be amenable to targeted therapy. less...

Glucocorticoid (GC)-responsive epithelial-mesenchymal interactions regulate lung development. The GC receptor (GR) mediates GC signaling. Mice lacking GR in all tissues die at birth of respiratory failure. In order to determine the specific need for epithelial GR in lung development, we bred triple transgenic mice that carry SPC/rtTA, tet-O-Cre and floxed, but not wild type, GR genes. When exposed to doxycycline in utero, triple transgenic (GRepi -) mice exhibit a Cre-mediated recombination event that inactivates the floxed GR gene in airway epithelial cells. Immunofluorescence confirmed the elimination of GR in Cre-positive, airway epithelial cells of late gestation GRepi - mice. Embryonic day (E) 18.5 pups had a relatively immature appearance with increased lung cellularity and increased pools of glycogen in the epithelium. Postnatal day 0.5, pups had decreased viability. We used quantitative RT-PCR to demonstrate that specific elimination of epithelial immunoreactive GR in GRepi - mice is associated with reduced mRNA expression for surfactant proteins (SPs) A, B, C and D; beta- and gamma �ENaC, T1alpha, the 10kD Clara cell protein (CCSP) and aquaporin 5 (AQP5). Western blots confirmed reduced levels of AQP5 protein. No reduction in the levels of the GR transport protein importin (IPO)-13 was observed. Our findings demonstrate a requirement for lung epithelial cell GR in normal lung development. We speculate that impaired epithelial differentiation, leading to decreased SPs, transepithelial Na and liquid absorption at birth, may contribute to the reduced survival of newborn mice with suppressed lung epithelial GR. less...

Endocrine therapies targeting estrogen action are pivotal for the prevention and treatment of ER-positive breast cancers. Previous studies sought to recreate hormone responsiveness by the stable expression of ERalpha in the ER-negative MDA-MB-231 breast cancer cells. Paradoxically, estrogen inhibits breast cancer cell growth when an exogenous ERalpha is expressed. In this study, we have built on previous studies by developing a Tet-off adenoviral system to express ERalpha in the ER-negative SKBr3 breast cancer cells that over-express both EGFR and HER2. This system efficiently delivers ERalpha and the expression level of ERalpha is controlled by doxycycline in a concentration-dependent manner. The growth of SKBr3 was inhibited by ERalpha expression and further inhibited in the presence of 1 nM 17beta-estradiol. SKBr3 cells were arrested at G0/G1 cell cycle upon ERalpha expression, which corresponded to an increase of p21Cip1/Waf1, hypo-phosphorylation of pRb and decrease of E2F1. Estrogen also reduced EGFR and HER2 expression in SKBr3 cells after ERalpha was expressed. Given that estrogen-induced increase of p21Cip1/Waf1 and decrease of E2F1 was also observed in MDA-MB-231 cells stably transfected with ERalpha, our results suggest that a common pathway might be shared by different breast cancer cell lines whose growth is suppressed by ectopic ERalpha and estrogen. less...

OBJECTIVE: Brucellosis is frequently seen in Mediterranean and Middle East countries, including Turkey. We report the medical and surgical management of 31 cases of native endocarditis. MATERIAL AND METHOD: Thirty-one patients were admitted to our clinic with suspected Brucella Endocarditis. The diagnosis was established by either isolation of Brucella species, or the presence of antibodies. Following preoperative antibiotic therapy patients underwent valve replacement with excessive tissue debridment. Patients were followed up with Brucella titers, blood cultures, and echocardiography. RESULTS: On admission all patients were febrile and mostly dyspneic (NYHA Class 3 or 4). The blood tests were normal except for elevated ESR, CRP and serological tests. The aortic valve was involved in 19 patients, mitral valve in 7 patients, and both valves in 5. After serological confirmation of BE, antibiotic therapy was maintained. Twenty-five of the patients received rifampicine, doxycycline, and cotrimaxozole; 2 of them received a combination of rifampicine, streptomycin, and doxycycline; and 4 of them received rifampicine, tetracycline, and cotrimaxozole. Tissue loss in most of the affected leaflets and vegetations were presenting all patients. Valve replacements were performed with mechanical and biologic prostheses. All the patients were afebrile at discharge but received the antibiotics for 101, 2+/-16, 9 days. The follow-up was 37, 1+/-9, 2 months. DISCUSSION: In our retrospective study, combination of adequate medical and surgical therapy resulted in declined morbidity and mortality rate. The valve replacement with aggressive debridement is the most important part of the treatment, which should be supported with efficient preoperative and long term postoperative medical treatment. Copyright (c) 2009 Wiley Periodicals, Inc. less...

ABSTRACT Based on the increase of resistance of genital mycoplasmas to effective antibacterials with worldwide significant differences, we compared the susceptibilities of a wide range of clinical isolates over several years. The susceptibilities of 469 M. hominis (n=290) and ureaplasma (n=179) isolates, collected during 1983 and 1989-2004, to eleven antibacterials were determined by agar dilution method. Additionally, the results from the routine testing (2005-2008) were considered. Doxycycline was the most active tetracycline against ureaplasmas and M. hominis (MIC(90)s, 1 and 8 mg/L, respectively). Significantly more M. hominis isolates ( approximately 10-13%) were resistant to tetracyclines compared to ureaplasmas ( approximately 1-3%). Ofloxacin was effective against both species (>95% susceptibility). Ciprofloxacin was moderately active against M. hominis and less active against ureaplasmas (70.3 and 35.2% susceptibility). Clarithromycin and josamycin were the most potent macrolides (MIC(90), 0.5 mg/L) against ureaplasmas. Erythromycin exhibited the lowest activity (MIC(90), 8 mg/L) like clindamycin, non-active against ureaplasmas, but the most potent drug against M. hominis. Cross-resistance was found between the antibacterials with the higher level between tetracyclines (53-93%), macrolides and erythromycin (70-100%) and between erythromycin and ciprofloxacin (43-55%). M. hominis has become more resistant to tetracyclines and fluoroquinolones over the years 1989-2004, with no changes in 2005-2008. Ureaplasmas have become more resistant to CIP in 1997-2004, showing high resistance to ERY over the time period (1989-2008). Doxycycline is still the drug of first-choice for ureaplasmal infections and may be used by co-infection with M. hominis. Finally, this study gives recommendation for alternatives in cases of resistance towards the commonly used antimicrobials. less...

Brill-Zinsser disease, the relapsing form of epidemic typhus, typically occurs in a susceptible host years or decades after the primary infection; however, the mechanisms of reactivation and the cellular reservoir during latency are poorly understood. Herein we describe a murine model for Brill-Zinsser disease, and use PCR and cell culture to show transient rickettsemia in mice treated with dexamethasone >3 months after clinical recovery from the primary infection. Treatment of similarly infected mice with cyclosporine failed to produce recrudescent bacteremia. Therapy with doxycycline for the primary infection prevented recrudescent bacteremia in most of these mice following treatment with dexamethasone. Rickettsia prowazekii (the etiologic agent of epidemic typhus) was detected by PCR, cell culture, and immunostaining methods in murine adipose tissue, but not in liver, spleen, lung, or central nervous system tissues of mice 4 months after recovery from the primary infection. The lungs of dexamethasone-treated mice showed impaired expression of beta-defensin transcripts that may be involved in the pathogenesis of pulmonary lesions. In vitro, R. prowazekii rickettsiae infected and replicated in the murine adipocyte cell line 3T3-L1. Collectively these data suggest a role for adipose tissue as a potential reservoir for dormant infections with R. prowazekii. less...

Light-directed gene patterning methods have been described as a means to regulate gene expression in a spatially and temporally controlled manner. Several methods have been reported that use photo-caged forms of small molecule effectors to control ligand-dependent transcription factors. Whereas these methods offer many advantages including high specificity and transient light-sensitivity, the free diffusion of the uncaged effector can limit both the magnitude and resolution of localized gene induction. Methods to date have been limited by the small fraction of irradiated cells that have expression levels significantly above uninduced background and have not been shown to affect a defined biological response. The tetracycline-dependent transactivator/transrepressor system, RetroTET-ART, combined with a photocaged form of doxycycline (NvOC-Dox) can be used to form photolithographic patterns of induced expression wherein up to 85% of the patterned cells show expression levels above un-induced regions. The efficiency and inducibility of the RetroTET-ART system allows one to quantitatively measure the limits of resolution and the relative induction levels mediated by a small molecule photocaged effecter for the first time. Well-defined patterns of reporter genes were reproducibly formed within 6-36 hours with feature sizes as small as 300 mum. After photo-patterning, NvOC-Dox can be rapidly removed rendering cells photo-insensitive and allowing one to monitor GFP product formation in real time. Patterned co-expression of the cell surface ligand ephrin A5 on cell monolayers creates well-defined patterns that are sufficient to direct and segregate co-cultured cells via either attractive or repulsive signaling cues. The ability to direct the arrangement of cells on living cell monolayers through the action of light may serve as a model system for engineering artificial tissues. less...

Clinical manifestations range from asymptomatic to fulminant in nature. We report three cases of ehrlichiosis in pediatric oncology patients, one of whom was a stem cell transplant recipient. Early symptoms included fever, malaise, and vague gastrointestinal symptoms. Laboratory abnormalities were initially attributed to chemotherapy toxicity. Illness was severe in all three patients and one patient died even after initiation of doxycycline. These cases emphasize the need for a high index of suspicion for tickborne illness in oncology patients, and the importance of a low threshold for starting empiric treatment before confirming the diagnosis. Pediatr Blood Cancer. (c) 2010 Wiley-Liss, Inc. less...

A total of 113 clinical Campylobacter strains (105 C. jejuni, 7 C. coli, 1 C. lari) were collected between 2006 and 2008 and tested for antimicrobial susceptibility to erythromycin, ciprofloxacin, doxycycline and meropenem. Of all the Campylobacter isolates, 52.2% were resistant to ciprofloxacin and 38.0% to doxycycline. None of them was resistant to erythromycin or meropenem. However, 51.3% of all Campylobacter isolates were intermediate susceptible to erythromycin (minimum inhibitory concentration 1-4 mg/l). These data confirm the value of macrolides as the drugs of choice for the treatment of severe Campylobacter infections. less...

LEFTY is expressed in normal endometrium in cells that decidualize. To understand the importance of this expression, we have studied the effect of LEFTY on decidualization in vitro and in vivo. Exposure of human uterine fibroblast (HuF) cells to recombinant LEFTY blocked the induction of the decidual differentiation-specific marker genes, IGFBP1 (IGF-binding protein 1) and PRL (prolactin) in response to medroxyprogesterone acetate, estradiol, and prostaglandin E2. The inhibitory effect was associated with decreased induction of the transcription factors ETS1 and FOXO1, both of which are essential for decidualization. Overexpression of LEFTY in decidualized HuF cells with an adenovirus that transduced LEFTY caused a marked decrease in IGFBP1 secretion, and withdrawal of medroxyprogesterone acetate from decidualized cells resulted in a decrease in IGFBP1 secretion and an increase in LEFTY expression. Moreover, overexpression of LEFTY in decidualized cells reprogrammed the cells to a less differentiated state and attenuated expression of decidual markers. Uterine decidualization was markedly attenuated and litter size was significantly reduced by retroviral transduction of LEFTY in the uterine horns of pregnant mice or by induction of LEFTY expression by doxycycline treatment in Tet-On conditional LEFTY transgenic pregnant mice. In addition, administration of the contraceptive agent drospirenone to ovariectomized mice induced a marked increase in LEFTY expression and inhibited decidualization. Taken together, these finding indicate that LEFTY acts as a molecular switch that modulates both the induction of decidual differentiation and the maintenance of a decidualized state. Because decidual cells express abundant amounts of LEFTY, the action of LEFTY on decidualization occurs by an autocrine mechanism. less...