s2fieldsofstudy: Biology Medicine

text:
Disruption of pro-oxidant and antioxidant systems with elevated expression of the ubiquitin proteosome system in the cachectic heart muscle of nude mice

Background Current research into the mechanisms of organ atrophy associated with cancer cachexia have centred on the loss of skeletal muscle, as it is one of the most profound physical changes of the disease. However, many patients with cancer cachexia also experience significant atrophy of the heart. The mechanisms causing cardiac tissue wastage in cancer cachexia are largely unknown. However, it is believed to involve a number of molecular interactions between the tumour and host. Increased levels of oxidative stress have been found in cancer cachectic skeletal muscle and has been linked to the activation of the ubiquitin proteosome system (UPS). The aim of the current study was to examine the role of oxidative stress and the UPS in the hearts of mice with cancer cachexia. Methods Oxidative damage to DNA (8-OH-2dG), mRNA levels of the ROS-producing enzymes NADPH oxidase (NOX), and xanthine oxidase (XDH), the antioxidant enzyme superoxide dismutase (SOD) and key components of the UPS was measured in the heart of mice with cancer cachexia. Protein expression levels of NOX enzyme subunits and SOD enzyme activity was also measured in the same heart samples. Results 8-OH-2dG levels were 1.5-fold higher in the heart of mice with cancer cachexia, and this was associated with a 1.7-fold lower level of NOX2 mRNA and twofold higher XDH mRNA in the same hearts. Cancer cachexia was also associated with a 1.5-fold lower level of SOD activity in the heart. Accompanying these pro- and antioxidant differences was a significantly higher level of mRNA for the key UPS elements MURF-1 (4.3=fold) and MAFbx (3.8-fold) in the hearts of mice with cancer cachexia. Conclusions The current study demonstrated that cardiac atrophy of cachectic mice is associated with oxidative damage to DNA in the myocardium. The higher levels of XDH mRNA in cachectic hearts suggest that xanthine oxidase may have an important role to play in producing oxidative stress. It appears that the combination of higher XDH expression and lower SOD enzyme activity are key contributors to oxidative stress and cardiac tissue damage in cancer-induced cardiac atrophy. Oxidative stress in the myocardium as with skeletal muscle may also induce increased expression of the E3 ligases MURF-1 and MAFbx as seen in this study. Electronic supplementary material The online version of this article (doi:10.1007/s13539-013-0116-8) contains supplementary material.

Introduction
Current research into the mechanisms of organ atrophy associated with cancer cachexia (CC) is centred on the loss of skeletal muscle mass as it is one of the most profound physical changes of the disease. However, studies have also established that CC causes significant atrophy of the heart muscle in animal models [1][2][3]. Decreases in heart weight by as much as 60 % have been reported in a rat model of CC [3]. Clinical research does not currently exist examining the pathologic consequence of cardiac atrophy in cancer cachexia. However, Tian et al. used echocardiography to analyse the function of cachectic hearts in a mouse model of CC [4]. The study found that, 14 days after the inoculation with tumour cells, the hearts of cancer cachectic mice had reduced heart rate and fractional shortening.
The mechanisms causing cardiac tissue wastage are largely unknown. However, it is believed to involve a number of molecular interactions between the tumour and host. Elevated cytokines in response to tumour presence and tumour-derived factors such as proteolysis inducing factor (PIF) and lipid mobilising factor together produce tissue wasting [5][6][7]. Host factors such as cytokines including IL-1, IL-6, TNF-α, INF-γ and PIF derived from the tumour have been shown to create a state of oxidative stress and net protein catabolism in skeletal muscle [8]. Similarly increased oxidative stress has been shown to be present in the cachectic heart [9], but how this is linked to the activation of protein breakdown pathways is currently unknown.
A key catabolic pathway known to contribute to both skeletal and cardiac muscle atrophy is the ubiquitin proteosome system (UPS). The UPS is the principle mechanism by which muscle proteins are degraded. The UPS has been found to be upregulated in both experimental models and patients with CC [10,11]. Enzymes which allow ubiquitin to tag proteins for destruction are termed E3 ligases. Currently, only three of the discovered several hundred E3 ligases have been found to be important in the process of muscle atrophy. These are termed E3 alpha, muscle-specific F-box (MAFbx), and muscle specific ring finger 1 (MURF-1) [12]. Examining a model of congestive heart failure, Adams et al. found a positive correlation between TNF-α and the E3 ligases MAFbx and MURF-1 [13]. Furthermore, the study found that TNF-α was able to bring about troponin I ubiquitinylation leading to impaired contractility of the heart.
Oxidative stress in a CC patient population may be an important link between the loss of tissue and initiation of key catabolic pathways [14]. A study by Kaynar et al. found that patients with either non-small cell or small cell lung cancer have elevated levels of xanthine oxidase (XO) activity compared with controls [15]. In the heart, XO is an important enzyme capable of catalyzing the oxidation of hypoxanthine and xanthine to uric acid whilst reducing O 2 to O 2 − and hydrogen peroxide (H 2 O 2 ). Traditionally, increases in XO in the failing heart are associated with ventricular remodelling, fibrosis, and hypertrophy [16]. The contribution of XO in cancer-induced cardiac atrophy has not been investigated, despite the enzyme being implicated in myocardial remodelling and dysfunction [17,18]. In terms of atrophy of the myocardium, like that of skeletal muscle, cardiomyocyte size is dependent on the relative rates of protein synthesis and degradation [19]. Increased levels of oxidative stress have been found in cancer cachectic skeletal muscle and has been linked to the activation of the UPS [20]. However, no such studies have examined the role of oxidative stress in the cancer cachectic heart. The current study aimed to test the hypothesis that increased levels of oxidative stress in the heart muscle of mice with cancer cachexia is associated with and up regulation of the UPS.
2 Materials and methods

Animals
All experimental procedures were carried out with approval from the Victoria University animal ethics committee (AEETH 07/05). Six-week-old BALB/c nu/nu nude mice purchased from the Animal Resource Centre, Western Australia, and were acclimatised for 1 week prior to injection of the cancer cell lines. Animals were maintained under controlled conditions of a 12-h light/dark cycle at 21°C. Food and water were provided ad libitum. Food and water were weighed before and after the experimental period to ensure weight loss in the CC group was not due to a decrease in caloric intake. Animals were randomised into three weight-matched groups consisting of a no cancer (control, n =8), cancer without cachexia control (cancer, n =12) and cancer with cachexia (cachexia, n =16).

Tumour model
Cancer and cancer cachexia were induced as previously described [25]. In brief, the murine adeno-carcinoma cell lines 13 (MAC13) and 16 (MAC16) were cultured in RPMI with 10 % FBS and 0.5 % penicillin/streptomycin (Invitrogen). Mice inoculated with the MAC13 cell line develop a malignant tumour without cachexia and are used as a cancer control (cancer). The MAC16 cell line produces a both solid tumour and cachexia (cachexia). Cells were kept in a humidified incubator at 37°C in 10 % CO 2 . Cells were grown to 80 % confluence and removed from cell culture flasks via trypsinisation. These cells were centrifuged at 500×g for 5 min at 4°C and isolated from the growth media. Cells were then resuspended in sterile PBS pre-incubated to 37°C and drawn up into a sterile syringe with a 25-gauge needle for subsequent injection into mice. Mouse body weights were recorded daily, and tumour size was measured using calipers. Animals were euthanised before weight loss in the cachexia group reached 25 % of their initial weight or before tumour growth exceeded 1,000 mm 3 ; this occurred at 30±3 days. Tissues were harvested and immediately placed in liquid nitrogen before storing in an −80°C freezer for later analysis.
In the case of the heart, it was removed intact, blotted to remove any blood, and weighed. The atria and any remaining portions of the great vessels were then removed prior to the ventricles being frozen.

SOD enzyme activity
Heart tissues were washed in heparinised PBS, and 15 mg of tissue was used for enzyme activity assays. Heart tissue was homogenized in 5 ml of 20 mM HEPES buffer (pH 7.2) containing 1 mM EGTA, 210 mM mannitol and 70 mM sucrose per gram of tissue. Samples were standardised so that 10 ul of sample with 10 mg of protein was added to each well on the 96-well assay plate. The assay kit detecting total SOD activity was undertaken as per the manufacturer's instructions (Cayman Chemical). The amount of activity was calculated and standardised for protein using the Bradford method (Bio-Rad).

DNA oxidation assay
An ELISA kit was used to measure the DNA oxidation byproduct 8-hydroxy-2-deoxy guanosine (StressMarq Biosciences). DNA was extracted from 15 mg of tissue using a DNA isolation kit (Promega). Each sample was then diluted so that 50 ug of DNA was used in the 8-hydroxy-2-deoxy guanosine assay. The competitive immunoassay involves the binding of free 8-hydroxy-2-deoxy guanosine to an antibody coated 96-well plate. The assay and sample concentration of 8-hydroxy-2-deoxy guanosine was carried out as per the manufacturer's instructions.

RT-PCR
RNA was extracted from frozen heart tissue samples using trireagent (Molecular Research Centre). Total RNA concentration yielded per sample was quantified spectrophotometrically at 260 nm. All samples were reverse-transcribed to cDNA using reverse transcription kit (Marligen). cDNA was stored at −20°C until further analysis. All primer sequences were designed using Primer 3 (primer sequences shown in Table 1). Real-time PCR was performed using a IQ 5 PCR detection system (Bio Rad). A supermix (Bio Rad) was prepared containing 10 ng of sample cDNA, forward and reverse primers diluted to a final concentration of 300 nM, in a total volume of 25 ul. Quantification of mRNA expression was achieved by running samples in duplicate and over a period of 40 cycles. Flourescence data were tabulated and mRNA levels quantified using threshold cycle values. 18S was used as a housekeeping gene to compensate for variations in input RNA amounts by normalizing values to this gene.

Protein quantification
Heart tissue was homogenized in 20 mM HEPES buffer. The homogenate was centrifuged, and the supernatant was collected and placed on ice. Protein content was assayed using the BCA Protein Assay Kit (Bio Rad). Fifty micrograms of total cellular protein was then loaded on 12 % polyacrylamide gels (NuSep) and separated by standard SDS-PAGE. Gels were then transferred to polyvinylidine fluoride membranes (Bio Rad). Blots were blocked with Tris-buffered saline including 0.1 % Tween 20 and 5 % nonfat dried milk. Primary antibody (Santa-Cruz) against p47 phox , NOX2, and GAPDH at a dilution of 1 in 2,000 was added to the membrane overnight at 4°C. Secondary donkey-antigoat antibodies (Santa-Cruz) conjugated to HRP at a 1 in 10,000 dilution was added to the membrane for 1 h at room temperature. Antigen-antibody complexes were analysed by chemiluminescence using the immune-star HRP detection kit (Bio Rad). Subsequent development and detection of the membrane was achieved with the Chemidoc system (Bio Rad). Protein expression data were then analysed using the Quantity One software (Bio Rad).

Statistical analysis
The reported values are expressed as mean±SEM. Data were analyzed using two-way ANOVA, with a Tukey's test post hoc analysis performed to determine differences between groups where appropriate. Significance was set at a P value equal to or <0.05

Results
The animals in this study were randomised to one of three groups being a no-tumour control, cancer or cancer cachexia. Animals in the cachexia group displayed all of the hallmarks of cancer cachexia including the loss of body weight (P = 0.003), skeletal (P =0.003) and cardiac (P =0.002) muscle with no significant differences in food and water intake between the groups ( Table 2). An analysis was then carried out to ascertain whether key oxidative and proteolytic pathways were upregulated in cardiac tissue.
To investigate the potential source of oxidative stress contributing to the oxidative DNA damage found in the cachectic hearts, we investigated the ROS producing enzymes, NADPH oxidase and XO. RT-PCR was performed on the key regulatory subunits (p40 phox and p67 phox ) and the catalytic core units (NOX1, NOX2 and NOX4) of NADPH oxidase. There were no changes in the key regulatory or catalytic core units of NADPH oxidase in the heart muscle samples except for NOX2 (Table 3). The current experiment found a significant 1.7-fold lower level of gene expression for NOX2 in cachectic hearts (P =0.03) compared with the cancer group (Table 3). To assess the possibility of posttranslational modification to the NADPH oxidase protein components, a protein expression analysis was undertaken on p47 phox and NOX2. There was no significant difference in either p47 phox and NOX2 protein abundance in the cancer cachectic hearts compared with both the control and cancer groups (Table 3).
Subsequently, the XDH enzymatic pathway known to produce ROS was investigated. The RT-PCR results showed a significant twofold higher level of XDH mRNA (P =0.04) in the heart of cachexia mice in comparison to control and cancer groups (Table 3). Other possible contributors to the state of   (Table 3). However, further investigation of total SOD enzyme activity showed a significant 1.5-fold lower activity in mice with cachexia compared with control (P =0.02) and cancer (P =0.001, Fig. 2). Accompanying a state of oxidative stress in cancer cachectic hearts were higher levels of expression of the major muscle proteolytic pathway, the UPS. MURF-1 and MAFbx mRNA expression was significantly higher, 4.3-fold (P =0.04) and 3.8-fold (P =0.04), respectively, in the hearts of the cachexia mice compared with the cancer group (Table 3).

Discussion
The cardiac atrophy secondary to CC observed in this study is associated with a pro-oxidative shift and consequential oxidative myocardial tissue damage. Furthermore, upregulation of the key ubiquitin ligases in cachectic hearts suggests induction of the UPS may be in response to oxidative stress and subsequent protein damage.
The current study demonstrated the importance of cardiac atrophy as a hallmark of the disease with an average 20 % decrease in heart muscle weight compared with controls.
The consequence of such cardiac atrophy was demonstrated by Tian et al. who used echocardiography to analyse the function of cachectic hearts in a mouse model of cancer cachexia [4]. Their study found that, 14 days after the inoculation with tumour cells, the hearts of cancer cachectic mice had reduced heart rate and fractional shortening [4]. To date, the mechanisms influencing cardiac tissue wasting in a state of CC are largely unknown. However, previous studies have shown that patients and animals with CC all show degrees of oxidative stress [21,22]. To elucidate whether a state of oxidative stress exists in the heart of mice afflicted with CC, the current study measured levels of 8-Oh-2dG and found a significantly higher levels in the cachexia group hearts. The higher 8-Oh-2dG suggests that the activation of pro-oxidant pathways in atrophied hearts of cachectic mice. Importantly, both the release of cytokines such as TNF-alpha and tumour-derived factors such as PIF have been shown to have pro-oxidant effects [20,21]. Gomes-Marcondes and Tisdale used myotubes in cell culture exposed to FeSO 4 and H 2 O 2 which showed that key components of the ubiquitin proteasome system were upregulated by oxidative stress [20]. NOX2 mRNA expression was lower in cachexia hearts compared with the control group, while the Western blot data were unchanged. The results suggest a decreased role for NADPH oxidase in contributing to cellular oxidative stress in cachectic hearts. The results are also in keeping with a previous study by Sullivan-Gunn showing a decrease in NOX2 levels in skeletal muscles of cancer cachectic mice [23]. The animals in this study were allowed to lose up to 25 % of their initial body weight, thus constituting late-stage cachexia. Considering that NADPH oxidase enzyme activity has been shown to degrade cultured myotubes in vitro [23], a time course study would be beneficial in understanding whether the current results are specific to cardiac atrophy in late cachexia. Another key pathway of ROS formation in the myocardium is XDH that was measured using RT-PCR. The results showed a twofold higher level of XDH in the cachexia group hearts. The activation of XDH has been implicated in a number of disease states involving the myocardium such as heart failure and myocardial dysfunction [16,17]. A study by Ekelund et al. highlighted the importance of XO in cardiac disease by showing that inhibition of the enzyme by allopurinol is capable of increasing left ventricular function and decreasing myocardial oxygen consumption in a dog model of heart failure [24].
In evaluating the potential contribution of antioxidant systems in the cachectic heart, SOD was analysed both at a gene and enzyme activity level. The SOD enzyme is a key endogenous antioxidant in the myocardium for the elimination of superoxide and has been implicated in the pathogenesis of CC [25]. Interestingly, while the cytosolic and mitochondrial SOD mRNA levels were unchanged in cachectic hearts, total SOD enzyme activity was significantly lower compared with controls. This suggests that the SOD enzyme is not compensating for increased levels of ROS. Thus, the concurrent higher levels of XDH and lower SOD activity may significantly contribute to a disruption in the balance between pro-oxidant and antioxidant mechanisms, leading to a state of oxidative stress as evidenced by greater oxidative damage to DNA (8- Oh-2dG) in the cachectic heart. With respect to how oxidative stress may contribute to cardiac atrophy in CC, it is currently unknown; however, evidence suggests induction of the UPS may be an important link between oxidative stress and atrophy in cachectic muscle [20]. Otsuka et al. found that in patients' hearts undergoing dilated cardiomyopathy, oxidative stress and the UPS activation are colocalised [26]. Increases in levels of XO and altered SOD enzyme function in the myocardium are traditionally linked to cardiac fibrosis and cardiomypathies [16,27]. However, the current study shows an association between oxidative stress and cardiac atrophy, suggesting an alternative pathologic role for oxidants in the myocardium. In elucidating the mechanisms by which ROS contributes to the loss of lean body mass, Gomes-Marcondes and Tisdale attempted to replicate the oxidative stress seen in cancer cachexia by using murine myotubes in cell culture [20]. The myotube cells were exposed to H 2 O 2 which induced oxidative stress. Their results showed that the induction of oxidative stress caused an increase in the expression of important UPS elements including the 20S proteasome and the E2 14k conjugating enzyme [20]. The UPS E3 ligases MURF-1 and MAFbx are expressed in cardiomyocytes and are an important component of cardiac protein turnover [28]. Increased mRNA expression of these ligases is linked to the degradation of cardiac troponin I, αactin-2 and MyoD leading to impaired contractility [13,29]. This study shows that both MURF-1 and MAFbx are upregulated at the mRNA level (Table 3) which is in keeping with results seen in studies analysing cachectic heart muscle tissue [30]. Interestingly, Cosper and Leinwand using CD2F1 hybrid mouse model of CC did not see any change in the UPS but found an upregulation in autophagy-related protein degradation [2]. However, the study by Tian et al. using a mouse model of CC similarly to this study found that mRNA of UPS E3 enzymes to be increased in cachectic hearts [30]. Furthermore, Tian and colleagues found that there was increased protein ubiquitinylation in the hearts of cancer cachexia mice compared with controls suggesting that indeed the UPS has an important role to play in cardiac atrophy secondary to CC [30]. A limitation of the current study is the changes in MURF-1 and MAFbx that are at the mRNA level and so correlative in nature. Future investigations would include the evaluation of UPS activation, the state of activation of other proteolytic systems and the phosphorylation levels of Akt and Foxo proteins upstream of ubiquitin ligase activation.

Conclusion
The current study used a mouse model of CC producing significant body weight loss and a reduction in heart mass by 20 %. The experimental data demonstrated that cardiac atrophy of cachectic mice is related to a state of oxidative DNA damage in the myocardium. Levels of XDH mRNA were higher in CC hearts, suggesting that XO may have an important role to play in producing oxidative stress. Most evidently, it appears that the combination of higher XDH expression and lower SOD enzyme activity are key contributors to oxidative stress and cardiac tissue damage in cancerinduced cardiac atrophy. Oxidative stress in the myocardium as with skeletal muscle may also induce increased expression of the E3 ligases MURF-1 and MAFbx as seen in higher mRNA levels of both in this study. Further research is needed in a timedependent manner to ascertain whether these changes are seen in early cachexia and are indeed initiating mechanisms.


extfieldsofstudy: Biology Medicine

text:
Roflumilast Inhibits Respiratory Syncytial Virus Infection in Human Differentiated Bronchial Epithelial Cells

Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H2O2 and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H2O2 formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus.

Introduction
Respiratory syncytial virus (RSV) is an enveloped negativesense single-stranded RNA virus of the Paramyxoviridae family. It is the main cause of severe lower respiratory tract infections (bronchiolitis and pneumonia) in infants and young children and it is also a pathogen of considerable importance in the elderly and in immunocompromised adults [1,2]. In addition, a substantial proportion of acute exacerbations in COPD or asthma have been related to RSV infections [3,4]. In COPD, acute exacerbations attributed to RSV may account for about 10.000 deaths per year among the elderly population (> 65 years of age) in the US [5].
The main target of RSV is the ciliated bronchial epithelial cells. RSV infection triggers a loss of ciliated cells and Goblet cell metaplasia associated with enhanced MUC5AC production [6][7][8][9]. Furthermore, RSV infection causes the release of numerous pro-inflammatory chemokines and cytokines from bronchial epithelial cells and entails an imbalance between an enhanced reactive oxygen species (ROS) production and a compromised antioxidant enzymatic armamentarium [10,11]. Collectively, given these numerous effects, bronchial epithelial cells are considered key players of RSV-induced lung diseases.
Among the new concepts in respiratory diseases, such as COPD and asthma, selective inhibitors of phosphodiesterase 4 (PDE4) are under scrutiny for more than two decades as new selective drugs. PDE4 is one out of the eleven families of cyclic nucleotide hydrolyzing phosphodiesterases in humans and uses cAMP as its specific substrate. As a corollary, inhibitors of PDE4 augment cellular cAMP content resulting in inhibitory effects on inflammation, oxidative stress and tissue remodeling [12][13][14]. PDE4 is expressed in human airway epithelial cells [15][16][17]. While initial studies largely failed to demonstrate significant effects of PDE4 inhibitors on airway epithelial cells [16,17], evidence is accumulating from more recent reports that selective inhibitors of PDE4 can: i) reduce EGF-stimulated MUC5AC expression [18], ii) enhance CFTR activity [19], iii) prevent TGFβ1-induced epithelial mesenchymal transition [20], iv) promote ciliary beat frequency and protect from tobacco smoke-induced loss of ciliated cells [21] and v) reduce the release of a number of cytokines or chemokines following different stimuli of primary human bronchial epithelial cells or established cell lines such as A549 or BEAS2B [22][23][24][25].
Historically the development of PDE4 inhibitors was intimately related to COPD and as the first-in-class PDE4 inhibitor roflumilast is currently in use for the treatment of severe COPD in patients with chronic bronchitis and frequent exacerbations. A major asset inherent to this remedy is its proven ability to mitigate the risk of acute exacerbations [26][27][28][29][30], which might be triggered by viral infections.
Evidence has been provided that a range of therapeutic compounds of potential interest in COPD such as statins [31], anti-oxidants such as N-acetylcysteine or L-carbocisteine [9,32], tiotropium [33], macrolides [34] and PPARγ agonists [35] are all capable to reduce RSV production in human bronchial epithelial cells.
However, effects of PDE4 inhibitors and specifically roflumilast on RSV-infected bronchial epithelial cells have not yet been explored. Therefore, the objective of the current study was to analyze the effects of the PDE4 inhibitor roflumilast-Noxide (RNO, the active metabolite of roflumilast largely governing clinical efficacy [36,37]) in an in vitro model of RSV infection in well-differentiated normal human bronchial epithelial cells (WD-HBE). In this context it was explored whether the PDE4 inhibitor influences viral load, ICAM-1 expression, markers of ciliated cells (β-tubulin, Foxj1, Dnai2) and Goblet cells (MUC5AC and CLCA1), a range of inflammatory cytokines (IL-13, IL-6, IL-8, TNFα) and the burden of oxidative stress and the anti-oxidative cellular armamentarium.

Cells, infections and incubations
Human lung tissue was obtained from patients subjected to surgery for lung carcinoma as previously described [18]. Procedures were approved by the local ethics committee. Te full name of this Commit is: "Comite Etico de Investigacion Clinica del Consorcio Hospital General Universitario de Valencia". Written informed consent of all donors were obtained. At the time of surgery, lung function was within the normal range (spirometry). WD-HBE cells were cultured and differentiated in 24 wells transwell inserts (0.3 cm 2 , Corning Costar, High Wycombe, UK) under air-liquid interface (ALI) conditions as previously described [9]. WD-HBE cells were infected with 2 × 10 6 plaque forming units (PFU) of RSV or mock in 100 µL of differentiation medium per insert in the presence or absence of RNO 0.1-100 nM. Cultures were incubated for 2 hours at 37°C and washed once with 500 µL of differentiation medium. RNO was added to the cultures for 1h prior to infections and remained present until the end of the experiment.
Cultures were maintained until day 15 post-infection and culture medium and analysed compounds were replaced every other day. Measurements were performed at the indicated time points over this period. In this study, cultures from three different donors were used.
RNO was obtained from Nycomed GmbH: A Takeda Company (Konstanz, Germany). It was diluted from a 10mM stock in DMSO to the final concentrations in 0.1% DMSO.

RSV production and determination of viral titters
RSV (Long Strain, ATCC VR-26) obtained from the ATCC (American Type Culture Collection; Rockville, MD, USA) was propagated in HEp-2 cells as previously described [38]. Viruses were purified from clarified culture supernatants by polyethylene glycol precipitation and centrifugation in a 30-45-60% discontinuous sucrose gradient in TNE buffer [39]. Viral titters were determined by plaque assay in HEp-2 cells with 0.5% low melting-point agarose overlaid (Conda Laboratories, Madrid, Spain). Virus inactivation (for mockinfection) was achieved by irradiation with UV light for 90 min and confirmed by plaque assay.

Quantification of viral infection
Viral infection in WD-HBE cultures was determined by immunocytochemistry of viral glycoproteins G and F and realtime RT-PCR as previously described [9].
Immunocytochemistry of viral glycoproteins was conducted with a pool of monoclonal antibodies (2F, 47F, 56F, 021/1G, 021/2G). For semi-quantitative Real Time RT-PCR, primers to amplify the nucleoprotein RNA of the human RSV (forward primer 5´ CATGATTCTCCTGATTGTGGGATGA3´, reverse primer: 5´ TCACGGCTGTAAGACCAGATCTAT 3´, probe: 5Ć CCCTGCTGCCAATTT3´) were designed [9]. The procedures for RNA extraction, cDNA synthesis and real time PCR were as described later. Ct values obtained for the RSV nucleoprotein RNA were related to GAPDH of the bronchial epithelial host cells and finally presented according to the standard 2-ΔΔCt procedure.

Functional ciliated cell analysis
Ciliary beat frequency and motility were evaluated as described previously by digital high speed videomicroscopy (DHSV) [9,40]. Three independent videos of each insert were recorded and the number of cells with cilia motility was assessed by manual counting by a blinded observer.

Immunofluorescence analysis of β-tubulin
Nitrocellulose membranes were removed from the inserts, placed in Tissue-Tek® OCT TM (Sakura Finetek, Europe, Alphen aan den Rijn, The Netherlands) and cut into 10 µm sections. Immunofluorescence was performed using a mouse monoclonal antibody against human β-tubulin and a secondary rhodamine labeled goat anti-mouse IgG (Sigma, St Louis, MO, USA). DAPI (Invitrogen Ltd., Paisley, UK) was used for nuclear staining. Ten sections of each group were analyzed and the results were shown as percent of mock-infected as previously described [41].

Analysis of Goblet cells by PAS staining
PAS staining served to identify Goblet cells in WD-HBE. Nitrocellulose membranes were removed from the inserts, fixed and paraffin-embedded as outlined before [18]. Inserts were cut in 5µm sections and stained using the PAS staining system (Sigma, St Louis, MO, USA). Finally, sections were mounted with DPX (Sigma, St Louis, MO, USA) prior to microscopical analysis.
WD-HBE were washed once with 100µL of sterile PBS, and 65 µL of fresh culture medium was added to the surface. After one hour of incubation, supernatants were collected and analyzed for the release of TNF-α, IL-8 and IL-6 using a multiplex cytometer-based method (Luminex DX100, Luminex Corp., Austin TX, USA) and ultrasensitive panels (Millipore, Billerica, MA, USA). Determinations were carried out following manufacturer instructions.

Determination of Total Antioxidant Capacity (TAC) and H 2 O 2 concentration
TAC was determined in whole cell lysates using the Total Antioxidant Assay kit (Cayman Chemical Company, Michigan, USA) following manufacturer's instructions. Intracellular levels of H 2 O 2 were determined using the Amplex Red Reagent (Invitrogen Ltd., Paisley, UK) as previously described [42].

Analysis of results
Data are shown as the mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA) followed by Tukey's multiple comparison test or t-test, as appropriate (GraphPad Software Inc., San Diego, CA). Significance was accepted at p<0.05. EC50 values were calculated from nonlinear regression analyses using GraphPad Prism Software.

Effects of roflumilast N-oxide on RSV replication and density of cells with cilia motility in WD-HBE
First, the effects of RNO on the viral load in RSV-infected well-differentiated normal human bronchial epithelial cells (WD-HBE) were investigated. WD-HBE were infected with RSV (2 x 10 6 PFU/insert, 2 h) in the absence or presence of RNO (0.1-100 nM) or mock-infected. At day 10 after RSV infection there was a very significant increase in intracellular viral antigens as visualized by immunocytochemical detection of viral F and G proteins ( Figure 1A, upper right versus left microphotograph). RNO reduced the number of RSV antigen positive cells in a concentration-dependent manner ( Figure 1A, lower panels). In parallel, WD-HBE infected with RSV showed a substantial increase in the expression of RSV nucleoprotein RNA that was reduced steadily by increasing doses of RNO (Figure 1  Next, the number of cells with motile cilia was evaluated. WD-HBE were infected with RSV in the presence of RNO (0.1-100nM) or vehicle. At day 10 after RSV infection, the number of cells with motile cilia decreased by about 55% compared to mock-infected cells. RNO prevented this reduction of ciliated cells in a concentration-dependent manner (EC50 of 5.9 nM) being significant at 1nM and fully prevented the effect of RSV at 100nM (Figure 1 panel D)

Effects of RSV and RNO on the number of β-tubulin labelled ciliated cells and mRNA levels of the ciliated cell markers foxj1 and Dnai2
Next it was addressed whether roflumilast N-oxide may reverse a loss in cilia following RSV [9] that may account for the effects on the number of cells with motile cilia. To this end, WD-HBE were infected with RSV (2 × 10 6 PFU/insert) in the absence or presence of RNO at 1 or 100nM, or mock infected. At day 10 after infection sections were immunostained to detect β tubulin IV. RSV reduced by 75% the apical β tubulin IV staining. RNO concentration-dependently rescued this reduction by 50% (Figure 2 panels A and B). This cilia loss was consistent with the results obtained for the gene expression of the ciliagenesis regulator FOXJ1 and the dynein DNAI2 ( Figure  2 panels C and D respectively).

Effect of roflumilast N-oxide on RSV-induced Goblet cell metaplasia and the enhanced expression of MUC5AC and hCLCA1, potential role of IL-13
RSV infection resulted in an increase of goblet cells and in a reduction of the thickness of the epithelial layer (verified by PAS staining, data not shown) and in an increase of MUC5AC and hCLCA1 expression. RNO prevented these alterations (Figure 3 panels A and B). EC50 values for MUC5AC and CLAC1 expression were of 0.25 and 3.8 nM respectively. As IL13 is implicated in these alterations, mRNA and protein levels were measured. RSV increased both expression and protein levels of IL13 and RNO restored them in a dose dependent manner (Figure 3 panels C and D).

Effects of roflumilast N-oxide on the expression and release of IL-8, IL-6 and TNF-α from RSV infected, differentiated WD-HBE
After RSV infection, we have found an increase of mRNA and protein release of IL-8, IL-6 and TNF-α in WD-HBE. This increase was inhibited by RNO concentration-dependently (EC50 of 0.6 (IL8), 0.4 (IL6) and 0.07 (TNF-α) nM for mRNA expression). Results are summarized in Figure 4.

Effect of RNO on the compromised anti-oxidant machinery and enhanced oxidative stress in RSVinfected WD-HBE
An imbalance between enhanced production of reactive oxygen species (ROS) and suppressed antioxidant enzymes and Nrf2 plays a critical role in RSV infection [9,11]

Figure 1. Roflumilast N-oxide (RNO) alleviated viral burden following RSV infection, reduced RSV-induced ICAM-1 expression and restored cilia motility in well-differentiated human bronchial epithelial cells (WD-HBE).
WD-HBE were infected with RSV (2 x 10 6 PFU/insert) or mock-infected (control) in the presence of Roflumilast N-oxide (RNO; 0.1-100nM) or vehicle and maintained in air-liquid culture over another 10 days for viral quantification or 15 days for ICAM-1 and cilia motility evaluation. RSV abundance in WD-HBE was determined by immunocytochemical detection of RSV F and G proteins (panel A) or real-time RT-PCR based quantification of RSV nucleoprotein RNA (panel B). In panel B viral nucleoprotein RNA was related to host cell GAPDH and normalized to RSV infection with vehicle (=1). In mock-infected WD-HBE, RSV nucleoprotein RNA was below detection limit. ICAM1 expression was determined by Real Time RT-PCR using mock-infected cells as controls (panel C). Cilia motility was evaluated by HSDV using three independent videos of each experimental replicate. Results represent mean ± SEM of three independent infections. Each condition was evaluated by triplicate in three independent wells. Cultures from three different patients were used. * p< 0.05 compared to mock-infected cells. # p<0.05 compared to infected cells.

Discussion
A key observation from the current study has been that RNO inhibits RSV infection in WD-HBE cultures which reproduce many aspects of normal human epithelium. RNO mitigates the decrease in ciliary activity as a reflect of the loss in ciliated cells, goblet cell metaplasia/hyperplasia and the increase in IL-13 and MUC5AC. RNO also inhibits the transcription and release of proinflammatory cytokines and ICAM-1 that follows RSV infection and also attenuates ROS by restoring the imbalance between its formation and a suppressed antioxidative capacity. Although our work has made use of a reference strain of RSV it would be interesting to carry out similar studies with a set of well characterized clinical isolates, even though Villenave et al. have reported qualitative similar effects (despite some quantitative differences) of the reference strain A2 and the clinical isolate BT2a in equivalent cultures of differentiated bronchial epithelial cells [8].
To our knowledge this is the first study that uses WD-HBE cultures generated from human lung tissue in an air-liquid interface protocol to study the protective effects of RNO against RSV infection. These cultures comprise ciliated cells, mucus producing cells and basal cells. Effects from RSV infection on WD-HBE in vitro as confirmed in the current work are reminiscent of the pathology of RSV-induced bronchiolitis in humans. (i) RSV target ciliated cells at their apical surface (ii). RSV infection is followed by a loss in ciliated cells and triggers goblet cell hyperplasia / metaplasia associated with an increase in MUC5AC (iii). RSV results in increased cytokines and chemokines supposed to orchestrate inflammatory cell influx characteristic of severe RSV bronchiolitis [8].
Although we haven't measured cAMP levels directly, the EC50 values used in this study support that in this in vitro model of RSV infected WD-HBE cultures RNO acts by inhibiting PDE4 with consequent increase in cAMP levels [37]. This increase in cAMP levels has been previously reported in cultured WD-HBE cells, alveolar type II cells, macrophages, smooth muscle cells, epithelial intestinal cells and cardiomyocytes cells using the same range of doses studied in this work [21,[43][44][45][46][47]. Concerning mechanisms involving in the reduction of RSV infection, speculative conclusions can only be established. RNO might interfere with mechanisms of RSV binding and entry to the (ciliated) bronchial epithelial cells. Earlier work points to ICAM-1, heparin, annexin II or TLR4 as potential "RSV receptors" on the host cell as they were found to interact with viral F (ICAM-1, TLR4) and G (heparin, annexin II) proteins [48]. Moreover ICAM-1 expression is controlled by ROS mediated events [49]. Data presented here indicate that after RSV infection there is a significant increase in the ICAM-1 mRNA levels in WD-HBE cultures which is inhibited by RNO. This inhibition could explain, at least in part, some of the antiinfective properties of RNO found and could be a direct consequence of the oxidative stress modulation observed in RNO treated cultures. TNFα (panel C) mRNA transcripts (upper panels) and released protein (lower panels) were performed at day 10 after infection. For quantitative mRNA expression analyses total RNA was extracted, reverse transcribed and analyzed by real time RT-PCR. For measurements of released cytokines apical supernatants from an one hour incubation as described in Methods were collected and analyzed using Luminex. Results are depicted as the means ± SEM of three independent infections. Each condition was evaluated by triplicate in three independent wells. Cultures from three different patients were used. * p< 0.05 compared to mock-infected cells. # p<0.05 compared to RSV infected cells.  Augmented ROS in RSV infection is attributed to an imbalance between (enhanced) formation by NADPH oxidases [50] and (suppressed) degradation by the anti-oxidative enzymatic apparatus governed by Nrf2 [11]. In the current and previous work from this laboratory [9] an initial increase in Nrf2 mRNA at about 4 days post RSV infection was followed by a later loss to about half the expression level in mock infected cells at ≥ 10 d. This kinetics has also been observed in A549 cells and small airway epithelial cells [51]. Diminished antioxidant enzymes following RSV infection as found for GPx in the current study have been described in vitro, in vivo and in human [51,52]. In RSV-infected mice reduced lung activities of SOD, catalase, GPx, GST are paralleled by a loss in nuclear Nrf2. In infants with severe RSV bronchiolitis anti-oxidant enzymes are compromised. RNO, in a concentrationdependent manner, rescued from the loss in HO-1 and GPx expression following RSV indicating a potential to support the anti-oxidative apparatus. These effects likely emanate from the ability of the PDE4 inhibitor to enhance Nrf2. RNO was not only reversing the loss in Nrf2 caused by RSV but increased Nrf2 transcripts by about 3 fold over (mock-infected) controls. Likely, this increase in Nrf2 over baseline cannot only be explained by reduced viral load. A direct effect of cAMP to enhance Nrf2 as described in human keratinocytes or melanocytes is not excluded [53]. Given that sulforaphane curbs viral load in RSV infected mice [54] the enhanced Nrf2 by RNO may support to alleviate RSV infection. RNO reduced the increased generation of H 2 O 2 in RSV-infected WD-HBE cultures. Aside from an inhibition of NADPH oxidases well described for PDE4 inhibitors [21] the enhanced GPx expression may contribute to mitigate H 2 O 2 release by roflumilast N-oxide [51]. Taming ROS may restore Nrf2 and the anti-oxidative armamentarium [9]. Collectively, the current data suggest that RNO may correct the imbalance between enhanced ROS generation and a suppressed anti-oxidative apparatus in RSV infection. These effects can explain the inhibitory at cilia activity, metaplasia, mucin production and inflammatory cytokine and chemokines expression and release reported here, and are in parallel with those previously described for the antioxidant N-acetyl-cysteine (NAC) [9].
RSV may cause a broad spectrum of (lower) respiratory diseases that includes life-threatening bronchiolitis and respiratory failure specifically in infants and young children. RSV is among the players in acute exacerbations of COPD and asthma [55]. Roflumilast, the most advanced of PDE4 inhibitors, has been recently approved by the European Medicines Agency (EMA) for the maintenance treatment of severe COPD disease (FEV1 post-bronchodilator less than 50% predicted) associated with chronic bronchitis in adult patients with a history of frequent exacerbations as add on to bronchodilator treatment. Data presented here indicate that pre-treatment of WD-HBE cultures with RNO, the active metabolite of roflumilast, inhibits RSV infection, which is one of the viruses related to exacerbations. Roflumilast, compared to other anti-inflammatory drugs, has shown effectiveness in therapeutic models of inflammation [56]. Although in principle roflumilast is not an antiviral drug, it would be interesting to explore if this compound could have any effect in a therapeutic scenario where the viral infection was well established in the airway epithelia. More studies are necessary to answer this question. It remains an open question, whether one or another from the growing list of different therapeutic drugs that have shown to reduce viral infection of human bronchial epithelial cells and their consequences in vitro, to which PDE4 inhibitors are now added, may translate into a favorable outcome in controlled clinical trials.


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BMSCs reduce rat granulosa cell apoptosis induced by cisplatin and perimenopause

Background The objective of this study was to evaluate the effect of bone marrow mesenchymal stem cells (BMSCs) on the apoptosis of granulosa cells (GCs) in rats. BMSCs and GCs were isolated from rats. GCs were separated into one of the following three groups: an untreated control group (control), a cisplatin (5 mg/L) treatment group (cisplatin), and group co-cultured with BMSCs and treated with cisplatin (BMSC). GC apoptosis was analyzed by annexin V staining and real-time PCR analysis for apoptosis-related genes. The effect of BMSCs was also determined in 9 to 10 month-old perimenopausal rats that were separated into the following groups: saline control, BMSC transplantation (1–2 × 106 cells), and estrogen treatment (0.158 mg/kg/d) groups. A young group consisting of 3 to 4 month-old rats that were treated with saline was also evaluated as a control. After 1 and 3 months, GC apoptosis was evaluated by TUNEL analysis. Results Cisplatin increased GC apoptosis from 0.59% to 13.04% in the control and cisplatin treatment groups, respectively, which was significantly reduced upon co-culture with BMSCs to 4.84%. Cisplatin treatment increased p21 and bax and decreased c-myc mRNA expression, which was reversed upon co-culture with BMSCs. As compared to young rats, increased apoptosis was observed in the perimenopausal rats (P < 0.001). After 3 months, the apoptosis rate in the BMSC group was significantly lower than that of the control group (P = 0.007). Conclusions BMSC therapy may protect against GC apoptosis induced by cisplatin and perimenopause. Further studies are necessary to evaluate therapeutic efficacy of BMSCs.

Background
Perimenopause or the permanent end of menstruation and fertility is characterized by reduced estrogen levels and disorder of the hypothalamus-pituitary-ovary/adrenal axis. Therefore, estrogen replacement therapy is commonly prescribed to increase the quality of life for individuals in perimenopause. However, significant risks have been associated with estrogen replacement therapy, including increased risk of breast cancer, endometrial cancer, and cardiovascular events [1][2][3]. Thus, better treatments without the associated risks are required.
Granulosa cells (GCs) are responsible for estrogen synthesis within the ovary and subsequent follicular atresia, and their numbers decrease in perimenopause [4]. Therapies that prevent their apoptosis may represent a novel treatment modality. Previous studies have shown that mesenchymal stem cells (MSCs), including those derived from bone marrow (BMSCs), are capable of repairing a variety of injured tissues [5,6] due to their potential to differentiate into osteoblasts, chondrocytes, adipocytes, cardiocytes, and neural cells [7]. In rats with chemotherapy-induced ovarian damage, BMSC transplantation improved ovarian structure and function [8]. However, little is known about the therapeutic effects of BMSCs on GC apoptosis associated with perimenopause.
The objective of this study was to evaluate the effect of BMSCs on the apoptosis of ovarian GCs in perimenopausal rats. BMSC and GC cultures were isolated from rats and characterized using microscopy, immunocytochemistry, and flow cytometry. In vitro GC apoptosis was induced by cisplatin in GCs co-cultured with and without BMSCs. In addition, GC apoptosis was analyzed in the perimenopausal rats treated with BMSCs and estrogen using TUNEL assays and compared to untreated controls and young rats. BMSC therapy may represent a novel treatment for perimenopause that may have less deleterious effects than estrogen replacement therapy.

Characterization of cultured BMSCs and GCs
Isolated BMSCs in culture were characterized by microscopy and flow cytometry analyses. Freshly isolated and cultured BMSCs were adherent, with most displaying polygonal morphology ( Figure 1A). By the third passage, BMSC morphology resembled fibroblasts with few contaminant cells ( Figure 1B). Most of the cultured BMSCs expressed CD29 (99.1% positive) and CD90 (98.2% positive) but were negative for markers of the hematopoietic lineage, including CD34 (1.01% positive) and CD45 (1.32% positive) ( Figure 1E-H), which is consistent with previous studies [9,10].
To determine the extent to which induction resulted in BMSC differentiation toward osteoblasts versus adipocytes lineages, cells were examined after Von Kossa staining. As shown in Figure 1C (black cells) and 1D (red cells), isolated BMSCs were capable of differentiating into both osteoblasts and adipocytes. Furthermore, the basal secretion of large amounts of angiogenic and antiapoptotic factors from BMSCs cells after three days in culture was observed and included 1674.22 ± 182.80 pg/mL vascular endothelial growth factor (VEGF), 22255.75 ± 3473.66 pg/mL hepatocyte growth factor (HGF), and 0.9237 ± 0.13 ng/mL insulin-like growth factor-1 (IGF-1).
Isolated GCs were also characterized by microscopy and immunocytochemistry. Cultured GCs were adherent and displayed polygonal morphology (Figure 2A). Cultured GCs also expressed intracellular follicle stimulating hormone receptor (FSHR), confirming their identity as GCs ( Figure 2B).

Effects of BMSCs on GC apoptosis in vitro
To determine whether BMSCs could protect GCs against cisplatin-induced apoptosis, GCs cultured in the absence or presence of BMSCs were treated with cisplatin. As shown in Figure 3, cisplatin increased apoptosis from 0.59% to 13.04% in the control and cisplatin groups, respectively ( Figure 3A and 3B). However, co-culture with BMSCs significantly reduced GC apoptosis to 4.84% ( Figure 3C).
Real-time PCR analysis was next undertaken to determine the effect of BMSC co-culture on the mRNA expression of apoptosis-related genes ( Figure 4, N = 6 for each group). Although no significant changes in survivin and bcl-2 mRNA expression were observed among the three treatment groups, differences in p21 (P = 0.033), c-myc (P = 0.029), and bax (P < 0.001) mRNA expression were detected. Co-culture of GCs with BMSCs significantly decreased p21 mRNA expression (2.944 ± 1.322 vs. 1.034 ± 1.492, P = 0.046). Cisplatin treatment decreased c-myc mRNA expression as compared to the control group (1.253 ± 0.789 vs. 0.314 ± 0.177, P = 0.028), and this effect was ameliorated with BMSC co-culture. Furthermore, a significant increase in bax mRNA expression was observed with cisplatin treatment as compared to the control group (2.934 ± 0.955 vs. 1.019 ± 0.211, respectively; P < 0.001); however, it significantly decreased upon co-culture with BMSCs (0.476 ± 0.167, P < 0.001; Figure 4).

Effects of BMSCs in an in vivo model of perimenopause
To determine if BMSC transplantation influences the GC apoptosis associated with perimenopause, an in vivo model was established in middle-aged rats ( Figure 5, N = 5 for each group). Both treatment (P < 0.0001) and time (P = 0.0008) had significant effect on the apoptosis rates. However, the interaction of time and treatment on the apoptosis rates was not significant (P = 0.638). As compared young rats, increased apoptosis was observed in the middle-aged rats after one month (1.53 ± 1.44% vs. 9.90 ± 3.23%, P < 0.001; Figure 5). After intervention with BMSC therapy or estrogen for 1 month, the apoptosis rate was lower than the control group although a significant difference was not observed. After 3 months, the apoptosis rate in the BMSC group (8.29 ± 3.35%, P = 0.007) was significantly lower than that of the control group (14.60 ± 3.51%). No difference in estrogen treatment was observed.

Discussion
In the present study, co-culture with BMSCs significantly reduced cisplatin-mediated GC apoptosis and cisplatin-induced changes in p21, bax, and c-myc mRNA expression. In an in vivo model of perimenopause, the GC apoptosis rate was significantly lower in rats receiving BMSC therapy as compared to the control group after three months.
BMSCs account for 1-10 per 100,000 nucleated cells within bone marrow [11]. Although their isolation is relatively simple and they amplify in abundance in vitro, an ideal marker of BMSCs has yet to be identified. In this study, BMSCs expressed CD29 and CD90, but not CD34 and CD45, which is in agreement with Stagg et al. [9]. Characterization of their differentiation potential confirmed their ability to differentiate into osteoblasts and adipocytes in vitro. In addition, BMSCs can differentiate into chondrocytes, tenocytes, and neural cells [7].
Cisplatin induces DNA crosslinking, which triggers apoptosis. In the present study co-culture with BMSCs reduced GC apoptosis induced by cisplatin, which is in agreement with a previous study [8]. Also, co-culture of GCs with BMSCs downregulated the mRNA expression of Bax and p21 and upregulated c-myc mRNA expression in GCs. Bcl-2 and Bax are both apoptosis regulating proteins that effect the mitochondria [12,13]; Bcl-2 competes with Bax for interaction with adenine nucleotide translocator (ANT) on the mitochondrial membrane, preventing apoptosis [14]. In addition, p21 overexpression may promote apoptosis [15][16][17]; it regulates the cell cycle through p53-dependent and independent pathways [18]. Finally, c-myc promotes cell cycle progression from the G0/G1 phase to the S phase [14]; its downregulation has been associated with Bax oligomerization and subsequent activation of caspase-9 and caspase-3, resulting in apoptosis [19]. Because BMSCs and GCs were separated by a semipermeable membrane, protection by BMSCs was elicited through secreted paracrine factors. However, further studies are necessary to identify the precise paracrine mediator(s) responsible for this protective effect.
Perimenopause is defined as occurring 12 months after the last menstrual period. At present, there are four animal models of perimenopause, including an ovarectomized animal model, a model that physically or pharmacologically disrupts of ovarian function, a 4-vinylcyclohexene diepoxide (VCD) follicle depletion model, and a natural aging animal model. The natural aging animal model was selected for this study as it most closely models the pathological changes associated with perimenopause.
BMSC transplantation reduced GC apoptosis, which is in accordance with two previous studies describing the restorative effects of MSCs after their transplantation into ovaries [8,20]. However, the mechanism by which the BMSCs influenced GC apoptosis remains unclear as they were introduced via injection into vena caudalis. For example, the transplanted BMSCs may have reached the site of injury within the ovary and replaced damaged tissue through integration into the ovarian tissue and differentiation into the required cell type. Alternatively, the BMSCs may have induced tissue repair and prevented apoptosis by secreting paracrine mediators, including glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), tumor necrosis factor (TNF), basic fibroblast growth factor (bFGF)   [11,[21][22][23], promoting functional recovery [24]. The latter mechanism is supported by the in vitro study that showed reduced GC apoptosis with BMSC co-culture, in which the cells were not in direct contact. Furthermore, analysis of cell culture supernatant revealed that BMSCs secreted IGF-1, HGF, and VEGF-A. VEGF promotes the formation of capillary networks, ensuring nutrition for GCs and oocytes [25]. In the ovary, IGF-1 regulates DNA replication of theca cells and GCs; it also regulates aromatase activity, secretion of inhibin, and generation of corpus luteum stimulating hormone receptor [26].
IGF-1 also prevents cell cycle progression from G0/G1 to S via PI3K/Akt signaling and inhibits Fas receptormediated apoptosis [27]. Finally, GCs express the HGF receptor, c-met [28]. Uzumcu et al. [29] reported that the level of HGF was inversely proportional to GC apoptosis, and HGF can reduce GC apoptosis in vivo and vitro. Further studies will be undertaken to elucidate the mechanism by which BMSCs influence GC apoptosis.
The present study has some limitations. For example, basic characterization of the perimenopausal rats was using vaginal smears to detect estrous cycle, estrous cycle disorders, and prolongation. However, the effects of perimenopause, estrogen therapy, or BMSC therapy on ovarian hormone levels in the serum were not determined. In addition, the in vivo study lacked a BMSC-only treatment group. Furthermore, the protein levels of c-myc, p21, survivin, Bcl-2, and Bax were not assessed to corroborate the data obtained using RT-PCR. Finally, the mechanism by which BMSCs influence GC apoptosis (i.e., integration and/or paracrine mediators) remains unclear and will be the subject of further studies.

Conclusion
BMSCs decrease the GC apoptosis in vitro and vivo. Further studies will evaluate the therapeutic efficacy of BMSC therapy for perimenopausal symptoms as well as the basis for these effects.

Experimental animals
Immature male (60-80 g), young female (3-4 months), and perimenopausal female (9-10 months) Sprague-Dawley rats were obtained from the Animal Center at Shanghai. All rats were kept under controlled temperature (30 ± 2°C) and light (14 h light, 10 h dark) conditions with rat chow and water ad libitum. All experiments were approved by the institutional animal committee of the Fuzhou General Hospital.

Isolation and identification of BMSCs
The femurs and tibiae were removed from immature male Sprague-Dawley rats. The bone marrow plugs were flushed with phosphate-buffered saline (PBS), layered over a Percoll solution (density 1.083), and separated Values are presented as mean ± SD. * indicates a significant difference from control; P < 0.05 (Two-way ANOVA: treatment effect, P < 0.0001; time effect, P = 0.0008). N = 5 for each group. by centrifugation at 2500 RPM for 20 min at room temperature. Mononuclear cells at the interface were recovered and washed twice with PBS. The cells were cultured with complete medium, consisting of DMEM/F12 (1:1) medium supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C and 5% CO 2 . At confluence, the cells were harvested for passage with 0.25% trypsin containing 0.02% EDTA. All experiments were performed using cells from the third to fifth passage.
BMSC surface expression of CD29, CD34, CD45, and CD90 was analyzed by flow cytometry. Briefly, cells were incubated with the R-phycoerythrin (PE)-labeled antibodies (BD Biosciences, San Jose, California, USA) and their corresponding isotype controls: PE-CD34 (no dilution), PE-CD29 (1:16 dilution), PE-CD45 (1:8 dilution), and PE-CD90 (1:8 dilution). After a 30-min incubation in the dark, the cells were isolated by centrifugation (800 g for 5 min) after which the supernatant was removed. After the cells were washed three times with 2 mL PBS, the cell suspensions were filtered with 200 μm mesh filters to make single cell suspensions. 1× 10 6 Cells were next analyzed for surface marker expression by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). Samples in which the primary antibody was omitted were used as the gate for the forward scatter (FSC) versus side scatter (SSC).
BMSCs were seeded in 6-well plates at a density of 1 × 10 5 cells/well. After 24 h, the supernatant in one of the plates was replaced with control DMEM-HG medium supplemented with 10% FBS, 10 -6 M dexamethasone, 0.5 mM 3-methyl-1-methyl xanthine, 10 μg/mL insulin, and 0.2 mM indomethacin. In the remaining plates, the supernatant was replaced with induction medium comprised of DMEM-HG medium supplemented with 10% FBS, 10 -7 M dexamethasone, 10 mM beta-glycerophosphate, and 50 mg/mL vitamin C. The medium was replaced every three days. The cells cultured in control medium were stained with Oil red after two weeks; the cells cultured in induction medium were identified by Von Kossa staining after three weeks.
To evaluate the secretion of growth factors, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1), by cultured BMSCs, cells were seeded in 6-well plates at a density of 1 × 10 6 cells/well. After 24 h, the supernatant was replaced with serum-free medium consisting of DMEM/F12 (1:1) medium, 100 U/mL penicillin, and 100 U/mL streptomycin. After three days, the supernatant was collected, and cytokine concentrations were determined using enzyme linked immunosorbent analysis (ELISA) using kits purchased from Uscn (Wu Han, Hubei, China).

Isolation and identification of GCs
Immature female rats (3-4 weeks) were injected once subcutaneously with 60 IU of pregnant mare serum gonadotropin (Animal Drugs Factory, Huangzhou, Zhejiang, China) to induce the development of multiple antral follicles. After 48 h, the rats were sacrificed, and the ovaries removed. The GCs were isolated by needle puncture under an inverted microscope. After obtaining single cell suspensions, they were washed twice with PBS and cultured with complete medium, including DMEM/F12 (1:1) medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C and 5% CO 2 . Cell of the third passage were seeded on microscope slides for immunocytochemistry analysis to confirm their identity. Briefly, single GC suspensions (1 × 10 5 cells/mL) were fixed with 4% paraformaldehyde and washed three times with PBS (5 min/wash) and then incubated with 0.5% Triton X-100 for 20 min at room temperature. After washing three times with PBS (5 min/wash), cells were incubated with 3% H 2 O 2 for 5 min at room temperature to eliminate endogenous catalase activity. Nonspecific binding was blocked by incubating the cells will normal goat serum diluted in PBS (1:50) for 20 min at room temperature. Next, samples were incubated with FSHR primary polyclonal antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) diluted in PBS in a humidified chamber at 4°C overnight. Controls consisted of samples incubated in PBS alone. After washing three times with PBS (5 min/ wash), secondary goat anti-rabbit lgG antibodies were added for 60 min at 37°C. After three washes with PBS (5 min/wash), samples were incubated with a horseradish peroxidase-labeled streptomycin solution for 30 min at 37°C prior to color development with the 3,3' Diaminobenzidine (DAB) chromogen. After the reaction was stopped by washing the samples with tap water, the samples were counterstained with hematoxylin for 20 s. Subsequently, the samples were washed, dehydrated, mounted with Permount (Fischer, Fair Lawn, NJ, USA), and analyzed using a Leica TCS-2 microscope (Leica Microsystems Ltd., North Point, Hong Kong, China) equipped with a D1X digital camera (Leica Microsystems Ltd., North Point, Hong Kong, China). Cultured GCs were separated into three groups: an untreated control group (control), a cisplatin (5 mg/L) treatment group (cisplatin), and group co-cultured with BMSCs and treated with 5 mg/L cisplatin (BMSC). GCs were seeded onto 6-well plates at a density of 5 × 10 5 cells/well. BMSCs were seeded on 6-well Millicell cell culture inserts (Millipore, Billerica, MA, USA) at a density of 2.5 × 10 5 cells/well. After 24 h, the supernatant was removed. In the control group, the medium was changed to 3 mL serum-free medium (DMEM/F12 medium containing 100 U/mL penicillin and 100 U/mL streptomycin). In the cisplatin group, cells were incubated with 3 mL serum-free medium with cisplatin (5 mg/L). In the BMSC co-culture group, cells were cocultured with BMSCs and incubated with 3 mL serumfree medium with cisplatin (5 mg/L). After 72 h, the GCs were harvested and analyzed for apoptosis using an annexin V/PI apoptosis detection kit (MultiSciences Biotech, China) according to manufacturer's protocols and flow cytometry (Beckman Coulter Epics XL Flow Cytometer; GMI, Inc.; Ramsey, Minnesota, USA).

Effects of BMSCs on GC apoptosis in vivo
Rats reach sexual maturity at 6 to 8 weeks and perimenopause at 10 to 15 months. Female rats (9-10 months) were randomly divided into the following three groups (n = 15 per group): control group, BMSC group, and estrogen group. In addition, 15 adult female rats (3-4 months) were included as the young group. Prior to initiation of the experiment, vaginal smears of the female rats were obtained at 8:30 am every morning to confirm the perimenopausal status of the older rats according to the estrous cycle. Upon confirmation, BMSCs (1-2 × 10 6 cells in 2 mL) were injected via vena caudalis of rats in the BMSC group; the control and young groups received a 2 mL saline injection. This treatment was repeated after one week. Rats in the estrogen group received 0.158 mg/kg/d nilestriol (Shang Hai New Hua Lian Pharmaceutical CO.LTD, China) for 8 consecutive days by gavage administration. Five rats from each group were randomly sacrificed one and three months after the second transplantation, and their ovaries were removed. The left ovary was fixed in formaldehyde and sectioned for analysis of GC apoptosis using a TUNEL apoptosis assay kit (Roche, Branford, CT). Tissue sections from each animal were examined by microscopy, and at least 100 GCs per field were counted in five randomly selected fields. The GC apoptosis rate was calculated as the number of apoptotic GCs divided by the total number of GCs. Cells were counted independently by two individuals who were blinded to the treatment group. The apoptotic index was defined as the average GC apoptosis rate from counted five randomly selected fields.

Statistical analysis
Quantitative data are presented as mean ± SD. Differences in gene expression among the three groups (control, cisplatin, and BMSC groups) were detected by one-way analysis of variance (ANOVA) with Tukey's range test. The GC apoptosis rates were analyzed by two-way ANOVA to evaluate treatment effect (control, BMSC, estrogen) and age as well as time effect (1 month vs. 3 months) along with their interaction. The statistical analyses were performed with SAS software version 9.2 (SAS Institute Inc., Cary, NC). A two-tailed P-value < 0.05 indicated statistical significance.


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High In Vitro Infectivity of a Doxycycline-Insensitive Strain of Orientia tsutsugamushi

We compared the infectivity and growth rates of 12 strains of Orientia tsutsugamushi in a cell culture system. ECV304 cells were infected with O. tsutsugamushi strains for 4 hr, and the culture was maintained for 72 hr. Immunofluorescence (IF) staining was performed at 4, 24, 48, and 72 hr after inoculation (hpi), and IF-positive foci were enumerated. The AFSC-4 strain, which is known to be insensitive to doxycycline, showed higher numbers of IF-positive foci than the other 11 strains at 4, 24, 48, and 72 hpi, which may explain its apparent insensitivity to antibiotics.

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higher than those in cultures of the Boryong strain, an antibiotic-susceptible strain, regardless of whether these cultures contained antibiotics. We hypothesized that if the AFSC-4 strain was capable of growing more rapidly than other strains, a higher amount of bacteria would be present at the time of antibiotic susceptibility testing. Thus, the rapidly growing strain would appear to be resistant to antibiotics, even when the antibiotic susceptibilities of these strains might not be different from one another. To verify this hypothesis, we compared the infectivity and growth rates of various O. tsutsugamushi strains.
Stock solutions of each O. tsutsugamushi strain were inoculated onto monolayers of ECV304 cells seeded in culture flasks (75 cm 2 ). When the infected ECV304 cells showed maximum cytopathic effects, the degree of infection of ECV304 cells as assessed by immunofluorescence (IF) staining was found to be approximately equal in each experiment. The cells were disrupted with glass beads and centrifuged at 350 × g for 5 min. To avoid the death of ECV304 cells by overgrowth of O. tsutsugamushi within 72 hr, the resultant supernatant was divided equally into 40 aliquots, of which 4 aliquots were used. The amount of each inoculum was one-tenth the amount that is usually used in antibiotic susceptibility tests in our laboratory. Each aliquot was inoculated onto monolayers of ECV304 cells grown in shell vials and incubated for 4 hr. At the end of the initial incubation period, the inocula were replaced with fresh medium and the cultures were maintained for 72 hr. Immunofluorescence staining was performed at 4, 24, 48, and 72 hr post-inoculation (hpi) with O. tsutsugamushi.
ECV304 cells infected with each strain of O. tsutsugamushi on a cover slip of the shell vial were fixed with acetone. The infected cells were then stained with mouse polyclonal antibodies in phosphate-buffered saline (PBS) for 30 min at 37°C, washed briefly with PBS Tween-20 (PBST), treated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (Jackson ImmunoResearch, West Grove, PA, USA.) for 30 min in a moist chamber, and finally washed 3 times with PBST. To clearly define O. tsutsugamushi, the host cells were counter-stained with 0.003% Evans blue in PBS. After the slides had been washed, they were placed in mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined at 400 × magnification under a fluorescence microscope (Zeiss, Germany) with a confocal laser scanning system (Bio-Rad, Hercules, CA, USA). The IF-positive foci per field were  Although the ECV304 cells were inoculated with approximately equal amounts of each O. tsutsugamushi strain, quantification of the bacterial number in each culture at 4 hpi revealed differences among the strains or isolates (Fig. 1). Interestingly, the bacterial count in a culture of the AFSC-4 strain was 2-5 times higher than that in cultures of other strains or isolates at 4 hpi. Additionally, the numbers of the AFSC-4 strain from 4 to 72 hpi were also larger than those of the other strains tested, but with the degree of difference decreasing over time. No consistent differences in the infectivity and growth rates were observed between laboratory-adapted strains and clinical isolates of the same genotype.
The present study revealed that the AFSC-4 strain of O. tsutsugamushi had more IF-positive foci than the other strains at 4, 24, 48, and 72 hpi. The bacterial count present at 4 hpi represents the number of O. tsutsugamushi successfully infecting ECV304 cells; and, therefore, is indicative of both its stability during detachment and the degree of infection of the ECV304 cells. The numbers of IF-positive foci at 24, 48, and 72 hpi represent the degree of multiplication. Therefore, the AFSC-4 strain showed either higher infectivity or stability and slower growth than the other 11 O. tsutsugamushi strains. Because the growth rate of O. tsutsugamushi decreases as the bacterium enters the stationary phase of growth from the exponential phase, it is not clear whether the slow growth of the AFSC-4 strain is an intrinsic property.
The present findings were also observed in a previous study [5], wherein the researchers used 3 indices to represent the number of O. tsutsugamushi organisms: the extent of infection, the percentage of maximally infected cells, and the intensity of infection. The first index, the extent of infection (expressed as a percentage of infected cells), was 84% in Karp versus 95% in AFSC-4 strain-infected cultures that did not contain antibiotics. The percentage of maximally infected cells was 60% in the Karp and 68% in AFSC-4 strain-infected cultures containing no antibiotics. The last index, the intensity of infection (measured by calculating the number of O. tsutsugamushi per infected cell), did not reveal any difference between the 2 strains; however, this index was calculated from the former 2 indices and rounding off values of > 50 to 51 during calculation may have influenced the result. In another study investigating antibiotic susceptibility in a cell culture system [4], 2 additional strains−C1 and C27−demonstrated higher infectivity (46% and 43%, respectively) than the Karp strain (39%) in cultures containing no antibiotics. In contrast to these strains, the C3 strain had a lower level of infection (24%) than the Karp strain. These 2 studies revealed that most doxycyclineinsensitive strains (with the exception of the C3 strain) were consistently present in larger numbers. Owing to differences in the study design such as cell type, inoculum size, observation period, and staining methods used, the magnitude of the differences in the previous studies are not as prominent as those observed in our study. Considering that we usually observed about 1.2-1.3 times larger numbers of bacteria in AFSC-4 strain-infected cells than that in Boryong strain-infected cells, the reduced inoculum size markedly influenced our present results. In a study comparing the antibiotic susceptibility of the AFSC-4 and Boryong strains, only slight differences in infectivity (63.8% and 63.5%, respectively) were observed [7]; however, infectivity was measured by a flow cytometric assay that might be not sensitive enough to detect small differences [8]. Nevertheless, the results of the present study are in line with those of a previous study performed by Groves and Kelly [9], which suggested that the rapid growth of O. tsutsugamushi strains might be the most important factor in determining pathogenicity.
The present study supports our previous report in which no doxycycline-insensitive strains were found among 37 O. tsutsugamushi isolates in Korea [10]. The strains tested in the present study included clinical isolates of Boryong, Yonchon, Je-cheon, Young-worl, and Neimeng-65 genotypes, which represent the most prevalent genotypes in Korea, with the notable exception of the Kawasaki genotype [11]. None of these strains had higher infectivity or more rapid growth in the present study, suggesting that doxycycline resistance by this mechanism was not present in Korea. Furthermore, if the delayed defervescence in scrub typhus is due to the density of O. tsutsugamushi rather its resistance to antibiotics, a delayed clinical response would be expected with other antibiotics such as azithromycin or rifampin. In actuality, a report by Watt et al [4] revealed that the C3 strain, and particularly C27, resulted in higher mortality rates in mice than the Karp strain when infections were treated with azithromycin. Although azithromycin has been considered efficacious even against infection by doxycycline-insensitive strains, these data suggest that the resistant strains are also azithromycin-insensitive. In addition, a clinical trial evaluating rifampin and doxycycline for the treatment of scrub typhus in Thailand showed that the fever clearance time was 52 hr in doxycycline-and 27.5 hr in rifampin-treated groups [12], compared to 19 hr and 17 hr, respectively, in Korea [13]. Although different study designs and regions might result in some variation in these results, fever clearance times in Thailand were prolonged in both the rifampin-and the doxycycline-treated groups. Taken together, these findings suggest that strains prevalent in northern Thailand are insensitive to rifampin and azithromycin, or alternatively, are only apparently resistant owing to factors independent of their antibiotic susceptibility, such as high infectivity.
Only 1 strain of doxycycline-insensitive O. tsutsugamushi was tested in the present investigation. Therefore, while this study provides an explanation for the apparent in vitro insensitivity to doxycycline, there could be multiple mechanisms responsible for delayed recovery from scrub typhus. Further studies measuring the growth rate of various antibiotic-insensitive strains of O. tsutsugamushi are able to resolve this limitation.