Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from 15N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.The presence of aspartic protease inhibitor in filarial parasite  Brugia malayi was identified and the recombinant protein was biochemically characterized. Aspins get fast gaining importance in the fields like medicine, agriculture and biotechnology. Hence, in this study, the inhibition ability of filarial Aspin against human aspartic proteases is attempted from structural biology point of view. This new knowledge may contribute to a better overall understanding of the mechanism that explains the versatility of human filarial parasite. Since filariasis is more often considered as the disease of poor countries, fight against filariasis is also a fight against poverty.Filariasis is a parasitic infectious tropical disease caused by thread like filarial nematodes. These worms occupy the lymph nodes and in chronic cases they lead to the disease “elephantiasis.” Over 120 million people have already been affected by it, and 40 million are seriously disfigured by this disease. These parasites in human, adopt numerous strategies to hamper the host immune system which can facilitate its survival. The ability of the parasite to modulate the host immune system is a concept which explains the versatility of human filarial parasites. One such interesting concept to understand is the secretion of protease inhibitors by filarial parasites. Recently, an Aspin from parasite  Wuchereria bancrofti, Brugia malayi or Brugia timori that affects 120 million people in 73 countries and another 1100 million people are at the risk of contracting this dreadful disease Lymphatic filariasis is a mosquito borne infection caused by Though the mass drug administration was initiated as a preventive measure, it had only a limited ability During the process of infection, all stages of the parasite are constantly exposed to various human proteases. It is interesting to understand how filarial parasites successfully evade or counteract the harmful effects produced by the various human proteases. Under this scenario, several lines of studies suggest that filarial parasites have evolved mechanism to neutralize the harmful effects produced by the human proteases. For example, filarial parasites produce three types of classical protease inhibitors viz., serine protease inhibitors (serpins), cysteine protease inhibitors (cystatins) and aspartic protease inhibitors (aspins) to overcome the harmful effects produced by the human proteases.taeniaformisBrugia malayi. The suggested function of serpins and cystatins was to contribute to the longevity of the parasite in the blood stream. Despite the pivotal role of Aspins in host pathogenesis, immune regulation and diagnostic marker The first evidence of protease inhibitors in parasite survival was Taeniaestatin from a non-filarial parasite Taenia Brugia malayi, termed as pepsin inhibitor homolog compared to the amino acid composition of PI-3 from Ascaris SuumIn this context, an Aspin from As these aspartic proteases are believed to play a key role in various physiological activities, understanding the aspartic protease inhibitory mechanism of Bm-Aspin becomes imperative to study its role in parasite survival and its immune evasion strategies. However, lack of Bm-Aspin three dimensional structure limit the ability of understanding aspartic protease inhibition mechanism. In order to unravel the structure-based protease inhibition mechanism, Bm-Aspin's, aspartic protease inhibition kinetics and solution structure determination has been initiated and presented in this study. This work happens to be the first report describing the feasibility of solution structure determination of filarial Aspin and understand its protease inhibitory efficiency from structural biology point of view.Ascaris Suum PI-3 and was believed to follow similar kind of pepsin inhibition. Hence pH 5.6 was considered. The sample conditions and the methods that were chosen to study the protease inhibition by Bm-Aspin were similar to those used by Abu-erreish and Peanasky to study the pepsin inhibition by PI-3 from Ascaris Suum2)-Phe-Val-Leu (4-pyridylmethyl) ester, the specific substrate for aspartic proteases from a stock of 1 mg/ml, was added The aspartic proteases pepsin, renin, cathepsin-E, and cathepsin-D purchased from Sigma, USA and the lab purified Bm-Aspin were used for the assays. Equimolar concentrations of Bm-Aspin and the proteases were mixed and incubated in 100 mM sodium acetate buffer at pH 5.6 for 10 min at 37°C separately. Bm-Aspin initially termed as “pepsin inhibitor homologue” The kinetics of aspartic protease inhibition by Bm-Aspin was determined by the UV- spectroscopic method mK value for Bm-Aspin was determined by linear regression method from plots of 1/V vs. 1/S, utilizing substrate concentrations of 7–80 µM. Three fixed concentrations of Bm-Aspin as explained above were used to determine the inhibition constant (iK) against six concentrations of substrate. Assays were carried out in triplicates and the kinetic constants for Bm-Aspin against the human aspartic proteases were determined using Graphpad Prism 2.0 .The In order to show the activity of the protease and its inhibition by Bm-Aspin under NMR conditions, the effects of SDS and pH on the protease were studied. We have performed protease activity assays using Casein Agar plate and also by the UV spectroscopic method. The inhibition by Bm-Aspin has also been looked at these conditions. Fine punch holes were made on Casein agar plate as described by Cheseseman 15N minimal medium using 15NH4Cl as the sole nitrogen source. The purification and the refolding protocols are the same that have been described earlier 2O (v/v). This NMR study was initially performed without any detergents and the resultant spectrum suggests the possibility of aggregation. Then the following chemicals/detergents were used to figure out the best one that helps to avoid aggregation: 0.5 M urea and 1% glycerol, 100 mM n-Dodecyl β-D-Maltopyranoside (DDM), 1% n-octyl-β-D-glucoside (OG), 1% triton-×100 and 100 mM SDS. The final goal is to obtain the most suitable condition for Bm-Aspin that could provide good quality NMR spectra. There after the protein sample for the NMR experiment was refolded and concentrated in the presence of the above mentioned detergents each time, respectively.The plasmid containing Bm-Aspin was expressed in 15N HSQC spectrum was acquired for Bm-Aspin at 298 K. Data was collected on the Bruker Avance III 700 MHz spectrometer available at Claflin University. The NMR experiments were recorded with the carrier position of 4.678 ppm for 1H and 117 ppm for 15N. All chemical shifts were referenced to internal D2O. The triple resonance NMR experiments like HNCO, HN(CO)CA, HNCA, CBCA(CO)NH and HNCACB on a uniformly 13C, 15N labeled Bm-Aspin were carried out as mentioned above. The data acquired were processed using NMRPipe 15N-HSQC spectra were collected by titrating with progressive additions of human aspartic proteases  to 15N-labeled Bm-Aspin to attain molar ratios of 0∶1, 0.1∶1, 0.5∶1 and 1∶1 respectively.2)-Phe-Val-Leu (4-pyridylmethyl) ester, successful inhibitions of all four proteases were observed.Inhibition effect of Bm-Aspin on the human aspartic proteases was examined . When asProtease digestion was observed in Casein agar plate by 5 µg of pepsin and 5 µg of pepsin preincubated (for 10 min at 37°C) with 100 mM SDS at both pH 5.6 and 7.0 . EncouraiK) for pepsin, renin, cathepsin-E and cathepsin-D inhibition by Bm-Aspin was found to be: 2.1 (±0.7) nM, 2.9 (±0.9) nM, 4.3 (±0.2) nM, and 6.5 (±0.6) nM respectively. The data clearly indicate the strength of inhibition among the four different proteases with Bm-Aspin.The double inverse Lineweaver-Burk plots depicted the inhibition of pepsin, renin, cathepsin-E and cathepsin-D by Bm-Aspin  possess appreciable peak movement indicating their involvement in the interaction phenomena. All the spectra are having the same trend in their protease interaction shifts with pepsin producing more appreciable shifts. The general inhibition characteristics that have been observed from the kinetic and inhibition assay methods are in perfect agreement with the NMR titration method, duly confirming the results. A visual look at the overlaid spectra clearly confirms the strong inhibitory characteristics of pepsin compared to the rest. There is strong evidence about the involvement of the C-terminal residues in the interaction process of all the proteases. Additionally, for pepsin, some glycines from the N-terminal domain are found to be affected as compared to the rest of the proteases, which could attribute to the more inhibitory nature of pepsin. From the binding studies of Bm-Aspin with the aspartic proteases, we have observed that there are five residues  of the C-terminal domain that are commonly affected in all the four aspartic proteases interactions. This provides an information that the same kind of interaction mode is followed in all the four protease interactions. Three of the N-terminal domain residues  are appreciably affected in interacting with Pepsin and Renin and they are smaller for CatD and CatE. These sites could be the aiding factor for the two former proteases to have higher activities compared to the latter two. These residue specificities for the proteases clearly indicate that they are the genuine interacting sites. As the HSQC spectrum has more peak overlaps, for simplicity, only the residues A213, I214, and Y215 that are having appreciable shifts when interacting with pepsin are shown here as an example.  and bound (Bm-Aspin+protease complex) states, respectively. The bar diagram has been drawn using the radial shifts data obtained for the earlier mentioned 10 residues, when each of the four proteases used and is as shown in Ascaris Suum inhibitor, PI-3. Hence, it is quite obvious to expect the role of Bm-Aspin C-terminal domain in the inhibitory actions against the aspartic proteases.On comparing the aspartic protease interactions with Bm-Aspin at their saturated conditions, the saturated sample spectra for each of the four proteases were overlaid on each other. Again, the same three residues considered in the previous example have been shown here . The peaBrugia malayi Aspin inhibits the important human aspartic proteases Filarial parasites while gaining entry into the mammalian host are exposed to a variety of hostile factors including human proteases. To circumvent the harmful effects of these proteases, parasites have evolved several counter attacking mechanisms. For example, filarial parasites are known to secrete serpins, cystatins, and aspins to neutralize the human proteases thereby contributing to the longevity of the parasite in the blood stream. Though the functional characterizations of serpins and cystatins have been reported Determination of inhibition constant for pepsin inhibition activity of Bm-Aspin was reported with Casein as a substrate in our previous studies Kinetic studies of Bm-Aspin were performed to find out its mode of protease inhibition. Our data clearly indicates that the protease inhibition by Bm-Aspin was found to be competitive for pepsin and cathepsin-E, non-competitive for renin and mixed for cathepsin-D. Similar trend of linear competitive inhibition was observed with Pepsin when Casein was used as a substrate Ascaris suum that infects the pigs Ascaris suum inhibitor, PI-3, was found to be ineffective against renin and cathepsin-D and in contrast an appreciable influence on all the four aspartic proteases was evident with Bm-Aspin. The role of PI-3 N-terminal end residues was discussed in the case of PI-3 - pepsin crystal structure complex The mode of pepsin and cathepsin-E inhibition by Bm-Aspin was found to be comparable to that of an Aspin, PI-3 from the non-filarial nematode iK) values determined by linear regression of 1/V vs 1/S plot suggests that renin, cathepsin-E and cathpesin-D inhibition by Bm-Aspin are 0.8, 2.2 and 4.4 times less specific than that of pepsin. This correlates with our previous iso-thermal calorimetric titration experiment results indicating that the preference of protease inhibition by Bm-Aspin is in the following order: pepsin>rennin>cathepsin-E>cathepsin-D The inhibition constant CA, HNCA, CBCA(CO)NH and HNCACB were carried out at 298 K on a uniformly double 15N edited NOESY spectra. The usage of CBCA (CO) NH/HNCACB was found to be extremely useful not only for the improved resolution in the carbon dimension, but more importantly, it provided the phase information which will be used in the assignment process. The initial assignment was simple with the easier identification of Glycine residues and its connectivity with the other easily identifiable Ala, Ser and Thr residues. Nearly 15 residue-stretches with sequential residue connectivity have been identified and assigned. The complete backbone and side-chain assignments for the other residues of Bm-Aspin is in progress and will soon be communicated elsewhere.The peak overlaps in the HSQC spectrum of Bm-Aspin with a large number of amide peaks make its resonance assignment extremely challenging. The backbone assignment of Bm-Aspin was initiated by establishing the neighboring residue connectivity using pairs of triple resonance spectra, HNCA and HNCOCA; HNCACB and CBCA(CO)NH. The sequential NMR spin system connectivities have been achieved by both intra- and inter-residue cross-peaks of Cβ and Cα respectively. Any ambiguities were resolved by going through the Before performing the NMR titration analysis between Bm-Aspin and human aspartic proteases, activity of pepsin in the above mentioned conditions (100 mM SDS and pH 7.0) was carried out. From these studies, it was clearly demonstrated that 100 mM SDS has no effect on pepsin activity and on the inhibitory activity of Bm-Aspin. The observation of protease digestion by pepsin at both pH 5.6 and 7.0 indicate a higher activity for pH 5.6, which is in agreement with earlier studies carried out on pH stability of pepsin The preliminary residue assignment of Bm-Aspin has been useful in the analysis of the Bm-Aspin interactions with the proteases. Our results clearly demonstrate that Bm-Aspin interacts with human aspartic proteases supporting our earlier results. The chemical shift perturbations were observed in the HSQC experiments upon the addition of aspartic proteases to Bm-Aspin. These observations provide the direct experimental proof that the Bm-Aspin contains the binding sites for human aspartic proteases. As expected, based on the displacement observed from the chemical shift perturbations in the HSQC of Bm-Aspin upon the addition of aspartic proteases, the inhibition slows down in the order of pepsin>renin>cathepsin-E>cathepsin-D. Similar findings were observed previously, when Bm-Aspin inhibition kinetic studies were carried out using UV-spectroscopy, suggesting the authentication of the experiments performed and the results obtained. However the detailed mechanism of protease inhibition by Bm-Aspin and its role in parasite survival can only be addressed further by structure-function studies with the determination of the solution structure of Bm-Aspin which would be initiated on completion of the assignment process.15N HSQC spectra of Bm-Aspin upon the addition of aspartic proteases, suggesting the possibilities of identifying the binding sites of human aspartic proteases on Bm-Aspin. This work happens to be the first attempt on the solution NMR studies of the protein involved in human lymphatic filariasis. Considering the physiological role played by these human aspartic proteases, understanding the protease inhibition mechanism of Bm-Aspin will be interesting to speculate Bm-Aspin, as an important drug target for human lymphatic filariasis. For which, the high resolution 3D structure determinations of Bm-Aspin in apo-form by solution NMR and the complex with human pepsin by X-ray crystallography methods are well underway in our laboratories. These high resolution structures will be instrumental in our understanding of the structure/function and mechanism of aspartic protease inhibition.We report here the aspartic protease inhibition kinetic studies and feasibility to conduct the solution NMR based structural studies on Bm-Aspin. Aspartic protease inhibition assay carried out using the specific substrate suggested that Bm-Aspin inhibits the important human aspartic proteases supporting our earlier published data. Inhibition kinetic studies suggest that, Bm-Aspin follows competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin, and mixed for cathepsin-D. The triple resonance NMR experiments conducted on Bm-Aspin with SDS detergent suggested the feasibility to carry out the NMR based structural studies. The activities of pepsin at pH 7.0 and in the presence of 100 mM SDS suggests that all the titrations carried out are in the biologically relevant form. The chemical shift perturbations were observed in the


Effectiveness of Internet-based self-management in patients with asthma has been shown, but its cost-effectiveness is unknown. We conducted a cost-effectiveness analysis of Internet-based asthma self-management compared with usual care.Cost-effectiveness analysis alongside a randomized controlled trial, with 12 months follow-up. Patients were aged 18 to 50 year and had physician diagnosed asthma. The Internet-based self-management program involved weekly on-line monitoring of asthma control with self-treatment advice, remote Web communications, and Internet-based information. We determined quality adjusted life years (QALYs) as measured by the EuroQol-5D and costs for health care use and absenteeism. We performed a detailed cost price analysis for the primary intervention. QALYs did not statistically significantly differ between the Internet group and usual care: difference 0.024 . Costs of the Internet-based intervention were $254  during the period of 1 year. From a societal perspective, the cost difference was $641 . From a health care perspective, the cost difference was $37 . At a willingness-to-pay of $50000 per QALY, the probability that Internet-based self-management was cost-effective compared to usual care was 62% and 82% from a societal and health care perspective, respectively.Internet-based self-management of asthma can be as effective as current asthma care and costs are similar.ISRCTN79864465Current Controlled Trials  Asthma is a chronic, inflammatory disorder of the airways clinically characterized by respiratory symptoms such as wheeze, cough, dyspnoea, chest tightness and impaired lung function In the past decade, the care for asthma patients has shifted from physician-managed care to guided self-management. Guided self-management includes asthma education, self-monitoring of symptoms and/or lung function and adjustment of treatment according to an action plan guided by a health care professional (not necessarily a physician). Self-management has been shown to improve asthma control and quality of life and reduce health care utilization and sometimes improve lung function Besides clinical effectiveness, the implementation of new disease management strategies requires an economic evaluation to determine whether the clinical benefits are gained at reasonable costs. Several cost evaluations have compared paper-and-pencil self-management plans to usual care in asthma Implementation of guided self-management programs may be enhanced by the use of Internet-based technologies, particularly in remote and underserved areas. In a recently conducted randomized controlled trial we have shown that Internet-based self-management is feasible and provides better clinical outcomes compared to usual physician provided care with regard to asthma related quality of life, asthma control, symptom-free days and lung function The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The study was approved by the Medical Ethics Committee of the Leiden University Medical Center. All participants gave their written consent.Two hundred patients participated in a 12-month multicenter, non-blinded, randomized controlled trial. Patients were recruited from 37 general practices  in the Leiden and The Hague area and the Outpatient Clinic of the Department of Pulmonology at the Leiden University Medical Center, The Netherlands over the period from September 2005 to September 2006 Details of the randomization and intervention have been described previously 1.61Utilities express the valuation of health-related quality of life on a scale from zero (death) to one . Patients described their health-related quality of life using the EuroQol classification system (EQ-5D) We obtained utility measurements at baseline, 3 and 12 months. For EQ-5D measurements 6.5%, 10% and 8.5% were missing and for visual analogue measurements 7%, 10% and 9% were missing at 0, 3 and 12 months, respectively. To correct for possibly selective non-response, missing measurements were replaced by 5 imputed values based on switching regression We distinguished three major cost categories: intervention costs, other health care costs and productivity costs Patients reported their use of health care resources and the hours of absence from work using a quarterly cost-questionnaire. We used Dutch standard prices for units of resource use  and hours of absenteeism, designed to represent societal costs and to standardize economic evaluations Cost-questionnaires were scheduled to be handed in at 3, 6, 9 and 12 months. Of these quarterly questionnaires, 10%, 14%, 19% and 9% were missing, respectively. Pharmacy records were available for 182 patients (91%). Missing cost-questionnaire and pharmacy record were imputed using multiple imputation, as previously described under ‘Utilities and QALYs’.Differences and statistical uncertainty of QALYs and costs were calculated using non-parametric bootstrap estimation with 5000 random samples (1000 from each of the 5 imputations). Differences in costs resulted from differences in volumes rather than differences in unit costs, since we used standard prices for units of resource use and hours of absenteeism. We estimated the intervention effect by a linear regression model with randomisation group as only independent variable, combining the 5 multiple imputation sets using Rubin's rules Analyses were carried out with Stata 9.0 .The base case cost-effectiveness analysis compared societal costs with QALYs gained based on the British EQ-5D over the period of one year. Because of the limited degree of modeling in this cost utility analysis, we carried out sensitivity analyses only on the use of different utility measures  and on the included cost categories .Statistical uncertainty of the cost-effectiveness was analyzed using the net benefit approach The Internet group and usual care group consisted of 101 and 99 participants, respectively. Mean age of the sample was 37 years and 70% of the participants were women . At baseAt baseline, the utilities according to the EQ-5D did not statistically significantly differ between the Internet group and the usual care group. EQ-5D utilities did not reach a statistical significant difference throughout the study. At 3 months and 12 months the difference in EQ-5D utility was 0.037  and 0.006 , respectively. Similarly, the difference in quality adjusted life years was not statistically significant: 0.024  .Visual analogue scale utilities were not statistically significantly different throughout the study. At 3 and 12 months the difference in visual analogue scale utility was 0.012  and 0.013 , respectively. The difference in quality of life years based on the visual analogue scale was estimated to be 0.007  .The total intervention costs were estimated at $25675, which is $254  per patient . The higThe difference in other health care costs was not statistically significant: $-217  . PatientPatients in the Internet group reported 114 hours of absence from work compared to 98 hours for patients in the usual care group. The 16 hours difference in absenteeism was estimated to be equivalent to $604  in monetary terms. The difference in societal costs  was therefore estimated at $641  in favor of usual care.The estimates of the cost differences and QALY differences were both not-statistically significant. The cost-utility ratio, based on these point estimates, was $26700 per QALY. The probability that Internet-based self-management was both more effective and less costly than usual care (dominant) was 30%. The probability that it was less effective, but more costly (dominated) was 10% . Due to From a health care perspective, the lower health care costs result in a cost-utility ratio of $1500 per QALY. The probability that Internet-based self-management is cost-effective from a health care perspective was 82% at $50000 per QALY and 86% at $100000 per QALY  and 2.QALYs gained, based on the visual analogue scale, were less than those based on the EQ-5D. The probability that Internet-based self-management is cost-effective based on visual analogue scale QALYs was 49% and 60% at $50000 and $100000 per QALY from a societal perspective and was 71% and 75% at $50000 and $100000 per QALY from a health care respectively.In this study we evaluated the cost-effectiveness of a new disease management strategy, Internet-based self-management, for patients with asthma. The QALY and cost differences, 0.024 and $ 641 respectively, between Internet based-self management and usual care were not statistically significant during a follow-up period of 1 year. Both the estimation of QALYs gained and the calculated expenses showed considerable uncertainty, which is displayed by the cost-effectiveness planes. The estimated cost-utility ratio was $26700 per QALY, which is generally considered acceptable We have previously shown substantial and statistically significant clinical effects in favor of Internet-based self-management with regard to asthma related quality of life, asthma control and lung function The intervention costs of $254 per patient were similar to intervention costs of a paper-and-pencil asthma self-management program Differences in other health care costs should be interpreted with caution, since almost all components showed statistically non-significant differences. Only the reduction in contacts with physiotherapists were statistically significant, suggesting that patients in the Internet group with better asthma control are less in need for physiotherapy. The cost of drugs for asthma show small decreases in short-acting β2-agonists and inhaled corticosteroids alone, but increases in combination therapy  and leukotriene antagonists in the self-management group. The increase in volumes and costs of asthma controller medication accompanied by a decrease in reliever medication might have contributed to improved clinical outcomes in favor of Internet-based self-management.Our study had several limitations. First, quality adjusted life year estimates were calculated from only two follow-up measurements. More measurements would possibly have resulted in more accurate QALY estimates, but we limited the number of follow-up measures in order to minimize the awareness of participating in a clinical trial among patients in the usual care group. Second, patients were inevitably aware of the allocated group, which may have influenced their utility ratings. Therefore, the effects observed may be due to unblinding. On the other hand, the influence of unblinded groups in pragmatic trials might be regarded as part of the intervention, since all interventions implemented in daily clinical practice are not blinded. Third, our economic evaluation was limited to one year. As pointed out above a longer duration would probably have resulted in reduced intervention cost estimates after one year. It is, however, unknown how EQ-5D utility scores will progress after one year.New cost-effective disease management strategies for asthma are required to face up to the global burden of asthma. Internet-based self-management is an innovative and effective management strategy in adults with asthma that improves clinical outcomes Protocol S1Trial protocol(DOC)Click here for additional data file.Checklist S1CONSORT checklist(DOC)Click here for additional data file.Flowchart S1CONSORT flowchart(TIF)Click here for additional data file.


Tailgut cysts ) are developmental abnormalities consisting of multiloculated cysts lined by squamous, transitional or glandular epithelium which, albeit rarely, may give rise to malignant transformations. Carcinoid tumours arising in the presacral region are extremely rare and usually benign, and only a few are described in the literature. Case 1: A 63-year-old female diagnosed as having bilateral ovarian cysts underwent surgery to remove a right adnexial mass that was histopathologically diagnosed as a well-differentiated carcinoid tumour. She is currently disease free after 18 months of follow-up. Case 2: A 41-year-old-female diagnosed with hepatic metastases and a solid pelvic mass arising from a moderately differentiated neuroendocrine carcinoma is currently alive with disease after having undergone surgical removal of the mass and several medical treatments. We here describe two different clinical histories of well- and moderately differentiated neuroendocrine tumours (NETs) arising from tailgut cysts in the prerectal space together with a review of the relevant literature. Neuroendocrine tumours (NETs) are a relatively rare and heterogeneous group of usually indolent malignancies, and there have been sporadic reports of NETs originating in the presacral region. Tailgut cysts  are very uncommon, generally congenital developmental abnormalities that consist of multiloculated cysts lined with squamous, transitional or glandular epithelium, and are usually incidentally identified in adult life as a result of radiological examinations performed for other reasons. Albeit rarely, they may give rise to malignant transformations, including NETs or adenocarcinomas. Here, we describe the clinical cases of two Italian female patients with NETs which developed within a tailgut cyst.A 63-year-old woman with a negative family history of malignancy had undergone a hysterectomy because of uterine fibromas when she was 45-years-old. She also had a radiologically diagnosed slipped lumbar disc that did not require pain medication. In July 2008, she underwent radiological examinations because of increased lumbar pain and the emergence of a peri-umbilical mass. Spine magnetic resonance imaging was negative for the presence of a neoplasm and/or suspected mass, but abdominal and transvaginal ultrasonography revealed bilateral ovarian cysts. Her blood carcinoembryonic antigen and cancer antigen (CA) 125 levels were normal, but she had above-normal levels of CA 19.9 . There were no other abnormalities.She underwent bilateral adnexectomy with the removal of a 58 × 35 × 43 mm cystic/solid mass located in the retroperitoneal region and loosely connected to the lower sacrum by means of fibrous attachments, leading to complete functional recovery. After being emptied, the cystic mass measured 40 × 30 × 20 mm. Macroscopically, the external wall was red and smooth, but the internal wall was multiloculated. A brown-red nodule with a diameter of 15 mm arising from the external wall was microscopically described as a NET (carcinoid) mainly with a trabecular architecture probably arising from a retro-rectal tailgut cyst. The histological material was reviewed by a pathologist (GP) at the European Institute of Oncology in Milan, who confirmed the diagnosis of a well-differentiated NET (based on the.2000 WHO classification). The trabecular architecture was compatible with a retro-rectal tail cyst origin. The immunophenotype was positive for cytokeratin AE1–AE3, synaptophysin, pancreatic polypeptide (PP), acid phosphatase and, focally, chromogranin A. The proliferation index (Ki-67) was <2%. Peritoneal washing did not reveal malignant cells. The patient received no medical therapy and after a follow-up of 25 months, is in good clinical condition with no clinical, biochemical or radiological signs of recurrence .In111-Octreotide scintigraphy showed several regions of uptake in the liver and hypogastric region. Blood chromogranin A and neuron-specific enolase levels were normal.This is the case of a 41-year-old woman who gave birth to twins in March 2003. In April of the same year, she underwent abdominal ultrasonography because of persistent rectal pain during evacuation; a solid mass was found close to the rectum. Subsequent colonoscopy excluded the presence of inherent lesions. A chest-abdomen computed tomography (CT) scan revealed a thickened rectal wall, the solid mass and multiple liver metastases. A total body The patient underwent posterior surgery with intersphincteric and pera-sacrococcygeal excisions. The surgical specimen reviewed by a pathologist (GP) at the European Institute of Oncology in Milan indicated a moderately differentiated (Ki-67 = 18%) neuroendocrine carcinoma (on the basis of the 2000 WHO classification) associated with residual epithelial cystic material. The immunophenotype was positive for chromogranin A, synaptophysin, cytokeratin AE1–AE3, and somatostatin receptor subtype 2. On the basis of the clinical, morphological and immunohistochemical findings, a clinically malignant, presacral carcinoid with liver metastases was diagnosed .In111-Octreotide scintigraphy scan revealed multiple suspected bone metastases. The patient received long-acting somatostatin analogue therapy and experienced stable disease until October 2009, when total body CT revealed the recurrence of multiple nodes. She is currently receiving PRRT with radiological stable disease.Between November 2003 and April 2004, the patient received six cycles of chemotherapy (carboplatin plus etoposide) because of CT evidence of progression in the number and size of the liver lesions. Subsequently, she received peptide receptor radionuclide therapy (PRRT) from June 2004 to November 2005 because of increasing liver lesions, which led to a partial tumour response. After the appearance of pleural lesions and a solid right adnexal mass, she underwent hysterectomy and bilateral adnexectomy, and the diagnosis of a neuroendocrine carcinoma was histologically confirmed only in the left adnexa. Simultaneously, a second total body Published reports of carcinoid tumours of the presacral region are rare and mainly involve direct extensions of a rectal mass . They arThe close association of presacral carcinoid tumours with tailgut cysts suggests that both probably originate in the hindgut and remnants of the tailgut or neuroenteric cord may be the origin of tailgut or presacral space carcinoid tumours. The cysts infrequently extend from the presacral space to involve the rectal wall and rarely extend laterally from the mid-line or into the postsacral space, or laterally and anteriorly to the rectum.To the best of our knowledge, only 28 cases of carcinoid tumours of the presacral region are described in the medical literature and of these 14 were associated with tailgut cysts. The rest were either associated with teratomas or with no other peculiar anomalies . AlthougHere, we describe the different behaviours of two well-differentiated NETs arising from RCHs. In the first case, despite the absence of residual epithelial material, the anatomic location of the NET and its benign behaviour make it very likely that it originated from a tailgut cyst. The second case demonstrates that not all NETs arising from tailgut cysts are benign. The second patient showed metastatic dissemination at the time of initial diagnosis and evidence of clinical recurrence over a 7-year observation period. The treatment of these malignancies may therefore vary from case to case. The time of the diagnosis, the radical nature of surgical resection and a histopathological diagnosis are the most important prognostic factors.Although NETs arising from tailgut cysts in the retro-rectal area are extremely rare and generally benign, they should be considered in the differential diagnosis of an adnexal mass evidenced by radiological and pathological means. The behaviour of our two cases are in line with their Ki-67 index values, thus indicating this as reliable predictors in NETs.


Cardiovascular diseases and cancers are leading causes of morbidity and mortality. Reducing dietary saturated fat and replacing it with polyunsaturated fat is still the main dietary strategy to prevent cardiovascular diseases, although major flaws have been reported in the analyses supporting this approach. Recent studies introducing the concept of myocardial preconditioning have opened new avenues to understand the complex interplay between the various lipids and the risk of cardiovascular diseases. The optimal dietary fat profile includes a low intake of both saturated and omega-6 fatty acids and a moderate intake of omega-3 fatty acids. This profile is quite similar to the Mediterranean diet. On the other hand, recent studies have found a positive association between omega-6 and breast cancer risk. In contrast, omega-3 fatty acids do have anticancer properties. It has been shown that certain (Mediterranean) polyphenols significantly increase the endogenous synthesis of omega-3 whereas high intake of omega-6 decreases it. Finally, epidemiological studies suggest that a high omega-3 to omega-6 ratio may be the optimal strategy to decrease breast cancer risk. Thus, the present high intake of omega-6 in many countries is definitely not the optimal strategy to prevent cardiovascular disease and cancers. A moderate intake of plant and marine omega-3 in the context of the traditional Mediterranean diet (low in saturated and omega-6 fatty acids but high in plant monounsaturated fat) appears to be the best approach to reduce the risk of both cardiovascular diseases and cancers, in particular breast cancer. Cardiovascular disease (CVD) is a leading cause of death in most countries. Reducing saturated fatty acid (SFA) intakes is still at the heart of dietary recommendations to reduce CVD, mainly because of its effect on blood cholesterol . This viThere is one exception: the Mediterranean diet , which iPreconditioning - that is, the ability of the myocardium to withstand an ischemia-reperfusion injury - is a major concept in cardiology . The extIn fact, two recent studies on rat models have provided major findings by comparing the effects of different dietary fat profiles on the induction of myocardial preconditioning ,20. In bThese data should help identify the optimal dietary fatty acid profile to reduce the risk and the complications of CVD. Thus, maintaining high  or increIn animal studies, omega-6 PUFAs have a strong mammary tumor-enhancing effect ,22. In oThus, there are several recent and concordant studies that strongly suggest that dietary omega-6 PUFAs - the consumption of which is encouraged worldwide to decrease blood cholesterol - increase the risk of breast cancers. In the same line of reasoning, it is important to recall that the most frequently prescribed cholesterol-lowering drugs (including statins) increase the blood concentration of arachidonic acid, the main omega-6 PUFA in cell membranes . Also, sIt could be said that most data regarding the effects of dietary omega-6 PUFAs on cancers are observational -30 and dAt the same time, omega-3 PUFAs were shown to have chemopreventive properties against various cancers and their complications, including colon and breast cancer ,36. It iThe association of oleic acid - the main fatty acid of olive oil, a major component of the Mediterranean diet - with breast cancer risk has been analyzed in several studies -46 and pFrom the most recent experimental and epidemiological studies, we conclude that the optimal dietary fat pattern to reduce the risk of both CVD and most cancers should include a low intake of SFAs and omega-6 PUFAs. Small amounts (1% to 2% of energy intake) of the essential linoleic acid - easy to find in most Western foods - are sufficient to prevent omega-6 deficiency ,51. The Finally, regarding the intake of oleic acid, it is critical to differentiate the food sources, since the health effects of oleic acid obtained from meat or from olive oil are different. To simplify the dietary advice aimed at protecting health - and help consumers to understand it - the best approach is probably the traditional Mediterranean diet model. No dietary pattern has been so extensively studied, and no other has been shown to provide so many benefits without any adverse effects.CVD: cardiovascular disease; LOX: 5-lipoxygenase; PUFA: polyunsaturated fatty acid; SFA: saturated fatty acid.The authors declare that they have no competing interests.MdeL drafted the manuscript. PS critically revised the manuscript and gave final approval for publication.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/50/prepub


C9ORF72 gene has recently been identified as an important cause of frontotemporal dementia and motor neuron disease; however, the phenotypic spectrum of this entity and its pathophysiologic basis have yet to be fully defined. Psychiatric features may be early and prominent, although a putative cortico-thalamo-cerebellar network has been implicated in the pathogenesis of the clinical phenotype. Differentiation of self from others is a core cognitive operation that could potentially link network disintegration with neuropsychiatric symptoms in C9ORF72-associated frontotemporal dementia.An expanded hexanucleotide repeat in the C9ORF72 expansion by using a novel paradigm requiring differentiation of the effects of self- and non-self-generated actions. The patient's performance was assessed in relation to two older male patients with bvFTD not attributable to the C9ORF72 expansion and four healthy older male subjects.We undertook a detailed behavioral analysis of self-other attribution in a 67-year-old male patient with behavioral variant frontotemporal dementia (bvFTD) due to the C9OFR72 mutation showed a deficit of self-other differentiation that was disproportionate to his otherwise relatively indolent clinical phenotype. The performance of the other patients with bvFTD was similar to that of healthy subjects.Compared with the healthy control group, the patient with the C9ORF72 expansion. We offer this preliminary observation as a stimulus to further work.We propose that impaired self-other differentiation is a candidate mechanism for neuropsychiatric decline in association with the  C9ORF72 gene has recently been identified as an important cause of frontotemporal dementia and motor neuron disease [C9ORF72 mutation spectrum, a culprit cortico-thalamo-cerebellar network has been identified as a potential substrate for certain clinical features, in particular, for neuropsychiatric symptoms [C9ORF72-associated frontotemporal dementia. Disordered self-other differentiation has been implicated in the pathogenesis of various psychiatric conditions, including schizophrenia, out-of-body autoscopic experiences, anxiety, and depression [An expanded hexanucleotide repeat in the  disease -3; howev disease ,3,4. Altsymptoms . Differepression -8. Neuropression ,9.C9ORF72 expansion, who exhibited an indolent phenotype of behavioral variant frontotemporal dementia (bvFTD) with relative preservation of many cognitive abilities. We designed a novel behavioral paradigm specifically to assess NT's ability to distinguish between the effects of self- and non-self-generated actions. We assessed NT in relation to a group of healthy older male control subjects and two older male patients (DC1 and DC2) with bvFTD not attributable to the c9ORF72 expansion.We recently had the opportunity to investigate this issue in a patient, NT, with the NT is a 67-year-old right-handed male retired information technology consultant with a postgraduate degree qualification. He presented with an insidious decline in personality and behavior extending over some 20 years. His increasing impassivity, impulsivity, and distractibility had led to his retirement from work. His wife reported that he was less gregarious and less empathic, more obsessional about money, and showed an increased preference for sweet foods. He made tactless remarks to strangers and had only limited insight into his difficulties. No concern had been expressed about his episodic memory or route-finding abilities, and his language, though somewhat tangential, remained articulate. No history suggested delusions, hallucinations, or other perceptual disturbance. A family history existed of behavioral decline in NT's mother from age 50, and dementia with parkinsonism in a maternal uncle. NT's general neurologic examination was unremarkable; in particular, no features of motor neuron disease were found. Longitudinal neuropsychological assessment over a 9-year interval demonstrated a largely stable profile with superior performance in most cognitive domains and only a mild relative weakness of aspects of executive function; NT's general neuropsychological data at the time of the experimental assessment are summarized in Table T1-weighted volumetric brain MRI demonstrated progressive, diffuse cerebral and cerebellar volume loss; brain MRI at the time of the behavioral assessment  gene.Serial registered DC2 is a 76-year-old left-handed male retired professor of English literature. He presented with an 11-year history of progressive prosopagnosia and personality change with social disinhibition and obsessionality. No history of delusions, hallucinations, or other perceptual disturbance was noted, with no known family history of dementia. The general neurologic examination was normal. Serial neuropsychological assessments over a 6-year interval demonstrated a largely stable profile with prominent anomia and recognition memory dysfunction; DC2's general neuropsychological data at the time of the experimental assessment are summarized in Table T1-weighted volumetric brain MRI demonstrated progressive cerebral volume loss involving the anterior temporal and frontal lobes, more marked on the right  and with similar educational and social backgrounds but no history of neurologic or psychiatric illness also were assessed.Four healthy right-handed male control subjects . Analyses of the three experimental conditions separately revealed that NT's performance was flawless for attribution of self-generated actions, whereas he performed significantly worse than healthy controls for attribution of both synchronous, non-self-generated actions  and asynchronous, non-self-generated actions . NT's errors were therefore entirely overattributions of the experimenter's actions on non-self trials as self-generated responses. Like NT, patients DC1 and DC2 correctly attributed all self-generated actions. However, in contrast to NT, DC1 and DC2 showed no significant differences in performance relative to healthy controls on either of the non-self conditions , and each patient's performance fell within the control range for every condition.Results on the experimental behavioral test are summarized in Table It is evident from the range of scores Table  that heaC9ORF72 expansion. Indeed, the deficit of self-other differentiation appeared disproportionate to NT's otherwise relatively mild cognitive phenotype. As this patient's cognitive evolution was otherwise so indolent, we hypothesize that impaired self-other differentiation may be a key feature in the development of the complex behavioral disturbances that accompany the C9ORF72 expansion, or may to lead the development of more-typical cognitive deficits. The specificity of this finding for C9ORF72-associated frontotemporal dementia remains to be resolved. However, the present data suggest that the ability to distinguish one's own from others' actions is not comparably affected in other forms of frontotemporal dementia (including MAPT-associated disease); nor does it appear to be simply a consequence of more severe disease, as DC1 and DC2 were both substantially more cognitively impaired than was NT. Together, these findings raise the possibility that impaired self-other differentiation is a behavioral signal of C9ORF72 mutations. It is of interest that NT tended to "overattribute" others' actions to his own agency. A similar overattribution bias to self for actions of ambiguous origin has been reported both in healthy individuals [C9ORF72 expansion here made self-overattribution errors in the synchronous non-self condition, but (unlike NT) were able to use the increased temporal delay in the non-self asynchronous condition to identify the external origin of the action. Although the overattribution to self of external actions may seem somewhat paradoxic in a condition such as schizophrenia with delusions of external control, it has been argued [Here we have shown that differentiation of the effects of one's own versus others' actions may become impaired in frontotemporal dementia associated with the ividuals ,13 and iividuals . Both hen argued  that sucC9ORF72 expansions. Although neuroanatomic correlation was not possible here, we hypothesize that dysfunction of the previously delineated cortico-thalamo-cerebellar network may have underpinned the behavioral deficit of self-other differentiation exhibited by NT [C9ORF72 mutations may index a generic mechanism of defective own-action modeling and representation that may be somewhat analogous to the deficit proposed previously in patients with schizophrenia (6). Such a pathophysiologic mechanism could potentially be expressed in a range of clinical neuropsychiatric phenomena.Previous theoretic accounts of the neurobiologic basis for sense of agency ,15 have C9ORF72 expansion, with both clinical and neuroanatomic correlation. This patient did not exhibit clinically overt psychotic symptoms ; it would be particularly pertinent to test our hypothesis in a group of patients who do exhibit such symptoms . The proposed role of the putative cortico-thalamo-cerebellar network could be directly assessed in a structural or functional brain-imaging paradigm. The use of other tasks designed to explore self-other differentiation in this population would further corroborate the present findings. The specificity of the deficit for C9ORF72-associated frontotemporal dementia requires further corroboration through comparison with larger cohorts of patients representing other forms of genetic and sporadic frontotemporal dementia. In addition, within the C9ORF72 expansion group, a need exists to establish the time course of development of the deficit in a longitudinal analysis. If our conjecture is correct, impaired self-other differentiation might manifest as an early feature, possibly even presymptomatically. The paradigm we propose is relatively straightforward and could be adapted to group longitudinal applications. We hope that our preliminary observation stimulates further hypothesis-led work directed to establishing the pathophysiologic basis of the complex behavioral disorders that characterize this newly discovered entity.We present this single-case analysis with a number of caveats and suggestions for future work. Foremost among these, the findings require replication in a cohort of patients with the BPVS: British Picture Vocabulary Scale; bvFTD: behavioral variant frontotemporal dementia; CBI: Cambridge Behavioural Inventory; D-KEFS: Delis-Kaplan Executive Function System; DS: digit span; GNT: Graded Naming Test; MRI: magnetic resonance imaging; NART: National Adult Reading Test; PIQ: Performance Intelligence Quotient; RMT: Recognition Memory Test; VIQ: Verbal Intelligence Quotient; VOSP: Visual Object and Space Perception; WASI: Wechsler Abbreviated Scale of Intelligence.We have no financial competing interests to declare. LED, CJM, SJC, and JDW receive salary and research support from the Medical Research Council, Alzheimer Research UK, and the Wellcome Trust. MNR sits on the Data Monitoring Committee for Servier DMC Phase 2B AD Study S38093, and also sits on the Bapineuzumab Independent Safety Monitoring Committee for Janssen Al/Pfizer.All authors read and approved the manuscript. LED was involved in study planning, design, and coordination, acquired and analyzed the behavioral data, and was involved in drafting and critically revising the article. CJM was involved in study design, acquisition of neuroimaging data, and drafting and critically revising the article. MNR assessed the patients clinically and was involved in study design and in drafting the article. SJC and JDW obtained funding for and supervised the study, and were involved in study planning and design, in data collection, and in drafting and critically revising the article.


Sex-related physiological differences result in different expressions of diseases for men and women. Data are contradicting regarding the increase in the female risk for cardiovascular disease (CVD) at mid-life. Thus, we studied possible sex differences in age-adjusted mortality for CVD and non-vascular diseases stratifying our findings by specific age groups.Over one million deaths (1 080 910) reported to the Finnish nationwide Causes of Death Register in 1986–2009 were analyzed. A total of 247 942 male deaths and 278 752 female deaths were of CVD origin, the remaining deaths were non-vascular. The annual mortality rates were calculated per 100 000 mid-year population, separately for men and women in 5-year age categories.The age-standardized risk of death from CVD was 80% higher for men (442/100 000) than for women (246/100 000). After age 45–54 the male CVD mortality rate elevated parallel to the non-vascular mortality, whereas in women the CVD mortality elevated considerably more rapidly than the non-vascular mortality from age 60 years onwards.Heart disease mortality in men accelerates at a relatively young age, but in women the risk shows a steep increase at approximately 60 years of age. These data emphasize the need to identify and prevent risk factors for CVD, especially in women in their mid-life years. Cardiovascular disease (CVD) is the leading cause of mortality in Western populations. It is also known that the life-long risk for CVD is higher for men than women Finland is known for the high rate of CVD mortality A total of 1 080 910 deaths from diseases and medical conditions reported to the Finnish nationwide Causes of Death Register in 1986–2009 were analyzed . This reThe annual mortality rates were calculated for each of the selected cause-of-death category per 100 000 mid-year population, separately for men and women in 5-year age categories. We used the Nordic Standard Population The age-standardized risk for CVD death in men (442/100 000) was 79.6% higher than the respective risk in women (246/100 000) . The malThe CVD and non-vascular mortality curves expressed on a logarithmic scale for men were parallel (p = 0.065), whereas for women the CVD mortality showed a steeper increase (p<0.001). The CVD mortality in both sexes demonstrates significant changes at mid-life . The incWe analyzed more than one million deaths of which approximately one half were classified as CVD deaths. The data covered whole Finland during 24 consecutive years (1986–2009). It is evident from our data that the male age-adjusted CVD mortality is higher than the female mortality throughout life. This difference is most conspicuous at 40–44 years of age. However, after 55–59 years of age the female age-specific CVD mortality increases significantly more rapidly than the mortality for non-vascular causes, while in men, the CVD and non-vascular mortality curves are parallel. This results in a significant difference in the behavior of age-adjusted CVD mortality between men and women after 60–64 years of age.The CVD mortality was much smaller in women than men of less than 45 years of age in Finland. These data are in line with previous ones from other countries There have been opposing interpretations whether CVD mortality is truly increased in women after menopause. Long-term follow-up data imply that menopause is followed by an increase in CVD mortality Our study has limitations. First, we do not have data on any other demographic characteristics than age and sex, and thus, we could not adjust mortality for factors, such as menopausal status, hormone and other therapy, socioeconomic status, smoking or body weight. We did the analysis separately for different periods of time and by five university hospital regions, and the results were similar to those shown here. Second, one can argue that CVD morbidity rather than mortality should have been studied. It is, however, known that CVD mortality closely reflects CVD morbidity Our study also has several strengths. First, our data included over million deaths from consecutive 24-year period. Second, our Cause-of-Death Register covers the entire country and is accurate In summary, we show that there are sex-related differences in CVD mortality. While men have acceleration in heart diseases mortality at relatively young age, in women this risk shows a steep increase at approximately 60 years of age. This is likely a consequence of risk factors appearing some 10–20 years earlier. Therefore, it is highly important to improve CVD risk factors, in women already at fertile age as well as in their mid-life years.


The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-), heterozygous (+/-) and wildtype (+/+) mice were subjected to the Barnes maze (BM) and the Morris water maze (WM), the latter being discussed as more aversive. Additionally, immediate early gene (IEG) expression, hippocampal adult neurogenesis (aN), and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of raised alertness, possibly impeding optimal learning performance in the more stressful WM. It has been shown that 5HTT is a principal target of various antidepressants such as the selective reuptake inhibitors (SSRI) as well as for drugs of abuse including MDMA (“ecstasy”) and cocaine )activity-regulated cytoskeleton-associated protein (Arc) also known as Arg3.1. The somatodendritic localization of Arc mRNA and protein is strongly regulated by synaptic activity , Arc  or cFos antibodies   for 48 h at 4 °C. Afterwards, sections were washed again three times for 5 min in 1x TBS and incubated for 1.5 h in biotinylated goat anti-rabbit or horse anti-goat secondary antibodies, respectively . Sections were then washed and processed with avidin-biotinylated horseradish peroxidase complex in 1x TBS  for 1 h at room temperature. After another washing step the binding sites of the antibodies were visualized using 3,3'-Diaminobenzidine . Transferring the slices into 1xTBS stopped the reaction. Subsequently, sections were mounted on slides, were left to dry at least overnight and were coverslipped with VitroClud . An Olympus BX51 microscope  coupled to the Neurolucida imaging system  was used to acquire representative images from the examined individuals and to quantify cFos and Arc immunoreactive (ir) cells found in the GCL as well as Ki67 and NeuroD immunostained cells detected in the SGZ. The volume of the GCL in each brain was also assessed via the Neurolucida system. For this purpose the GCL in each brain was surrounded by a closed line. The area inside this line was measured automatically by the system. With the thus resulting area from each brain slice, the slice thickness and the number of series, we were able to extrapolate the GCL volume for each brain. 5-HTT genotype did all the assessments. Immunopositive cells were counted at 20 x 1600 magnifications using the serial section manager function of the Neurolucida software. Labeled cells were counted in both hippocampi in every eighth 50 μm thick section, recording the suprapyramidal and infrapyramidal layer of the SGZ/GCL separately. This resulted in an analysis of an average of 8 sections per mouse brain. Experimenters blind to the treatment and For quantitative evaluation a fictive coronal separation plane along the septotemporal axis of the hippocampus was used to divide the data obtained from septal and temporal sections. The first section that showed the corpus callosum disconnected inside the two hemispheres was declared as the first section of the temporal part of the hippocampus. All sections before that point were considered to hold the septal part of the hippocampus . Some sections were lost in the process of cutting or during free-floating immunohisto-chemistry, which resulted in an irregular number of sections. Therefore, those brains that had less than three sections within the septal or temporal part of the hippocampus were excluded from the evaluation of the septal and temporal hippocampus, respectively. After this adjustment, the mean number of immunopositive cells in the suprapyramidal and/or infrapyramidal layer of the SGZ/GCL was calculated per septal or temporal hippocampus per section for each brain.5-HTT genotype as the between subject factor. For further comparison of 5-HTT genotypes the areas under the learning curves (AuCs) of the acquisition phase of the tests  were calculated for each individual. The calculated AuCs were subjected to one-way ANOVAs with 5-HTT genotype as the between subject factor. Post hoc analyses were performed using Bonferroni corrected t-tests. Spearman's rank correlation coefficient was used to assess the correlation between learning performance, represented by the AuCs and numbers of immunoreactive cells in the DG. Corticosterone concentrations were analyzed using a two-way ANOVA with 5-HTT genotype and treatment  as the between subject factors. As the corticosterone levels of the two hormone cohorts had been measured in two separate assays, they had to be statistically analyzed separately. Graphics presented and statistics carried out for behavioral and hormonal measures were done using the statistical software Graph Pad Prism. Behavioral data from the spatial learning tests [the acquisition phase of the BM  and the WM  as well as the reversal trials ] were analyzed using repeated measures (RM-)ANOVA with trial as the repeated measure and Quantitative immunohistochemistry data were analyzed using two-way ANOVA with genotype and treatment  as the between subject factors. Post hoc analyses were performed using Bonferroni corrected t-tests. For immunohistochemical measures, statistical evaluation was performed with SPSS . For all presented data, significance was defined as p≤0.05. P-values of ≤0.01 were considered to be highly significant and 0.05<p<0.1 was defined as a trend.5-HTT genotype effect . Post hoc analysis revealed no significant differences . During the acquisition phase of the BM, an overall reduction in the latency to find the correct hole was found, indicated by a highly significant trial effect . Bonferroni corrected post hoc analysis however revealed no significant differences . In the acquisition phase of the WM, a reduction in the latency to find the hidden platform was detected, as indicated by a highly significant trial effect  was found. However, no significant genotype effect was found . A weak trend towards a trial by genotype effect was detected , post hoc tests however did not reveal any significant effects.Statistical analysis of the plasma corticosterone levels from mice of the first hormone cohort that had lived under control conditions  or experienced one BM or one WM trial, revealed no significant genotype effect . However, a highly significant treatment effect was found . Such a trend was found neither for the 5-HTT+/- nor for the 5-HTT+/+ mice.Despite the fact that there was no significant gene by treatment interaction, uncorrected t-tests revealed significantly higher corticosterone levels in 5-HTT-/- mice versus 5-HTT+/+ mice in the BM  as well as the WM . 5-HTT+/- mice generally exhibited intermediate corticosterone levels. An uncorrected t-test showed that 5-HTT+/- had significantly higher levels than 5-HTT+/+ mice in the BM  but not in the WM. Noteworthy, 5-HTT-/- mice tested in the WM tended to show higher corticosterone stress hormone levels compared with mice of the same We examined the brains of 5-HTT+/+ and 5-HTT-/- mice, which had been subjected either to the control condition (undisturbed in home cage) or to one of the two spatial learning tests (BM or WM). In addition to the quantitative assessment of cells immunoreactive for IEGs and neurogenesis markers, we also measured the volume of the GCL in each of the brains. Statistical evaluation of these measurements however revealed neither a significant treatment effect  nor a significant genotype effect  as well as no significant interaction effect . For this reason, we refrained from normalizing cell counts with the volume. 5-HTT genotype effects irrespective of the treatment. Cell counts for cFos and Arc were found to be higher in 5-HTT-/- compared to 5-HTT+/+ mice in any of the examined hippocampus subregions  and the temporal hippocampus  . 5-HTT genotype effects could be revealed in the suprapyramidal blade of the SGZ  of the septal hippocampus as well as in the infrapyramidal blade of the SGZ  and in both blades of the SGZ  of the temporal hippocampus. There was however no main effect of treatment. Yet, a significant 5-HTT genotype by treatment interaction of Ki67-ir cells in both blades of the SGZ of the septal hippocampus could be found , tendentially increased in the infrapyramidal blade  and significantly increased in both blades of the SGZ  in the temporal part of the hippocampus . s) =-0.72, p=0.036,) as well as in both blades of the SGZ  of the SGZ. Moreover, a trend towards a negative correlation could also be found in the infrapyramidal blade of the SGZ . Applying the Spearman`s test the only linear dependence between the escape latency  and the number of new-born immature neurons immunoreactive for NeuroD could be found in the temporal hippocampus of 5-HTT-/- mice tested in the WM . In moreThe aim of this study was to evaluate whether mice with altered levels of brain 5-HT as a consequence of 5-HTT deficiency perform differently in two spatial memory tests, the WM and the BM, prospectively differing in aversiveness. Moreover, we asked whether neurobiological equivalents to possible learning differences exist. For this purpose, we investigated the phenomenon of adult neurogenesis and IEG expression as a marker for neuronal activity in the hippocampus in addition to blood corticosterone levels as a marker for stress reactivity of these mice. 5-HTT genotypes showed similar learning performance. These results unambiguously point to a comparable spatial learning capacity of 5-HTT-/-, 5-HTT+/- and 5-HTT+/+ mice, but that there are some time-dependent performance differences in the WM, but not in the BM.From numerous studies, it is known that the lifelong reduced or absent 5-HTT function is associated with multiple changes at the behavioral level compared to 5-HTT+/+ littermates (for review see :). The bIncreased floating in the water maze could be explained in the light of a generally hypoactive phenotype of 5-HTT-/- mice as described in home cage behavior as well as in a number of different behavioral tests ,27,86-89Interestingly, we did not detect initially more floatings in 5-HTT /- mice compared to 5-HTT+/- and 5-HTT+/+ mice, but the number of floatings increased in the course of three subsequently performed trials per day  during the first three days of the WM training session. This resembles the results of two different studies performed by the group of Wellman  who show5-HTT genotype after BM experience in both stress hormone cohorts. Moreover, repeatedly experiencing any of the two spatial learning tests resulted in 5-HTT genotype differences with highest plasma corticosterone levels in 5-HTT-/- mice, while 5-HTT+/+ mice seemed to habituate regarding their stress response. These 5-HTT genotype differences are in accordance with former studies analyzing glucocorticoid and Adrenocorticotropic hormone (ACTH) levels in mice of different 5-HTT genotypes in response to stressors such as handling and injection [5-HTT genotype differences after repeated spatial learning test performance can be regarded as a result of gene by stress experience interaction and point to an altered stress sensitivity of 5-HTT deficient mice. Therefore, the assumed different levels of aversiveness of BM and WM in combination with an increased stress sensitivity of the tested 5-HTT deficient animals might explain inferior learning performance in the WM as an altered responsiveness of 5-HTT-/- mice towards a seemingly inescapable aversive WM environment without actually affecting spatial learning abilities per se. In contrast to the WM the BM does not utilize the fear of drowning as a strongly aversive stimulus, and therefore was already shown to result in lower blood plasma stress hormone levels . Moreovenjection ,96. Furtnjection , whereasnjection ,98-100. 5-HTT gene exhibit a greater reactivity to stress. This relation is supported by human imaging studies, which have found that carriers of the S-allele display greater amygdala activation in response to fearful stimuli [5-HTT genes [Comparable to the results of 5-HTT deficient mice or rats, human individuals carrying the S-allele of the  stimuli , that is stimuli . MoreoveTT genes ,104. We were aware that the chosen time-point was not ideal for IEG expression analysis related to effects of the exposure to the different test apparatuses as in immunocytochemistry as well as in mRNA-studies IEG protein/mRNA expression had been shown to peak within the first hours (protein) or even minutes (mRNA expression) after different learning tests or stress exposure and then return to baseline after a maximum of 24 hrs ,105-107 5-HTT genotype effects with significantly higher number of Arc-ir cells in the DG of 5-HTT-/- mice compared to 5-HTT+/+ mice. In contrast to our results, significantly reduced Arc mRNA expression was detected in the whole hippocampus of 5-HTT deficient rats, irrespective of hippocampal subregions and different layers [5-HTT genotype dependent alterations in baseline Arc expression may be explained by a compensatory mechanism in response to the lifelong altered extracellular and intracellular 5-HT-levels, which could be the neurobiological correlate of heightened anxiety. This notion is supported by the fact that serotonergic signaling and Arc thus have opposing effects on AMPA type glutamate receptor (AMPAR)-trafficking. While serotonergic signaling enhances trans-synaptic signaling efficiency via the insertion of AMPARs into synapses at postsynaptic neurons [As expected, we did not detect differences between the number of Arc positive cells in mice of the different treatment groups , but overall t layers . Additiot layers . These r neurons , Arc exp neurons . The latRegarding cFos, significantly increased numbers of cFos-ir cells were found in the septal hippocampus of 5-HTT-/- compared to 5-HTT+/+ mice. From a number of older studies it is known that cFos is upregulated in the DG in response to various acute stressors including immobilization, noxious stimulation and hyper osmotic stress ,112 as wFinally, the increased baseline expression of the two IEGs Arc and cFos shown in this study may have promoted the pronounced stress sensitivity of 5-HTT-/- mice. Thus, elevated Arc and cFos protein levels could represent a “stressed” brain phenotype in consequence of lifelong 5-HTT deﬁciency, thereby presenting the neurobiological correlate of increased stress sensitivity and heightened anxiety of 5-HTT-/- animals. 5-HTT genotype effects regarding proliferation (Ki67) and neurogenesis (NeuroD) comprise 5-HTT-/- mice displaying more immunoreactive cells than 5-HTT+/+ animals. This result is quite feasible when keeping in mind that, first, the lack of 5-HT clearance in these mice results in a persistent 5 to 13-fold increase of 5-HT concentrations in the extracellular space [ar space ,17, and ar space -125. Schar space . In thisar space . Thus, iBeyond the overall increased aN levels in 5-HTT deficient compared to 5-HTT+/+ mice our statistical tests revealed a significant gene by environment interaction of Ki67-ir cells in the SGZ of total DG. The number of Ki67-ir cells was shown to be different between naïve and WM-tested 5-HTT+/+ mice, with those tested in the WM displaying more proliferating cells in the SGZ than naïve animals. Moreover, naïve 5-HTT-/- mice already exhibited a trend towards higher numbers of proliferating cells compared to 5-HTT+/+ mice, whereas there was neither a significant difference between the two genotypes after BM or WM nor a significant effect of treatment within 5-HTT-/- mice. Considering that stress decreases aN ,127,128,There were two studies dealing with the impact of WM-testing on hippocampal aN in the rat, which resulted in contradictory outcomes. The first one, which is also probably the most well known study to convey the enhancing effects of spatial learning on aN, was carried out by Gould and colleagues in 1999  In theirThe finding that there was no significant difference between the differently treated 5-HTT-/- mice, including the fact that there was already a trend towards higher numbers of proliferating cells in naïve 5-HTT-/- vs. 5-HTT+/+ mice suggests that this embodies a ceiling effect, that 5-HTT-/- mice are closer to a “stress threshold” than 5-HTT+/+ mice. A comparable ceiling effect was already described by Nietzer and co-workers, who found higher spinogenesis in the amygdala in naïve 5-HTT-/- vs. 5-HTT+/+ mice . As strein vivo and in vitro showed a severe learning and memory impairments [Regarding the number of NeuroD-ir cells, a positive correlation between the number of NeuroD-ir cells in 5-HTT-/- mice subjected to the WM and their overall performance (AuC) could be found revealing that mice with lower numbers of NeuroD-ir cells performed worse in the WM. The modulation of aN by WM test experience and the detected correlation between the number of newborn neurons and the learning performances point to an important role of this neuroplasticity phenomenon in the handling of stress and in learning and memory processes. This discovery is consistent with a study of human hippocampal neurogenesis by Coras and colleagues showing that patients with low numbers of proliferating progenitor cells and new-born neurons airments . 5-HTT genotype effects regarding cell activation (Arc/ cFos) or neurogenesis (Ki67/ NeuroD) were found in the suprapyramidal blade of the DG. However, only environmental factors like spatial experience, transient forebrain ischemia, or kainic acid administration, have been found to cause region specific effects regarding IEG-expression, aN and or even morphological changes [Moser and Moser suggested that the hippocampus is functionally different along its septotemporal axis . In rode changes -143. So 5-HTT genotypes, but the performance differences of 5-HTT deficient mice in the WM task support a role for the 5-HTT in the hippocampally-mediated interaction between stress and spatial learning performance. Moreover, we suggest that increased IEG expression and aN levels observed in the hippocampus of 5-HTT deficient mice can be the neurobiological correlate of emotion circuit dysfunction and heightened anxiety as 5-HTT-/- animals per se display a “stressed” phenotype as a consequence of long-life 5-HTT deﬁciency. The experience of BM and WM tests results in either altered or even absent neuroadaptive increases in IEG and aN marker expression in 5-HTT-/- animals reducing adequate compensation processes and resulting in exaggerated behavioral responses e.g. in the more aversive WM. In summary, our BM and WM results point to comparable spatial learning capabilities of mice with different


Pleomorphic hyalinizing angiectatic tumors (PHATs) are rare non-metastasizing tumors of uncertain lineage. The current study presents a case of PHAT arising in the thigh of a 68-year-old female and examines the clinicopathological characteristics of the tumor. Magnetic resonance imaging (MRI) revealed an intramuscular mass located in the adductor longus. The tumor was surrounded by lipomatous tumor. Wide resectioning was performed for the internal tumor, whereas intralesional resectioning was performed for the external tumor. Histopathologically, the internal lesion was diagnosed as a PHAT and the external lesion was diagnosed as an hemosiderotic fibrolipomatous lesion (HFLL). No recurrence or metastases were identified during the 6-year follow-up period. As the adipose tissue surrounding the PHAT resembled a HFLL, therefore, the association between ‘early PHAT’ and HFLL is discussed. Although PHATs may represent low-grade sarcomas, HFLLs may be benign tumors. Folpe a14 cases  describeA 68-year-old female presented with a solitary asymptomatic tumor of the left medial thigh that had been rapidly growing for ~2 months. There was no history of trauma and a physical examination revealed an elastic soft tumor measuring ~12×7 cm in size.67Gallium scintigraphy showed significantly abnormal uptake in the tumor. Furthermore, chest radiography and computed tomography found no evidence of lung metastasis, and the laboratory findings were normal.Radiography showed an expansion of the soft tissue on the medial aspect of the thigh, but no bony changes or calcifications. Magnetic resonance imaging (MRI) revealed that the tumor mass was isointense compared with the muscle on T1-weighted images . On T2-wBased on a presumed diagnosis of a benign fibrous tumor or schwannoma, tumor excision was performed. The surgical findings concluded that the tumor was located in the femoral abductor muscle, with the femoral artery penetrating into the tumor. As a benign tumor was suspected according to the intraoperative frozen section examination, the majority of the mass was excised and the remaining portion was left attached to the artery. The gross appearance revealed two layers of structures: An internal lesion with a hard, white-tan colored tumor and an outer lesion composed of lipomatous tumor tissue . A wide Histopathologically, the stroma revealed loose fascicles that were arranged in whorls or a haphazard, patternless fashion. This lesion contained a number of enlarged, thin-walled blood vessels with rims of a fibrinous or hyalinized material and organized thrombi and foci of hemosiderin deposition . The parImmunohistochemistry revealed a number of tumor cells were positive for vimentin, cluster of differentiation (CD)34, CD99 and B-cell lymphoma 2, whereas staining for S-100 protein, α-smooth muscle actin, desmin, AE1/AE3 and epithelial membrane antigen was negative. Mitotic figures were rare and the labeling index of Ki-67 was <3%. Based on these clinical and histological findings, a PHAT was diagnosed.No recurrence or metastases were identified during the 6-year follow-up period.PHATs are non-metastasizing soft-tissue tumors of uncertain lineage occurring within the superficial subcutaneous tissues and muscles ,4. PreseDue to its rarity, PHATs may be misdiagnosed as other soft-tissue lesions. Malignant fibrous histiocytoma and schwannoma should be considered within the differential diagnosis, as cellular pleomorphism and ectatic, hyalinized blood vessels with infiltration by variable chronic inflammatory cells is present in those types of tumors –7. The cCurrently, PHATs are a benign condition according to the World Heath Organization classification  and therA previous study reported an association between ‘early PHAT’ and HFLL . An HFLLThe published case studies of PHATs have primarily discussed the pathological findings of the disease. To the best of our knowledge, only one study has described the imaging characteristics of this tumor on MRI . In the Due to the considerable potential for local recurrence, surgical excision with a tumor-free margin is the preferred treatment for PHAT . Low-dos


Tobacco smoking is a well-recognised risk factor for many diseases [1]. This study assesses the extent of smoking-attributable hospitalisation in the Northern Territory (NT) Aboriginal and non-Aboriginal populations, and examines smoking-attributable hospitalisation trends for the years 1998/99 to 2008/09.Hospital discharge data were used for the analysis. The proportion of conditions attributable to tobacco smoking was calculated using the aetiological fraction method. Age-adjusted smoking-attributable hospitalisation rates were calculated to describe the impact of tobacco smoking on the health of Territorians. A negative binominal regression model was applied to examine trends in smoking-attributable hospitalisations.Aboriginal Territorians were found to have higher rates of smoking-attributable hospitalisation, with Aboriginal males more than three times and Aboriginal females more than four times more likely to be hospitalised for smoking-attributable conditions than their non-Aboriginal counterparts. The age-adjusted hospitalisation rate for Aboriginal males increased by 31% and for Aboriginal females by 18% during the study period. There were more modest increases for NT non-Aboriginal males and females (5% and 17% respectively). The increase among Aboriginal males occurred up until 2005/06 followed by moderation in the trend. There were small reductions in smoking-attributable hospitalisation rates among all populations in younger age groups (less than 25 years).Aboriginal Territorians experience much higher smoking-attributable hospitalisation rates than non-Aboriginal Territorians. The scale of the smoking burden and suggestion of recent moderation among Aboriginal men reinforce the importance of tobacco control interventions that are designed to meet the needs of the NT’s diverse population groups. Preventing smoking and increasing smoking cessation rates remain priorities for public health interventions in the NT. Tobacco smoking is a well-recognised risk factor for respiratory and cardiovascular diseases and many cancers . It contTo assist health promotion efforts aimed at reducing smoking prevalence in the NT, and to help reduce the excess burden attributable to tobacco smoking, up-to-date information on the effects of smoking on morbidity is needed. The aim of this study is to estimate the extent and time trend of smoking-attributable hospitalisations in NT Aboriginal and non-Aboriginal populations for the period 1998/99 to 2008/09.Inpatient de-identified unit record level discharge data for NT public hospitals were obtained from the NT Department of Health for the period from 1 July 1998 to 30 June 2009 for persons who were residents of the NT. Data were coded using the International Classification of Diseases and Related Health Problems, 10th revision (ICD-10-AM) . The infNT population estimates were based on 2006 Census data from the Australian Bureau of Statistics (ABS) .The estimation of attributable risk of tobacco smoking in causing lung cancer was first introduced by Morton Levin. in 1953 and it formed a foundation of the aetiological fraction method . It is aSmoking-attributable conditions included in this study are partially based on the Australian Burden of Disease and Injury (ABOD) report . In the The relative risks for smoking-attributable conditions used in this study are largely based on English et al . with suFor conditions in which tobacco smoking is a contributory cause, estimates of the relative risk are combined with smoking prevalence data to derive the aetiological fraction, according to the formula: whereAF = aetiological fractione = proportion of smokers in a populationpRR = relative risk of developing a disease or condition For a small number of conditions  directly derived aetiological fractions were used.Data on smoking prevalence by age and sex for Aboriginal Territorians were obtained from the Australian Bureau of Statistics, 2004/05 National Aboriginal and Torres Strait Islanders Health Survey , and forStata software  was used for all statistical analyses. Annual smoking-attributable hospitalisation rates by sex and Indigenous status were calculated for the years 1998/99 to 2008/09 and age-adjusted using the 2001 Australian estimated resident population.The annual change in smoking-attributable hospitalisation rates by sex and Indigenous status were estimated by means of negative binomial regression models, in preference to Poisson regression, to adjust for data overdispersion. Age-specific hospitalisation rates and time trends were further calculated to determine variations within these rates between different age-groups and different periods.The study was approved by the Human Research Ethics Committee of the NT Department of Health and Menzies School of Health Research (HREC-2010-1335).The number and proportion of NT smoking-attributable hospitalisations during the years 1998/99 and 2008/09 are presented in Table Across all years, the age-adjusted smoking-attributable hospitalisation rate in the NT Aboriginal population was 285 per 10,000 population, three times higher than the rate for non-Aboriginal people . The rate among NT Aboriginal males was 330 per 10,000 and among NT Aboriginal females, 252 per 10,000. Among NT non-Aboriginal males the age-adjusted rate was 97 per 10,000 and 62 per 10,000 among non-Aboriginal females.Comparison of smoking attributable hospitalisation rates was made between Territorians aged under 25 years and Territorians aged 25 years and above  for the years from 1998/99 to 2008/09, compared to an increase of 0.8%  among Aboriginal females. For non-Aboriginal males we observed a decrease in annual average smoking-attributable hospitalisation rates of 0.7% . No meaningful trend was observed for non-Aboriginal females .For Territorians aged under 25 years, there was a decrease in annual average smoking-attributable hospitalisation rates among both Aboriginal and non-Aboriginal people. For young Aboriginal people this decline was mainly driven by a decrease in lower respiratory disease. For non-Aboriginal Territorians aged under 25 years, the conditions behind the decline were antepartum haemorrhage, followed by asthma and otitis media. Table The most common cause of smoking-attributable hospitalisations for Aboriginal Territorians was chronic obstructive pulmonary disease  followed by Ischaemic heart disease IHD, 14.5%). For non-Aboriginal Territorians COPD (24.3%), IHD (16.2%) and low birth weight (8.3%) were the most common causes of smoking-attributable hospitalisations (Table .5%. For Of the ten most common conditions the greatest increase in hospitalisations during the study period was for lung cancer, which increased more than three times among Aboriginal Territorians and by almost three times among non-Aboriginal Territorians. Among non-Aboriginal people smoking-attributable hospitalisations for asthma almost halved and also decreased for low birth weight.Despite vigorous anti-smoking campaigns initiated by both government and non-government agencies, Territorians experience much higher smoking-attributable hospitalisation rates than other Australians ,16. ThisOf specific concern from this study is that despite focus in the NT on improvements in antenatal care there was only a marginal decrease of smoking-attributable hospitalisation rates among Aboriginal females aged under 25 years between 1998/99 and 2008/09. This is of particular concern as these young women may be pregnant or raising children. Smoking in pregnancy is known to be a major risk factor for preterm delivery, complications in childbirth, fetal growth restriction, stillbirth, low birth weight and infant mortality [The high smoking prevalence in the NT, particularly among the Aboriginal population poses an ongoing challenge, not only for health and health services, but also economically. There is clear evidence for the benefit of health promotion interventions targeted at prevention, legislation and smoking cessation, preventing smoking and increasing cessation rates and these need to remain priorities for public health professionals. However, the differential impact of such interventions for Aboriginal people remains poorly understood . Higher Because factors that contribute to smoking prevalence occur at both, the community level, as well as at an individual level ,22, a coThere are a number of recognised limitations to this study. Firstly trends in hospitalisation rates should be interpreted cautiously, as they are influenced by changes in admission practices and disease management. Secondly, data on admissions to the single NT private hospital were not available at the time of this study. While most hospital services to Aboriginal Territorians are provided by public hospitals , the absThere are also limitations to the measures of prevalence used in this study. Apart from differences in survey methods, factors such as age at which smoking began, duration of smoking, the number of cigarettes smoked per day, nicotine content or filter type, and different behaviours influence the results of surveys in smoking prevalence . Due to Aboriginal Territorians experience much higher smoking-attributable hospitalisation rates than non-Aboriginal Territorians. The scale of the smoking burden and suggestion of recent moderation among Aboriginal men reinforce the importance of tobacco control interventions that are designed to meet the needs of the NT’s diverse population groups. Preventing smoking and increasing smoking cessation rates remain priorities for public health interventions in the NT.The authors declare that they have no competing interests.SLMPhas made substantial contributions to the design of the study, analysis and interpretation of the data, drafting the manuscript. SQL has made substantial contributions to the design of the study, supervised the data analysis and interpretation of the results, drafting the manuscript. SLG has made substantial contributions to the design of the study, interpretation of the result and editing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/545/prepubAppendix A. Causes of death and principal diagnoses identified as tobacco-related conditions.Click here for file


Processes such as cell proliferation, angiogenesis, apoptosis, or invasion are strongly influenced by the surrounding microenvironment of the tumor. Therefore, the ability to change these surroundings represents an important property through which tumor cells are able to acquire specific functions necessary for tumor growth and dissemination. Matrix metalloproteinases (MMPs) constitute key players in this process, allowing tumor cells to modify the extracellular matrix (ECM) and release cytokines, growth factors, and other cell-surface molecules, ultimately facilitating protease-dependent tumor progression. Remodeling of the ECM by collagenolytic enzymes such as MMP1, MMP8, MMP13, or the membrane-bound MT1-MMP as well as by other membrane-anchored proteases is required for invasion and recruitment of novel blood vessels. However, the multiple roles of the MMPs do not all fit into a simple pattern. Despite the pro-tumorigenic function of certain metalloproteinases, recent studies have shown that other members of these families, such as MMP8 or MMP11, have a protective role against tumor growth and metastasis in animal models. These studies have been further expanded by large-scale genomic analysis, revealing that the genes encoding metalloproteinases, such as MMP8, MMP27, ADAM7, and ADAM29, are recurrently mutated in specific tumors, while several ADAMTSs are epigenetically silenced in different cancers. The importance of these proteases in modifying the tumor microenvironment highlights the need for a deeper understanding of how stroma cells and the ECM can modulate tumor progression. Genetic alterations in tumor cells are essential for tumor development but not sufficient to generate malignant tumors. The tumor stroma resulting from an evolving crosstalk between tumor cells and different host cell types is required to create a permissive environment for the invasion of genetically altered tumor cells  and the associated cell-surface receptor (uPARAP/endo180) specifically involved in interstitial collagen remodeling. We also describe novel findings generated by the collaborative EU-FP7 funded network, MicroEnviMet (No. HEALTH-F2-2008-201279). This project has shed light on novel functions of membrane-associated MMPs in the control of cell apoptosis and angiogenesis, as well as on the complex tumor-host interplay in which proteinases can either boost cancer progression or protect the host against malignancy.It is now recognized that proteinases contribute to all stages of tumor progression  and are produced not only by the tumor cells themselves, but mainly by the different non-malignant host cells composing the tumor. Among the different classes of proteinases implicated during different stages of cancer progression, the MMPs constitute a family of 24 human zinc-binding endopeptidases that can degrade virtually all ECM components and have a growing number of substrates belonging to all important families of cell regulators: integrins, cell-surface receptors, kinases, chemokines, and cytokines  are composed of three polypeptides α-chains (homotrimers or heterotrimers) assembled into a triple-helical structure forming the collagenous domain. The N-terminal non-collagenous domain of these fibrillar collagens is proteolytically removed by ADAMTS-2  is associated with focal expression of MMP13 mRNA in stromal fibroblasts  has emerged as an important collagenase that cancer cells use to degrade and invade in a collagen-rich environment , lipoprotein receptor-related protein, proTNF-alpha, and the aggrecanase ADAMTS-4  has been established to elucidate the in vivo functions of ADAMTS-12  are applied to these knock-out mice. In the absence of ADAMTS-12, both the angiogenic response and tumor invasion into host tissue are increased. This finding is in line with the anti-angiogenic functions reported for other ADAMTS family members such as ADAMTS-1, ADAMTS-2, and ADAMTS-8  to be targeted in each case. Discerning which MMP(s) to target and when to inhibit are major issues that are facing researchers in the field. In addition, the design of highly selective MMP inhibitors is mandatory to overcome the failure of broad spectrum MMP inhibitors in clinical trials (Table The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.