The aims of this study were to examine the alternation in serum exosome concentrations and the levels of serum exosomal miR-9 and miR-124, two brain-specific miRNAs, in acute ischemic stroke (AIS) patients and to explore the predictive values of these miRNAs for AIS diagnosis and damage evaluation. Sixty-five patients with AIS at the acute stage were enrolled and 66 non-stroke volunteers served as controls. Serum exosomes isolated by ExoQuick precipitations were characterized by transmission electron microscopy, nanoparticle-tracking analysis and western blotting. The levels of exosomal miR-9 and miR-124 were determined by real-time quantitative PCR. Compared with controls, the concentration of serum exosomes and the median levels of serum exosomal miR-9 and miR-124 were significantly higher in AIS patients (p<0.01). The levels of both miR-9 and miR-124 were positively correlated with National Institutes of Health Stroke Scale (NIHSS) scores, infarct volumes and serum concentrations of IL-6. The areas under the curve for exosomal miR-9 and miR-124 were 0.8026 and 0.6976, respectively. This proof of concept study suggests that serum exosomal miR-9 and miR-124 are promising biomarkers for diagnosing AIS and evaluating the degree of damage caused by ischemic injury. However, further studies are needed to explore the potential roles of the exosomes released from brain tissues in post stroke complications. Stroke is a leading cause of death and long-term adult disability worldwide, and ischemic strokes account for more than 80% of stroke cases. Ischemic stokes occur when a vessel supplying blood to the brain is blocked. Early and accurate diagnosis of ischemic strokes is of critical importance for clinical therapy and would greatly improve the outcome and prognosis . In the MicroRNAs (miRNAs), which are endogenous small (~22nt) single-stranded non-coding RNAs, play important roles in regulating various biological processes by complementarily binding to 3`untranslated regions (UTRs) of targeted mRNAs . In addiExosomes are 30–100nm membrane-derived vesicles released by most cell types and exist in almost all body fluids. Exosomes contain a variety of proteins, nucleic acids and lipids derived from their host . These sMiR-9 and miR-124 are two brain-specific miRNAs that are highly expressed in the developing and adult vertebrate brain . They arThe ethics committee of the Affiliated Hospital of Nantong University approved this research protocol, and participants or their legally authorized representatives provided written informed consent.AIS patients were recruited among those admitted to the Department of Neurology of the Affiliated Hospital of Nantong University between September 2014 and August 2015. Either magnetic resonance imaging (MRI) or computed tomography (CT) imaging of the brain confirmed ischemic stroke. Experienced neurologists determined the neurological deficits by using the NIHSS within 24 hours after the stroke onset. Patients with intracerebral hemorrhage or unknown etiology were excluded. The mean time of enrollment blood draw was 16.5 hours. Non-stroke controls were recruited from those who underwent an annual medical examination at our hospital. The demographics and clinical characteristics of the 65 AIS patients and 66 non-stroke controls are provided in MRI was performed with a 3.0-T clinical whole-body MR scanner. The edge of the hyperintense signal on each slice was manually outlined, and the volume was determined by multiplying the area of diffusion hyperintensity by the slice thickness plus the interslice gap. The total lesion volume was calculated by summing the lesion volume on each slice. Two independent observers measured the DWI lesion volumes, and the results were averaged for further analysis.Five milliliters of venous blood were collected into plain tubes, allowed to clot at 37°C for 30 min and refrigerated at 4°C for 2 h, and then were centrifuged at 1,600 g for 10 min. Hemolysis samples were excluded at this step. Clear serum was transferred to fresh tubes and centrifuged at 20,000 g for another 10 min at 4°C. This step was used to remove cell debris, apoptotic bodies and blood platelets that were packed with large amounts of miRNAs , 12. TheTo isolate exosomes, -80°C stored serum was thawed on ice and centrifuged at 21,000 g for 15 min at 4°C, and the supernatant was transferred to a new tube. According to the recommended protocol, a one-fourth volume of ExoQuick Solution  was added and incubated at 4°C for 2 hours after brief up-and -down mix. Then, the mixture was centrifuged at 1,500 g for 30 minutes and the supernatant was removed by careful aspiration. Centrifugation for another 5 min was performed to remove the residual liquid. The exosome-containing pellet was subsequently re-suspended in nuclease-free water.8 exosomes/ml, and they were then injected into the NanoSight sample chamber. The size distributions and vesicle concentrations were assessed with Nanoparticle Tracking Analysis (NTA) software.We used a NanoSight NS300 Instrument with a 405-nm laser  to analyze the size distribution and quantify the exosome preparations. Isolated exosomes were diluted 1,000 times in particle-free PBS to obtain approximately 5 × 10For TEM morphology investigation, samples were prepared as described by Thery et al. . BrieflyExosomal protein was extracted with RIPA lysis buffer . The total protein concentration was quantified with a BCA protein assay kit . Approximately 40 μg of total protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked for 1 h at room temperature with 5% bovine serum albumin, then immunoblotted with anti-CD9, anti-CD81 , and anti-CD63 antibodies . The proteins were detected by using enhanced chemiluminescence .miRNA qRT-PCR kits, containing miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer, were purchased from Riobio . Total exosome RNAs extracted from 250 μl serum were polyadenylated and reverse-transcribed with a poly (T) adapter into cDNAs. The expression of miR-124 and miR-9 was detected by using Perfect Real Time kit  and a CFX96 PCR system  according to the manufacturer’s instructions and all reactions were run in triplicate.Total RNA was extracted from the serum exosomes using an Exosome RNA Purification Kit  according to the manufacturer’s protocol. Twenty-five fmol synthetic cel-miR-39  was added as a spike-in control. The concentration and integrity of isolated RNA were evaluated with a Bioanalyzer 2100 instrument . miDETECT A Track™ normalized = Target raw—(Control raw—Control median run), where “Target” is the miRNA to be normalized; “Control” is cel-miR-39, the mean Cq of cel-miR-39; “raw” is the instrument Cq result for that sample, and “median run” is the median Cq of all 131 raw results for the mean Cq of cel-miR-39.The raw qPCR data of miRNAs were normalized to spiked cel-miR-39 as described previously . Target The serum concentration of IL-6 was measured with a high-sensitivity ELISA kit . The recommended serum concentration of IL-6 in non-stroke individuals is less than 6.4 pg/ml. Values below the detection limit (0.08 pg/ml) were assigned a value equal to half the detection limit.Statistical analysis was performed by using GraphPad Prism 5 . Categorical variables expressed as numbers and percentages were obtained for demographic data, and a chi-square test was used to compare categorical variables between groups. Continuous variables are presented as medians (with the interquartile range), and a Wilcoxon test was used to compare continuous variables. Correlations between the variables were assessed by using the Spearman's correlation coefficient. Receiver-operating-characteristic (ROC) curves were constructed to assess the sensitivity and specificity of the miR-9 and miR-124 measurements and to compare their ability to diagnose acute ischemic stroke. All hypothesis testing was two-tailed, and p-values of less than 0.01 were considered to be statistically significant.Obtaining a sufficient amounts of "real" exosomes is a prerequisite for an exosome study. Ultracentrifugation remains the golden standard for exosomes isolation; however, owing to the limited volume of serum and the lower yield after repeated ultracentrifugation, we chose ExoQuick, which is a method based on precipitation, to isolate serum exosomes . AccordiConsistently with the characteristics of exosomes , precipiSerum microvesicles (MVs) concentrations are modulated by multiple factors, such as inflammation and injury ; howeverNext, the relative abundance of serum exosomal miR-9 and miR-124 was detected by using real time qPCR. Because there is a lack of housekeeping genes that are suitable for use as normalization control in studies of circulating exosomal microRNAs, we established a practical workflow to ensure that the PCR data obtained from different experiments and individuals were comparable. First, total RNA was extracted from exosomes precipitated from the same volume serum (250 μl) and was eluted in the same volume of RNase free water. The same volume of RNA solution was then used for 3`-extension with a poly (A) tail and subsequent reverse transcription and qPCR. Second, 25 fmol of Cel-miR-39 was spiked in as an external control and for the following data normalization. Finally, in our initial experiment we determined the relative stability of miR-451, an abundant microRNA present in red blood cells, which was monitored as an internal control.As illustrated in To determine the diagnostic value of miR-9 and miR-124 in the acute stage of ischemic stroke, the correlations between the levels of these two microRNAs and NIHSS scores and infarct volume were examined. Consistently with results from previous report , NIHSS sIL-6 is a pro-inflammatory cytokine that is released early after the onset of brain injury. The level of serum IL-6 can reflect the degree of brain ischemic damage and correlates with stroke lesion volume –20. To fTo evaluate the diagnostic value of exosomal miR-9 and miR-124 for AIS, ROC curves were generated to discriminate AIS patients from non-stroke controls. The areas under the curve (AUC) for exosomal miR-9 and miR-124 were 0.8026 (95% CI: 0.7235–0.8816) and 0.6976 (95% CI: 0.6506–0.7895), respectively. These findings suggested that both miR-9 and miR-124 may be sensitive biomarkers that can identify the AIS patients from non-stroke individuals  and miR-In the present study, we successfully isolated and characterized serum exosomes from AIS patients and explored the effects of acute ischemic brain injury on serum exosome concentrations and levels of serum exosomal miR-9 and miR-124, two brain-specific microRNAs. Compared with non-stroke controls, in patients with acute ischemic injury, we found that both the concentrations of serum exosomes and the levels of serum exosomal miR-9 and miR-124 were significantly elevated. Furthermore, this increase was coordinated with NIHSS scores, infarct volume and the overexpression of pro-inflammatory cytokine IL-6, thus suggesting a close relationship between the levels of serum exosomal miR-9 and miR-124 and the degree of brain damage. To our knowledge, this is the first study exploring the serum exosomal miRNAs in AIS patients.The number of MVs released by many cell types often increases after cellular activation, and is considered to be a novel biomarkers for neurological disorders . Using fOwing to their high stability, relative tissue specificity, association with disease states, presence in readily detectable concentrations, ease of manipulation and measurement sensitivity, circulating miRNAs are considered to be promising sources for the discovery of sensitive and specific biomarkers of a broad spectrum of diseases . A numbeMiR-124 is the most abundant miRNA in the brain and is expressed in almost all brain regions . MiR-9 iCirculating miRNAs exist in two different package forms, either being encapsulated in microvesicles (exosomes and shedding vesicles) or being associated with proteins of Argonaute family or HDL particles. In 2007, Veladi and colleagues demonstrated that miRNA and mRNA can be exchanged between cells by exosomes and subsequently exert their particular biological functions , but theGiven the important roles of exosomes in mediating intercellular communication, and the functions of miR-9 and miRNA-124 in anti-inflammatory action by inhibiting the production of pro-inflammatory cytokines , 38, exoAltogether, in this proof of concept study, our data revealed significantly elevated levels of serum exosomal miR-9 and miR-124 in AIS patients, and they are promising biomarkers for diagnosing AIS and evaluating the degree of damage caused by the ischemic injury. However, multicenter studies are needed to confirm the specificity and sensitivity of these circulating miRNAs. In addition, it will be worthwhile to explore the potential biological effects of these sudden intruding brain-derived exosomes, which transfer information from the central nervous system to the peripheral system, and their involvement in post-stroke complications.S1 Fig(TIFF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.


Osteosarcoma, a bone cancer most commonly seen in adolescents and young adults, is usually a high-grade malignancy characterized by a very high risk for the development of pulmonary metastases. High-grade osteosarcomas are usually treated by preoperative and postoperative chemotherapy and surgery, with a very limited number of active agents available. Rarer lower-grade variants such as parosteal and periosteal osteosarcoma or low-grade central osteosarcoma are treated by surgery only. Imaging to search for possible metastases focuses on the lung. Computed tomography is the most sensitive method but cannot reliably distinguish small metastases from benign lesions. Advances of local imaging and surgical reconstruction now allow the use of limb-salvage in an ever-increasing proportion of patients. While still troubled by complications, non-invasive endoprosthesis-lengthening mechanisms have led to an increased uptake of limb-salvage, even for young, skeletally immature patients. Radiotherapy is employed when osteosarcomas cannot be removed with clear margins, but very high doses are required, and both proton and carbon-ion radiotherapy are under investigation. Unfortunately, the past 30 years have witnessed few, if any, survival improvements. Novel agents have not led to universally accepted changes of treatment standards. In patients with operable high-grade osteosarcomas, the extent of histological response to preoperative chemotherapy is a significant predictive factor for both local and systemic control. Attempts to improve prognosis by adapting postoperative treatment to response, recently tested in a randomized, prospective setting by the European and American Osteosarcoma Study Group, have not been proven to be beneficial. Many agree that only increased knowledge about osteosarcoma biology will lead to novel, effective treatment approaches and will be able to move the field forward. Unfortunately, the only time period since then during which significant prognostic gains were achieved was from the late 1970s until the early 1980s, when combining multi-agent chemotherapy with surgery revolutionized treatment2. Unfortunately, the decades since have witnessed no further improvements of survival in North America3 or Europe6. Nevertheless, there have been numerous advances in the management of osteosarcoma which merit review and discussion.A 1.6–1.8-million-year-old hominin metatarsal from the South African Swartkrans paleoanthropological cave site makes osteosarcoma the earliest documented cancer of humankind10 and 20–30% for patients with primary metastases or axial primaries11. Most patients are treated using a neoadjuvant approach, and histologic response to preoperative chemotherapy has emerged as an independent prognostic indicator7. While combined preoperative and postoperative chemotherapy has never been shown to provide survival benefits over adjuvant chemotherapy alone (as long as both contain the same cumulative doses)12, it offers time to prepare for surgery and allows anin vivo evaluation of the effects of systemic treatment. These may be estimated by a variety of imaging methods, but histologic assessment for the proportion of viable tumor remaining at surgery is the gold standard. Patients whose primaries respond well to chemotherapy, usually defined as <10% tumor viability, generally suffer fewer local13 and systemic10 recurrences and achieve greater survival probabilities7 than others. This manuscript will try to highlight recent advances achieved in this context of first-line treatment.Osteosarcoma is a rare bone cancer which mainly affects adolescents and young adults. Though lower-grade variants exist, most are high-grade malignancies with a high propensity for lung metastases. Current standard treatment consisting of surgery plus chemotherapy leads to long-term, disease-free survival in approximately 60% of patients with localized extremity disease14, and its role remains to be defined. The importance of accurate imaging at initial diagnosis and after preoperative chemotherapy, however, cannot be overstated. A detailed review of local imaging would be beyond the scope of this article and the reader is referred to the recent literature19.Imaging of bone sarcomas was revolutionized by magnetic resonance imaging (MRI), which, for the first time, allowed detailed assessment of tumor extent within the bone marrow cavity and into soft tissues, as well as its relation to surrounding structures such as joints, nerves, and vessels. In addition, MRI may also be used to predict histologic tumor response to preoperative chemotherapy, as may positron emission tomography (PET)/computed tomography (CT), sequential bone scans, and others. PET/MRI has entered the scene more recently20. Unfortunately, even modern CT scanning cannot reliably discriminate small lung metastases from small benign lesions. A recent study of 283 CT-identified lesions which led to 123 thoracotomies in 70 osteosarcoma patients found 234 of the lesions to be metastases. An additional 31, 14 of those metastases, were identified only upon thoracotomy. Lesion size ≥6 mm was suggestive for metastases, but many smaller lesions were also malignant. While most metastases were nodular and calcified, atypical findings were common21. This analysis again confirms that chest CT has its limitations. Given the dire consequences associated with incomplete resection of metastatic osteosarcoma11, we believe that any lung lesion detected by CT should be viewed with a high index of suspicion and treated as if it could be a metastasis. However, as highlighted by unrelated surveys among leading study groups22 and members of the Connective Tissue Oncology Society23, the jury is still out and considerable variability surrounds the management of pulmonary lesions.Chest CT remains the gold standard for imaging lung metastases99mTechnetium bone scans have long been part of the standard diagnostic workup. Some years ago, whole-body MRI with short time inversion recovery (STIR) imaging was found to be more sensitive for detecting bone metastases in children with suspected multifocal bone lesions than bone scans, but also less specific24. Similar observations were made for PET/CT: in a recent series of 39 osteosarcomas investigated by 40 paired bone scans and PET/CTs and of whom five had bone metastases, PET/CT detected all, while bone scans missed two. On the other hand, three PET/CTs were falsely positive25. It seems that histologic confirmation with a biopsy is often required before an osseous lesion suspected by whole-body STIR–MRI or PET/CT is considered a true bone metastasis but that bone scans will usually not detect additional lesions in patients investigated by either of those techniques.A minority of osteosarcomas will present with synchronous bone metastases;26. CNB does not seem to be nearly as reliable in telangiectatic osteosarcoma: in one series, nine of 26 were misdiagnosed as aneurysmal bone cysts27.Osteosarcoma must be confirmed histopathologically before initiating tumor-directed therapy. Biopsies were traditionally performed via incisional procedures. Even though scientists may lament a paucity of tissue for research, less invasive core needle biopsies (CNBs) are now assuming an ever-increasing role. These have been shown to be very effective as long as adequate cores can be sampled. A French analysis of CNB in 73 osteosarcomas reported an overall sensitivity of 93.1%, specificity of 100%, and positive and negative predictive values of 100% and 99.9%, respectively, as long as the specimen was adequate28 remains the mainstay of curative local therapy. Spurred by major advances of imaging and of surgical reconstruction opportunities, recent years have witnessed a major shift from amputations towards limb-saving procedures29. Limb-salvage, however, poses challenges, particularly in growing individuals. Earlier models of expandable endoprostheses required additional surgery for every lengthening. Various non-invasive lengthening mechanisms are now available, including incorporated engines or magnetic devices30. However, these are still associated with frequent complications and needs for revisions33. Several papers emphasize that further technical advances are direly needed: in one series, 10 patients experienced 37 implant-related complications31, and in another, 42% of 38 patients experienced complications, including 10 prosthesis revisions and two amputations34. A third reported an average 2.5 revisions for complications in 71 patients32; a fourth even questioned whether complications associated with a particular, rather popular, expandable prosthesis were acceptable for its continued use33. Given that such devices are obviously still immature, one might look back to the bygone age of rotationplasties with (never expected) nostalgia. Their function remains quite good even with long observation periods. A recent Italian series evaluated 25 patients living with rotationplasties for a mean of 15 years35. While arthritis of the tibiotalar, subtalar, and talonavicular joints was radiographically present in most, they showed improved gait parameters as adults compared with previously reported findings for children with rotationplasty35.Surgery with wide margins37. Results of a meta-analysis suggest that debulking may no longer be required when radiation doses of 70 Gy or higher are administered. Local control probabilities after radiotherapy were lower for craniofacial osteosarcomas than for those of other sites37.Any osteosarcoma that can be operated on should be operated on to maximize the chance for local control and hence survival. However, not all osteosarcomas are operable. Several series have confirmed that selected patients may achieve permanent local control with radiotherapy, particularly if this is combined with effective chemotherapy and gross total resection37. Among 78 patients with inoperable osteosarcoma of the trunk irradiated with a median of 70.4 Gy carbon-ion radiotherapy (CIRT) by Japanese investigators, the 5-year local control rate was 62%38. Osteosarcomas were also included in an array of sarcomas of the spine39 or extremities40 treated with CIRT. While the observed results were also encouraging, further research is required before such techniques can be considered standard. A systematic review of clinical outcome studies published between 2007 and 2015 concludes that there is insufficient evidence on the long-term effectiveness and harm of protons to either support or refute their use in children with osteosarcoma or basically any other pediatric cancer41.The high radiation doses required to sterilize osteosarcoma are difficult to achieve with conventional techniques, so that proton and heavy-ion radiotherapy have come into focus. In probably the largest series of 55 osteosarcomas treated with protons, the mean total radiation dose was 68.4 Gy. At 5 years, the local control rate was 72% and overall survival was 67%42. The MAP combination of HD-MTX, doxorubicin, and cisplatin45 is frequently used, but similar results have been achieved with other protocols employing several of the mentioned agents10. A meta-analysis of published osteosarcoma trials concluded that using three of the drugs led to better results than using only two but that administering all four did not lead to further improvements46.Systemic therapy for osteosarcoma has changed very little for over 30 years and still relies on varying combinations incorporating several of the same four “old” drugs, namely high-dose methotrexate (HD-MTX), doxorubicin (Adriamycin), cisplatin, and ifosfamide43 as well as a second44 concluded that there was no benefit of adding ifosfamide. The two papers differed in their conclusions regarding L-MTP-PE: while the first stated that analysis was prevented by an interaction between ifosfamide and L-MTP-PE43, the authors no longer detected a statistically significant interaction 3 years later and decided to examine each intervention separately, as originally planned. They now reported a non-significant trend toward better event-free survival (EFS) and improved overall survival with L-MTP-PE44. Commentators voiced interaction concerns and questioned whether INT0133’s results met generally accepted standards for practice-changing conclusions. They called for additional clinical evaluations to define the role of the drug and to demonstrate whether any potential benefit requires concurrent use of ifosfamide47. We along with others have also argued for additional randomized comparative evaluation to substantiate the utility of the drug48. Since then, however, little new evidence concerning its potential efficacy has emerged. Results from the metastatic cohort of INT0133 pointed in the same direction as in non-metastatic patients but were not statistically significant49. Come 2016, there is additional evidence that L-MTP-PE has a favorable safety profile: a patient access study of 200 patients reported 3,679 infusion-related adverse events after 7,482 infusions, commonly chills, fever, headache, and fatigue, but only rarely severe50. However, there have been no further trials which shed more light upon the potential efficacy of the drug, so uncertainties remain regarding its potential role.Several prospective trials have attempted to introduce additional agents for either all patients or certain risk groups. Some years ago, the prospective randomized INT0133 trial addressed two potential additions to MAP using a randomized two-by-two factorial design: the cytotoxic agent ifosfamide and the macrophage activator liposomal muramyl tripeptide phosphatidylethanolamine . A first publication51), interferon alpha-2b, was investigated in the largest prospectively randomized osteosarcoma study to date, the European and American Osteosarcoma Study Group (EURAMOS)-1 trial53. A total of 716 patients whose resectable localized or primary metastatic osteosarcomas responded well to preoperative MAP were randomized after surgery to four additional cycles of MAP either with or without maintenance pegylated interferon alpha-2b53. Of 357 patients randomized to receive the study drug, 271 actually started, of whom 105 stopped early. As expected, for patients whose osteosarcomas responded well to chemotherapy, 3-year EFS for all randomized patients was favorable at 76%. The hazard ratio from an adjusted Cox model was 0.83, but the 95% confidence interval (CI) included 1, meaning that MAP plus interferon alpha-2b was not statistically different from MAP alone. Interpretation of the data is, of course, complicated to a certain extent by the relevant proportion of patients who never started or who prematurely stopped interferon alpha-2b. Nevertheless, the results do not argue for its inclusion in standard osteosarcoma treatment53.Another drug with immunological properties  was investigated in the poor responder cohort of EURAMOS-152. In this trial, MAPIE patients were to receive an additional three courses of 14.000 mg/m2 ifosfamide with 500 mg/m2 etoposide and two courses of ifosfamide at 9.000 mg/m2 added to doxorubicin. MAPIE lasted 11 weeks longer than MAP. The study sought to detect absolute improvements of 10% from 45% to 55% in 3-year EFS and 5-year overall survival (hazard ratio 0.75)56. Of 618 randomized patients, 310 were allocated to postoperative MAP and 308 to MAPIE; 3-year EFS rates were 55% (95% CI 49–60) and 53% (95% CI 47–59), respectively. MAPIE was more toxic and fewer patients received their intended chemotherapy doses. MAPIE was also associated with higher risk of secondary malignancy, predominantly leukemia, mostly with cytogenetic abnormalities associated with the administration of alkylating drugs  or etoposide . Therefore, the EURAMOS consortium argues against adding ifosfamide and etoposide to the MAP backbone of MAP therapy for patients whose osteosarcoma shows a poor response to preoperative treatment56.Encouraging results with the combination of high-dose ifosfamide and etoposide were reported from phase II trials of primary or relapsed metastatic osteosarcoma57. A Scandinavian–Italian study investigating postoperative high-dose carboplatin/etoposide with ASCT involved 71 patients with primary metastatic or axial osteosarcoma, of whom 29 received two and 10 one course of HDCT; 5-year EFS and overall survival were 27% and 31%, respectively. When patients not receiving HDCT owing to disease progression were excluded, there were no differences in outcomes between patients who received HDCT or not58.Anyone arguing that the alkylator doses used in EURAMOS-1 were not sufficient and that high-dose chemotherapy (HDCT) with autologous blood stem cell transplant (ASCT) was a better idea should be duly cautioned by results from recent uncontrolled prospective trials: in an American study, 18 patients with newly diagnosed localized high-grade osteosarcoma and poor histologic response received HDCT/ASCT with melphalan and cyclophosphamide; 5-year EFS and overall survival were 28% and 48%, respectively59. Chemotherapy used in this trial varied by age. Among 318 patients, 55 with primary metastases, 160 were randomized to zoledronate. The trial was stopped for futility after the second planned interim analysis when 3-year EFS was 57% for the zoledronate group and 63% for controls (p=0.094)59. While the use of different chemotherapy backbones for different patients might confound interpretation to a certain degree, these results argue against zoledronate's ability to improve oncologic outcomes in osteosarcoma.A completely different drug, the bisphosphonate zoledronate, was investigated in the prospective, randomized French multi-center OS2006 trial, which asked whether 10 courses of zoledronate added to chemotherapy and surgery might improve EFSOS2006 as well as EURAMOS-1 exemplify that prospective randomized trials are essential to adequately assess whether treatments which show promise in the lab or in early phase studies will truly increase cure rates. They also demonstrate that such trials are feasible, even in very rare cancers such as osteosarcoma.In summary, there is currently no evidence whatsoever that altering postoperative treatment in patients whose osteosarcomas respond poorly to preoperative chemotherapy or that modifying standard systemic treatment for other reasons will lead to anything but additional side effects and risks. The use of such approaches should be limited to prospective trials and otherwise discouraged.60. Similarly, a Norwegian nationwide cohort which included 29 low-grade central osteosarcomas, four of those with areas of de-differentiation, reported 5-year sarcoma-specific survival of 93% and confirmed that low-grade osteosarcoma has an excellent prognosis when resected with a free margin61. These series suggest that low-grade osteosarcomas with small high-grade foci may still be treated by surgery only. The numbers are too small to draw conclusions for low-grade central osteosarcomas which contain larger high-grade areas.While multi-modal treatment consisting of surgery and chemotherapy is the undisputed treatment standard for patients with high-grade central (arising within the affected bone) osteosarcoma of the extremities or axial skeleton, some osteosarcoma variants deserve special consideration. As for all osteosarcomas, surgery should strive to achieve wide margins. The role of additional systemic treatment, however, varies: low-grade central osteosarcoma arises within bone, just like high-grade central osteosarcoma. These tumors may carry areas of de-differentiation, and the decision to employ chemotherapy is often made based on the most malignant component. An Italian series of 132 low-grade central osteosarcomas included 33 in which high-grade (grade 3) areas were detected in the resected specimen, and postoperative chemotherapy was given to 22 of these 33. High-grade areas accounted for less than 50% in 20/33, among whom only one in nine patients not receiving chemotherapy (unrelated causes) and one in 11 receiving chemotherapy (metastatic recurrence) died. Among 13 of the 33 patients with >50% grade 3 component, 12 received adjuvant chemotherapy, two had local recurrences, and four had metastatic recurrences. The only patient from this cohort treated by surgery only survived disease free8. The already-mentioned Norwegian series also included 20 parosteal osteosarcomas, eight with signs of de-differentiation, and reported 90% 5-year sarcoma-specific survival61. A retrospective British analysis of 80 parosteal osteosarcomas observed overall survival of 92% and 88% at 5 and 10 years, respectively. Local recurrences were associated with intralesional surgery, were de-differentiated in 80%, and were associated with inferior survival. The authors observed neither medullary involvement nor the use of chemotherapy to correlate with survival62. One may conclude that, similar to low-grade central osteosarcoma, adequate surgery is the treatment of choice for parosteal osteosarcoma, that it is imperative to avoid local failure, and that there is no proven role for chemotherapy.Parosteal osteosarcoma is a low-grade surface osteosarcoma which may also contain high-grade areas8. While sometimes treated with chemotherapy in addition to surgery, the currently available retrospective evidence suggests that treatment should be surgery only. Corroborating similar findings from a 2005 European survey63, an Italian series reported a 10-year overall survival of 84% for 33 patients, 14 of whom received chemotherapy. The authors did not find survival to be influenced by chemotherapy64.Periosteal osteosarcoma is a surface osteosarcoma of intermediate malignancy65.Craniofacial osteosarcomas carry a comparatively high local recurrence risk. The benefit of adding systemic treatments to surgery is not as well defined as for other sites, and no prospective data on adjuvant therapy has recently emerged. Nevertheless, current guidelines suggest using the same multimodal approach as for other high-grade osteosarcomas67. The aim of tumor-directed follow-up is therefore to detect local recurrences or metastases while surgery is still feasible68. Surveillance usually includes chest X-rays or chest CTs in addition to history, physical, and imaging of the former primary tumor site. The wide variability of surveillance protocols actually employed is exemplified by a Musculoskeletal Tumor Society (MSTS) survey, where the number of first-year surveillance visits ranged from three to six, chest X-rays from zero to three, chest CT scans from one to four, and X-rays of the former primary site from three to six69. Imaging guidelines developed by the Children’s Oncology Group (COG) suggest a schedule which heavily relies on repeated CT scanning of the lungs20. However, conventional chest CT is associated with considerable radiation exposure, which led to criticism of these guidelines70.Osteosarcoma recurrences may still be cured as long as they are operable68. A randomized follow-up study from India investigated 495 patients operated on for seemingly localized primary or recurrent extremity sarcomas (359 of these bone sarcomas). Chest X-ray was compared with CT scanning and 6-monthly with 3-monthly follow-up71. The authors concluded that chest X-rays were not inferior to CT scans in terms of detecting pulmonary metastases and did not lead to inferior survival; 3-year overall survival was 64% with 6-monthly and 69% with 3-monthly follow-up, respectively71.Several recent studies have attempted to lend more of an evidence base to osteosarcoma-directed follow-up. A retrospective single-center analysis of 101 patients with routine chest X-ray surveillance reported 34 recurrences. All eight local recurrences were noted clinically, and only two of all recurrences developed beyond 5 years. The authors propose more frequent surveillance visits during the first 2 years and chest X-ray instead of chest CT72, so this may change should further studies demonstrate benefits for this technique in the follow-up of bone sarcoma.Our interpretation of the currently available evidence is that routine follow-up for lung metastases can usually be performed with chest X-rays. Ultralow-dose CT, which limits radiation exposure to the equivalent of chest X-rays in two planes, has shown promise for lung cancer screening73, or to augment the usability of known effective agents by mitigating toxicities, exemplified by adding the cardioprotective agent dexrazoxane to increase doxorubicin exposure74, are ongoing. It would be very optimistic to expect anything but limited improvements from such approaches.Members of the generation of doctors who saw osteosarcoma cure rates rise within their professional lifetimes have by now reached retirement or are close. How do we move forward? The optimal “conventional” chemotherapy regimen remains to be defined, and efforts to identify additional effective cytotoxic combinations, as exemplified by the demonstration of activity for the gemcitabine/docetaxel combination78. As exemplified in 29 patients with refractory/relapsed osteosarcomas registered between 2009 and 2013 within the French Sarcoma Group–Bone Tumor Study Group (GSF–GETO) who received 33 treatment lines of targeted therapies, off-label use is already quite common79. Prospective trials will be essential to define their role or that of any other new treatments which may arise.Like in most other cancers, immunotherapy and the so-called targeted therapies are current hot topics80. Other trial designs and endpoints have been called for81, and COG has constructed baseline EFS outcomes – including 12% EFS at 4 months for patients with measurable recurrent or refractory disease – that could be used as comparators for future phase II trials80. EFS-based outcomes have already been employed in sequential phase II trials of sorafenib given alone82 or in combination with everolimus83 and have demonstrated some activity. Antagonists of the insulin-like growth factor type-1 receptor (IGF1R) are examples of other agents which have shown limited activity in several trials85; however, their development was more or less terminated after they failed in common cancers.Unfortunately, osteosarcoma tumor matrix often prevents capturing the effects of investigational treatments by conventional radiologic Response Evaluation Criteria In Solid Tumors (RECIST): lesions simply cannot shrink, even if the tumor cells are killed. Accordingly, a retrospective analysis of seven COG osteosarcoma phase II trials found all drugs inactive on the basis of radiographic response86. The identification of the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway as a central vulnerability for therapeutic exploitation and subsequent detection of responsiveness of osteosarcoma cell lines to PI3K/mTOR inhibition88 or the detection of BRCAness in a substantial subset of osteosarcomas89 with the subsequent demonstration of susceptibility of osteosarcoma cells with a BRCAness signature to poly(ADP-ribose) polymerase (PARP) inhibition90 may serve as current examples of preclinical endeavors which deserve clinical evaluation.Both sorafenib and IGF1R inhibitors were tested because the focus of osteosarcoma research has shifted towards gaining a better understanding of the driving forces behind tumor development and progression and then hypothesis-driven drug discovery and developmentIt can only be hoped for that not only will we manage to learn more about the biology of osteosarcoma but also this will lead to further steps towards its eradication. Only time, dedicated pre-clinical research, and well-designed clinical trials will tell.ASCT, autologous blood stem cell transplant; CI, confidence interval; CIRT, carbon-ion radiotherapy; COG, Children’s Oncology Group; CNB, core needle biopsy; CT, computed tomography; EFS, event-free survival; EURAMOS, European and American Osteosarcoma Study Group; HDCT, high-dose chemotherapy; HD-MTX, high-dose methotrexate; IGF1R, insulin-like growth factor type-1 receptor; L-MTP-PE, liposomal muramyl tripeptide phosphatidylethanolamine; MAP, methotrexate, Adriamycin, and cisplatin; MAPIE, methotrexate, Adriamycin, cisplatin, ifosfamide, and etoposide; MRI, magnetic resonance imaging; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; PET, positron emission tomography; STIR, short time inversion recovery.


Major depressive disorder (MDD) is a chronic condition that affects one in six adults in the US during their lifetime. The current practice of antidepressant medication prescription is a trial-and-error process. Additionally, over a third of patients with MDD fail to respond to two or more antidepressant treatments. There are no valid clinical markers to personalize currently available antidepressant medications, all of which have similar mechanisms targeting monoamine neurotransmission. The goal of this review is to summarize the recent findings of immune dysfunction in patients with MDD, the utility of inflammatory markers to personalize treatment selection, and the potential of targeting inflammation to develop novel antidepressant treatments. To personalize antidepressant prescription, a c-reactive protein (CRP)-matched treatment assignment can be rapidly implemented in clinical practice with point-of-care fingerstick tests. With this approach, 4.5 patients need to be treated for 1 additional remission as compared to a CRP-mismatched treatment assignment. Anti-cytokine treatments may be effective as novel antidepressants. Monoclonal antibodies against proinflammatory cytokines, such as interleukin 6, interleukin 17, and tumor necrosis factor α, have demonstrated antidepressant effects in patients with chronic inflammatory conditions who report significant depressive symptoms. Additional novel antidepressant strategies targeting inflammation include pharmaceutical agents that block the effect of systemic inflammation on the central nervous system. In conclusion, inflammatory markers offer the potential not only to personalize antidepressant prescription but also to guide the development of novel mechanistically-guided antidepressant treatments. Major depressive disorder (MDD) affects one in six adults in the United States during their lifetime ,2. For mThe current practice guidelines recommend prescribing antidepressant medications based either on clinical characteristics  or non-clinical factors  . This isLarge community trials such as the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial have shown that less than a third of patients adequately respond to the initial antidepressant medication trial . In factSeveral lines of investigation implicate inflammation in the pathophysiology of depression in a sub-group of patients with MDD ,33. PatiPatients with MDD also have elevated markers of non-specific inflammation, such as c-reactive protein (CRP). Produced by the liver, CRP is a pentameric protein that increases thousands of folds in response to acute infection or injury and is hence also referred to as acute-phase reactant . ChronicAmong specific inflammatory markers, elevated levels of interleukin 6 (IL-6) have been most consistently reported in patients with MDD as compared to healthy controls. Several meta-analyses have found elevated levels of IL-6 in peripheral circulation in depressed patients as compared to controls with moderate to large effect size ,46,47,48Recent evidence also implicates interleukin 17 (IL-17) in the pathophysiology of MDD. One of the downstream consequences of IL-6 elevation is the differentiation of native T-helper (Th) lymphocytes into IL-17 producing Th17 lymphocytes, thus promoting secretion of IL-17 . The rolChemokines have been implicated in depression by facilitating migration of peripheral immune cells into the central nervous system . A recenWhile the brain has been considered an immune-privileged organ, emerging evidence implicates the role of peripheral inflammation in the pathophysiology of depression . The facl-3,4-dihydroxyphenylalanine, the rate-limiting step of dopamine synthesis [The putative mechanisms underlying the role of IL-17 involves its effect on the BBB. Peripheral IL-17 binds to the IL-17 receptors on the BBB leading to the generation of reactive oxygen species (ROS), which in turn increase BBB permeability . Increasynthesis . Reducedynthesis ,72,73,74It is widely acknowledged that antidepressant treatments affect the immune system. A recent meta-analysis found significant reductions in IL-4, IL-6, IL-1β (specific only to SSRIs) and IL-10 after antidepressant treatment along with no changes in IL-2, TNF-α, IFN-γ, and CRP . These fBupropion, a dopaminergic noradrenergic antidepressant, has been shown to suppress the Th1-  and Th17Inflammatory biomarkers are poised for widespread application to profoundly change the current clinical practice. Systematic reviews have found that elevated inflammation predicts poor response to commonly used antidepressant medications . Two recWhile CRP is a clinically pragmatic biomarker for treatment assignment, its level may be elevated due to a multitude of acute and chronic factors. Thus, there is a need to identify more specific factors which can guide differential treatment selection among currently available antidepressant medications. Among specific inflammatory markers, IL-17 has emerged as a potential candidate. In two separate samples, Hennings et al. recently demonstrated that lower pre-treatment levels of ROR α mRNA, a transcription factor involved in differentiation of naïve CD4+ T cells into Th17 cells , were asn = 106). The rates of symptomatic remission  were significantly higher (p < 0.05) with brodalumab 140mg q2week (47%) and 210 mg q2week (43%) as compared to the placebo (9%) [n = 320), defined as scores ≥ 11 on the Quick Inventory of Depressive Symptomatology Self-Report [p < 0.01) than the remission rate of 17.8% with placebo [While personalizing the prescription of currently available antidepressant medications can improve clinical outcomes for over half the patients with MDD in 12 weeks , novel aebo (9%) . Notablyf-Report . Remissi placebo . However placebo ,104,105.Future studies are needed to test the superiority of inflammatory marker-based antidepressant prescription relative to the current practice of clinical decision-making. A CRP-matched treatment assignment offers the most pragmatic choice in this regard . In indiIn the search for novel antidepressants targeting inflammation, l-leucine, an essential amino acid is also a substrate for the LAT-1 transporter, harmful effects of peripheral inflammation can be mitigated by oral administration of l-leucine. A pilot double-blind placebo-controlled study is currently underway to test this hypothesis (NCT03079297).A parallel approach, as outlined by Remus et al., targets transport of kynurenine at the BBB to reduce the effect of peripheral inflammation on the central nervous system. This approach is based on the earlier work by Dantzer et al., which showed that peripheral inflammation leads to the induction of IDO, which in turn diverts tryptophan away from the synthesis of serotonin to that of kynurenine . KynurenDue to the poor outcomes of depressed patients in clinical practice, there is a pressing need to identify newer treatments and better strategies to personalize currently available antidepressant treatments. In the search for biomarkers to personalize antidepressant medication selection, inflammatory biomarkers, such as CRP, have emerged as a robust and pragmatic option. However, as over a third of patients with MDD are resistant to currently available medications, novel antidepressants that target the pathophysiology underlying depression are also needed. Anti-cytokine treatments have emerged as potentially selective agents to target proinflammatory cytokines in those patients with markers of systemic inflammation. Additional strategies include targeting of the BBB to mitigate the CNS effects of peripheral inflammation. In conclusion, inflammatory markers present clinically useful targets for both personalizing antidepressant prescription and for identifying novel antidepressants.


Existing evidence on the effects of smoke-free policies on respiratory diseases is scarce and inconclusive. Spain enacted two consecutive smoke-free regulations: a partial ban in 2006 and a comprehensive ban in 2011. We estimated their impact on hospital admissions via emergency departments for chronic obstructive pulmonary disease (COPD) and asthma.Data for COPD  came from 2003–2012 hospital admission records from the fourteen largest provinces of Spain and from five provinces for asthma (ICD-9 493). We estimated changes in hospital admission rates within provinces using Poisson additive models adjusted for long-term linear trends and seasonality, day of the week, temperature, influenza, acute respiratory infections, and pollen counts (asthma models). We estimated immediate and gradual effects through segmented-linear models. The coefficients within each province were combined through random-effects multivariate meta-analytic models.The partial ban was associated with a strong significant pooled immediate decline in COPD-related admission rates , sustained over time with a one-year decrease of 13.6% . The association was consistent across age and sex groups but stronger in less economically developed Spanish provinces. Asthma-related admission rates decreased by 7.4%  immediately after the comprehensive ban was implemented, although the one-year decrease was sustained only among men .The partial ban was associated with an immediate and sustained strong decline in COPD-related admissions, especially in less economically developed provinces. The comprehensive ban was related to an immediate decrease in asthma, sustained for the medium-term only among men. Ample evidence links environmental exposure to second-hand tobacco smoke (SHS) to substantial harmful health effects, particularly to cardiovascular and respiratory systems ,2. NeverA wealth of research has evaluated the heath impact of the implementation of smoke-free laws. Several studies have examined outcomes based on changes in several respiratory symptoms among hospitality workers before and after the implementation of smoking bans. Based on questionnaire data –6 or pulst of January of 2006  dictating a complete smoking ban at the workplace and a partial ban in hospitality venues  where partial restrictions applied according to venue size. Venues larger than 100m2 had to be 100% smoke-free or establish a physically separated smoking section (with its own ventilation system) not to exceed 30% of the total surface. Owners of smaller venues could continue allowing tobacco consumption, just as before the ban, or designate their venue as 100% smoke-free. This regulation achieved an important reduction in workplace-related SHS exposure but the impact was very modest in bars and restaurants [In Spain smoke-free regulations have been introduced consecutively. The first law came into force on the 1taurants .st of 2011 banning tobacco consumption in virtually all public spaces (comprehensive ban), including hospitality venues regardless of size. The new regulation eliminated all exceptions for hospitality venues drastically reducing SHS exposure in bars and restaurants [To further reduce SHS exposure, a reform of the smoking legislation took effect on January 1taurants . Contrartaurants .The main aim of the present work was to assess the joint association of two consecutive smoking bans and hospital admissions via emergency departments due to COPD and asthma using a large sample of the Spanish population.st, 2003 through November 30, 2012 was obtained from the Hospital Discharge Records Database of the Spanish National Health System (CMBD-H for its acronym in Spanish), which receives discharge notifications from all public hospitals and private institutions within the consortium. We examined the number of daily hospital admissions for COPD as primary diagnosis  in individuals 40 years or older, and for asthma (ICD-9 493) regardless of age. Daily population estimates in each province were calculated by linear interpolation from population figures at the beginning of each year provided by the Spanish National Institute of Statistics.The study was conducted in the fourteen largest provinces of Spain for COPD, and in five provinces for asthma (see http://www.aemet.es/), and influenza data came from the Spanish Influenza Sentinel Surveillance System (http://vgripe.isciii.es/gripe/inicio.do). The National Health System’s Hospital Discharge Records Database (CMBD) (https://www.msssi.gob.es/estadEstudios/estadisticas/cmbdhome.htm) provided the information on acute respiratory infections. Finally, the Spanish Society of Allergology and Clinical Immunology , the Spanish Aerobiology Network , and the Asturias Regional Health Council  provided the data on specific pollen counts. Relevant pollen count information was only available from five provinces in the country, limiting the asthma admissions-related analyses to these provinces.Daily temperatures were collected from the National Institute of Meteorology . To guarantee patient confidentiality, clinical records data were anonymized and de-identified prior to analysis.t in province p was assumed to follow a Poisson distribution with mean ϕp; where t was the day indexed from day 1  to day 3,622 , t1 and t2 were the days at which the partial and comprehensive smoking bans came into effect on January 1st, 2006, and January 2nd, 2011, respectively, and I(t ≥ t1) and I(t ≥ t2) were indicators for time periods after each smoking ban implementation. The coefficients st, 2006; whereas nd, 2011, whereas We adopted a two-stage hierarchical approach ,27 to suar model log(λtp)Based on previous work , all Poihttp://www.R-project.org/).To assess potential heterogeneity in the effect of smoking bans by sex and age, additional province-specific analyses were performed separately for men and women, as well as by age groups for COPD patients (40–64 years and ≥ 65 years) and asthma patients (< 15 years and ≥ 15 years). All Poisson additive models were fitted using penalized quasi-likelihood methods, as implemented in the function gam from the R package mgcv ′ determining population-average linear trends for each ban period summarizing the association across all provinces. The pooled percent changes in hospital admission rates at the implementation of the partial ban and one year afterwards were estimated as 100{exp(α2)−1} and 100{exp(α2 + 365α3)−1}, respectively. Similarly, the pooled percent changes at the implementation of the comprehensive ban and one year afterwards were defined as 100{exp(α4)−1} and 100{exp(α4 + 365α5)−1}, respectively [Initially, a random-effects multivariate meta-analysis  with no ectively .xp = ′ in separate random-effects multivariate meta-regression models [To explore potential differences in the association by province-level characteristics we included several variables as single second-stage covariates n models . These vI2 statistic. The I2 statistic describes the proportion of total variation in estimates The overall heterogeneity in the association across provinces, as well as the residual heterogeneity beyond that explained by province-level characteristics, were detected with the multivariate extension of the Cochran chi-squared test and quantified with the extended ogeneity ,32. The During the entire study period there were 431,797 hospital admissions for COPD in the fourteen largest Spanish provinces, as well as 95,411 hospital admissions for asthma in five provinces. By province, the average number of daily hospitalizations varied from 1.3 to 33.1 for COPD, and from 0.9 to 9.6 for asthma . The proI2 = 98.2% for COPD and 93.8% for asthma; all P values < 0.001). The pooled segmented linear trends across all provinces are displayed in Linear trends in hospital admission rates during the pre-ban, partial-ban, and comprehensive-ban periods varied widely across provinces . Over 90The partial smoking ban was associated with a strong significant pooled immediate decline of 14.7% in COPD-related admission rates, which was sustained over time with a one-year 13.6% decrease. There was no subsequent effect of the comprehensive ban on COPD-related admission rates . After the partial ban, both sexes experienced the aforementioned strong pooled decline in COPD-related admissions , though Asthma-related admission rates increased by 12.1% immediately after the partial ban, but decreased by 7.4% after the comprehensive ban, though these pooled estimates were highly imprecise. Also, the pooled rise in asthma-related admissions after the partial ban (immediate increases of 14.8% in men and 10.3% (ns) in women) was fully offset in men with the implementation of the comprehensive ban (an immediate decrease of 17.4%) but not in women (an immediate decrease of 3.5% (ns)).P for effect modification = 0.04), percentage of population unemployed (P = 0.05), and percentage of rural population (P = 0.02). Both the immediate and one-year pooled declines in COPD-related admission rates reached 16.6% following the partial ban in provinces with a relatively low human development level (25th percentile). In provinces with a high unemployment rate (75th percentile), immediate and one-year rates declined 18.3% and 20.1%, respectively. Finally, in provinces with a relatively high percentage of rural population (75th percentile) rates decreased 19.2% and 18.4%, respectively. However, these factors contributed little to the high heterogeneity observed in COPD-related admission trends across provinces . Effect modifications for asthma-related admissions were not explored due to the limited number of meta-analyzed provinces.The implementation of the first smoking regulation, a partial ban, was associated with a strong immediate decline in COPD-related hospital admissions. This reduction persisted all throughout the regulation years experiencing only modest changes when the second smoking law, a comprehensive ban, came into force. The observed association is consistent across age and sex groups but stronger in less economically developed Spanish provinces.Existing evidence of the impact of implementing smoke-free legislation on COPD-related hospital admissions remains scarce and results are inconsistent. Some studies report a decrease in admissions –13, wherOur segmented-linear models allowed us to differentiate between the associations observed right after the law´s execution (immediate association) and those taking place over the medium-term. Thus, we were able to observe that the onset of the partial ban was associated with a rapid reduction in COPD-related hospital admissions. These results are consistent with the fast-acting harmful effects of SHS such as reduced pulmonary function and increased inflammatory processes, which have been observed even at low levels of exposure .The current project´s pilot study evaluated the effect of the partial smoking ban on hospital admissions in the two largest Spanish cities, Madrid and Barcelona . That stRegarding asthma-related hospital admissions, most authors report a decline in hospital admissions ,12,16–21It is worth highlighting here the great variability in estimates across provinces for both COPD- and asthma-related admissions. This heterogeneity has been previously described by Tan and colleagues when comparing studies performed in different countries  as well Nevertheless, residual variance remains very high, and it is likely that other variables, such as regional variability in compliance with the bans, may be related to differing rates of respiratory conditions-related hospital admissions. Estimates of police reports and sanctions by region varied widely from the time the partial ban was put in place, though compliance with the law cannot be compared due to the absence of official statistics .Overall, more benefits on cardiovascular and respiratory diseases are observed in regions with comprehensive smoking laws banning smoking at the workplace as well as in bars and restaurants . In factHowever, despite both diseases being highly prevalent at the population level, not only COPD-related hospital admissions are far more common than asthma-related ones but one third of hospitalized patients is at risk of re-admission within 90 days, especially those with severe disease and high comorbidity . Thus, COur results should be interpreted in the context of the study’s limitations. The main one pertains to the ecological nature of the study which precludes the inference of causality. Additionally, simultaneous country-wide implementation of the regulations eliminated the possibility of a control group for comparisons.Other limitations include the lack of information on patients´ smoking status on admission records. Further, despite adjusting for highly relevant covariates, changes in other factors not accounted for such as improvements in disease management during the period under study may have influenced our estimates, although any improvements are likely to have been too gradual to have any significant effect. Similarly, there might have been registry-related administrative factors affecting hospital admissions regarding these diseases. However, we are unaware of any such changes. Regarding asthma-related results for children, we only had the required data on specific pollen counts for five provinces, thus, limiting the statistical power necessary for sex-stratified analyses for those under 15 years of age. Larger samples are needed to further research in this area. Finally, meta-regression results are prone to low power issues and ecological bias when examining aggregate measures of individual-level characteristics.Major strengths of this study include the analysis of a large sample including the fourteen largest Spanish regions and the availability of a large number of covariates modeled based on daily observations. The richness of these data allowed the creation of separate models for each province as well as by age and sex groups.The implementation of the first partial smoking ban is associated with a 14.7% decrease in COPD-related hospital admissions. This reduction remained steady and, basically, unaffected by the introduction of the comprehensive smoking ban. This association is consistent across sex and age groups and stronger in less economically developed provinces than in those with stronger economies. Unfortunately, asthma-related results are not conclusive. An immediate reduction in hospital admissions was observed after the implementation of the comprehensive ban. However, the reduction was sustained only for one year and only in men.


Mode of delivery  is thought to affect gut microbiota, which in turn may affect psychological well-being. As such, mode of delivery is potentially a modifiable factor for psychological well-being. Here we examined the association of mode of delivery with child and adolescent psychological well-being. We used multivariable linear regression in a population-representative Hong Kong Chinese birth cohort, “Children of 1997,” to examine the adjusted associations of mode of delivery with behavioral problems assessed from parent-reported Rutter score at ~7 (n = 6294) and ~11 years (n = 5598), self-esteem assessed from self-reported Culture-Free Self-Esteem Inventory score at ~11 years (n = 6937) and depressive symptoms assessed from self-reported Patient Health Questionnaire-9 score at ~13 years (n = 5797). Cesarean Section (CS) was associated with children born in private hospitals, boys, and firstborns, higher maternal body mass index, higher maternal age, preeclampsia, higher socioeconomic position (SEP) and maternal birth in Hong Kong. CS was unrelated to behavior, self-esteem and depressive symptoms adjusted for infant characteristics , maternal characteristics (mother’s age and place of birth) and SEP. In a developed non-Western setting, mode of delivery was not clearly associated with childhood or early adolescent psychological well-being. Infants born vaginally initially have more microbes from the Firmicutes family and fewer from the Bacteroidetes family4. Although gut microbiota eventually normalize5, early life is a sensitive period when exposures may have lifelong consequences. Children born via CS have a higher risk of allergies6, asthma7 and diabetes8 later in life, with differences in gut microbiota during early life as a possible explanation. Gut microbiota are also associated with psychological well-being, through neural, endocrine and immune pathways, i.e., the gut-brain axis, which may affect brain function and behavior9. The idea that the gut could affect psychological well-being has been recognized since the middle of the nineteenth century and is generally accepted in the biological sciences10, and has been shown in several animal studies13.Childhood psychological well-being is a growing concern in public health as mental health issues often emerge in adolescence and extend throughout adulthood with many other related adverse effects affecting health and social functioning. Cesarean section (CS) is a potentially modifiable factor that may be associated with the child’s psychological well-being. A growing body of evidence suggests that mode of delivery affects the infant’s gut microbiota. Infants born vaginally acquire gut microbiota from the maternal vaginal and fecal microbiota while infants born by CS acquire their gut microbiota from the environment15 or cognitive delay15, and inconsistent associations for behavioral disorders20 and autistic spectrum disorder (ASD)22. Two recent Swedish cohort studies reported associations of CS with higher risk of ASD and psychosis, but the lack of associations in the sibling-matched analyses indicated that the observations were likely due to unmeasured genetic and environmental  factors23. Another Swedish sibling-matched study found that birth by CS was associated with higher risk of attention-deficit/hyperactivity disorder (ADHD), but only elective CS had positive sibling associations, thus also implying that findings were confounded18. Curran et al. (2015) reported null associations of mode of delivery with ADHD and ASD in the Millennium Cohort in the United Kingdom19. However, the association of mode of delivery with other indicators of psychological well-being, as self-esteem during childhood and adolescent depressive symptoms during early adolescence has not, to our knowledge, previously been examined. CS rates tend to be higher than recommended by the World Health Organization (WHO) and thus represent a modifiable intervention target. To clarify the role of mode of delivery in psychological well-being, we examined prospectively in a large population-representative birth cohort, Hong Kong’s “Children of 1997,” the association of mode of delivery with child and adolescent psychological well-being, including measures of behavior, self-esteem, and depressive symptoms.The association of mode of delivery with cognitive development has previously been examined, with no association found for intelligenceAs shown in Fig. About a quarter of the births were CSs (26.8%), 56.6% were unassisted vaginal birth and 16.6% assisted vaginal birth , as opposed to clinical records, which could introduce measurement error. However, type of delivery was obtained shortly after delivery and is quite memorable. Second, screening tools were used to assess behavioral problems, self-esteem, and depressive symptoms as opposed to clinical diagnosis. However, these questionnaires have been validated in Chinese populationsLactobacillus or Bifidobacterium genera33, may have a role in the treatment of depression, as seen in some preliminary randomized controlled trials32. Differences may have occurred for a number of reasons. Mode of delivery may affect proportions of bacteria from the Firmicutes and Bacteroidetes families, but may only be associated with short-term differences in gut microbiota, rather than representing differences that continue into adolescence, although early life is a key phase for neurological development. Alternatively, any effects of events at birth may operate via different mechanisms, such as cortisol from a stress response36 leading to child behavioral problems. For example, Li et al. (2011) found elective cesarean delivery associated with fewer behavioral and emotional problems compared to unassisted vaginal delivery while assisted vaginal delivery was associated with more problems17. However, we found no associations of assisted vaginal delivery with behavioral problems. Furthermore, Li et al. (2011) did not examine the differences between the implications for CS, thereby not allowing further examination of how cortisol may mediate the pathway between mode of delivery and psychological well-being.Our study is not consistent with a link between gut microbiota, established early in life, and psychological well-being. In adults, probiotics, of the Mode of delivery does not appear to be associated with the psychological well-being of children and adolescents. However, a clearer understanding of the long-term effects of mode of delivery on gut microbiota will help elucidate the impact of CS on health, delineate any possible pathways involving the gut-brain-axis and identify where modification of gut microbiota in infants could potentially be beneficial.Since our participants are children, informed consent was obtained from the parents, next of kin, caretakers or guardians (informants) on behalf of the participants by completing the questionnaire at enrollment as approved by The University of Hong Kong Medical Faculty Ethics Committee. Ethical approval for further studies was obtained from the University of Hong Kong-Hospital Authority Hong Kong West Cluster, Joint Institutional Review Board and/or the Ethics Committee of the Department of Health, Government of the Hong Kong SAR as appropriate. All methods were performed in accordance with the relevant guidelines and regulations.37. The study was initially established to investigate the effects of secondhand smoke exposure on infant health. Families were recruited at the first postnatal visit to one of the 49 Maternal and Child Health Centers (MCHCs) in Hong Kong, which parents of all newborns were encouraged to attend. Characteristics obtained using a self-administered questionnaire in Chinese at recruitment and subsequent routine visits included maternal characteristics (mother’s birthplace and age at birth), infant characteristics  and socioeconomic position (SEP).The Hong Kong “Children of 1997” birth cohort is a population representative Chinese birth cohort  that covered 88.0% of all births in Hong Kong from April 1, 1997 to May 31, 1997, and has been described in detail elsewherePassive follow-up via record linkage was instituted in 2005 to obtain routinely collected information including bi-annual assessments of emotional and behavioral problems using the Revised Parent’s Rutter Scales in Chinese ) and self-esteem using Form A of the Culture-Free Self-Esteem Inventories for Children by Battle in Chinese (grade 4 (age 9-10 years) onwards) from the Student Health Service, Department of Health, which provides free annual check-ups for all school students. Active follow-up via direct contact was instituted in 2007, including postal surveys. Maternal height and weight, gestational diabetes and preeclampsia were obtained from surveys I (conducted in 2008 and 2009) and III (conducted in 2011 and 2012). Survey II, including the Patient Health Questionnaire-9 (PHQ-9), was first sent in February 2010 then re-sent a second and third time as necessary over the following 5 months. Non-responses were followed-up via two-waves of telephone interviews from November 2010 to April 2011 and from July 2011 to June 2012 and during pilot studies for in-person follow-up (June to August 2011 and June to August 2012). The study obtained ethical approval from the University of Hong Kong-Hospital Authority Hong Kong West Cluster Joint Institutional Review Board.Mode of delivery was categorized as vaginal delivery or CS. Mode of delivery was obtained from the baseline survey completed by the primary caregiver . Mode of delivery was recorded as normal vaginal birth without instrumentation, vaginal birth with suction or forceps or CS. We considered vaginal births together . Given that unassisted and assisted vaginal deliveries would be expected to have the same effect on gut microbiota, we also classified mode of delivery as unassisted vaginal delivery, assisted vaginal delivery or CS to check whether any associations were specific to CS. We did not collect information on the possible indications for CS or whether the CS was primary versus repeat.38. The scales consist of a set of 31 items describing emotional and behavioral difficulties, with each item scored 0 for does not apply, 1 for applies somewhat or 2 for certainly applies. A total score (range 0–62) and subscores for conduct problems , emotional problems  and inattention/hyperactivity  were calculated, where a higher score indicated more emotional and behavioral problems. When considering the presence of emotional and behavioral problems, a total score of 13 or more was considered as overall emotional and behavioral problems39. Presence of conduct problems, emotional problems or hyperactivity was defined as having the corresponding subscore above the sex-specific 97th percentile  for this population.Emotional and behavioral problems at ~7 years (6- <9 years) and ~11 years (9- <13 years) were assessed from the Revised Parent’s Rutter Scales in Chinese for parents40. Assessment inventories developed in Western countries may be less valid in non-Western settings. However, the items of the SEI were chosen with concern for content that is least sensitive to change across cultures41. The SEI has good validation in assessing self-esteem in adolescents and has been used in Hong Kong Chinese42. Responses are of the forced-choice variety that the respondent checks either “yes” or “no” for each item. A total score (range 0–50) and subscores for general self-esteem  , social self-esteem  , academic self-esteem (perception of ability to academic success) , and parent-related self-esteem (perception of status at home)  were calculated, where a lower score indicated lower self-esteem. Based on the findings from the same study, we excluded unreliable scores with a lie scale of 2 or less (n = 158), although a lie scale of 5 or more had been used to indicate a lack of defensiveness and a reliable self-esteem inventories40. Presence of low self-esteem was defined as having a total score of 19 or less40. Each subscale score can be classified into one of five categories . A ‘very low’ subscale score indicated low self-esteem in the respective subscale category 40.Self-esteem at ~11 years (9- <13 years) was assessed from the Form A of the Culture-Free Self Esteem Inventories (SEI) in Chinese for children43 in Chinese. The PHQ-9 scale has good sensitivity and specificity in detecting depression in youth44, is an effective screening tool for the risk of depression among adolescents45 and has been validated in Chinese adolescents46. The scales consist of a set of 9 items describing symptoms and functional impairment, with each item scored 0 for not at all, 1 for several days, 2 for more than half the days or 3 for nearly everyday. A total PHQ-9 score (range 0–27) was calculated, where a higher score indicated more depressive symptoms. Presence of PHQ-9 Major Depressive Disorder was defined as having a total score of 11 or more mapping on the DSM-IV-TR44.Depressive symptoms at ~13 years (12- ≤15 years) were assessed from the PHQ-9We used multivariable linear regression to estimate the adjusted associations of mode of delivery with Rutter, SEI and depressive symptom scores from which estimated beta-coefficients with 95% confidence intervals (CIs) are presented. We adjusted for potential confounders, which are presented in three models. Model 1 adjusted for sex, age at assessment and survey mode for depressive symptoms since for PHQ-9 was obtained by postal questionnaire, telephone interview and in-person interview. Model 2 additionally adjusted for SEP , mother’s birthplace and birth hospital. Model 3 additionally adjusted for birth weight, gestational age, parity, maternal age, maternal body mass index (BMI), gestational diabetes and preeclampsia.47 as multiple imputation is the gold standard in this situation48. We used the ‘Hmisc’ package in R 3.0.1  for the multiple imputation. We summarized the results from 20 imputed datasets into single estimated beta-coefficients with CIs adjusted for missing data uncertainty. The results were based off 20 imputations due to fraction of missing information (γ) and the preventable power falloff of less than 1%. We also performed a complete case analysis for comparison using Stata version 10 .Among the 7,681 birth cohort members included, 3.6% were missing mode of delivery. Overall, 13.2% of data points were missing, mainly maternal BMI (58.8% missing) and gestational diabetes (23.6%) because these were obtained from Survey I or Survey III. We used multiple imputation to predict missing values of exposures and confounders incorporating data on the primary outcomes , exposures (mode of delivery), confounders and other factors potentially associated with mode of delivery based on a flexible additive regression model with predictive mean matchingData are available upon request from the “Children of 1997” data access committee: aprmay97@hku.hk.Supplementary Tables


Abnormal decision making is a behavioral characteristic of drug addiction. Indeed, drug addicts prefer immediate rewards at the expense of future interests. Assessing the neurocognitive basis of decision-making related to drug dependence, combining event-related potential (ERP) analysis and source localization techniques, may provide new insights into understanding decision-making deficits in drug addicts and further guide withdrawal treatment. In this study, EEG was performed in 20 abstinent heroin addicts (AHAs) and 20 age-, education- and gender-matched healthy controls (HCs) while they participated in a simple two-choice gambling task (99 vs. 9). Our behavioral results showed that AHAs tend to select higher-risk choices compared with HCs . ERP results showed that right hemisphere preponderance of stimulus-preceding negativity was disrupted in AHAs, but not in HCs. Feedback-related negativity of difference wave was higher in AHAs than HCs, with the P300 amplitude associated with risk magnitude and valence. Using source localization that allows identification of abnormal brain activity in consequential cognitive stages, including the reward expectation and outcome evaluation stages, we found abnormalities in both behavioral and neural responses on gambling in AHAs. Taken together, our findings suggest AHAs have risk-prone tendency and dysfunction in adaptive decision making, since they continue to choose risky options even after accruing considerable negative scores, and fail to shift to a safer strategy to avoid risk. Such abnormal decision-making bias to risk and immediate reward seeking may be accompanied by abnormal reward expectation and evaluation in AHAs, which explains their high risk-seeking and impulsivity. It largThe gambling task has been widely used to examine abnormal decision-making behavior in addicted subjects  and expectancy toward outcomes with the inconsistency of expectancy eliciting more negative-going FRN  and reward valence (win vs. loss). Based on previous neurobehavioral evidence  = 1.613, p = 0.115] or education level . All subjects were right-handed, with normal or corrected-to-normal visual acuity, with no history of neurological problems. None of the subjects was taking any psychotropic, neurological, or psychiatric medications at the time of experiment. Potential participants were excluded when they had any history of other addictions or any neurological or psychological disorders. All participants provided written informed consent before enrolment in the study, which was approved by the Ethics Commission of Institute of Psychology of the Chinese Academy of Sciences .Forty participants were involved in the current study, including 20 AHAs  and 20 HCs . AHAs were recruited from the Gansu Compulsory Isolated Detoxification Center in China, meeting the criteria of Diagnosis and Statistics of Mental Disorder 5th edition (DSM-V) for heroin dependence. The average duration of drug abuse and weekly dosage of AHAs were 6.56 ± 4.54 years and 3.20 ± 2.25 g, respectively. The AHAs who participated in our study were abstinent from heroin and other dependent drugs for at least 4 month (abstinent period: 9.00 ± 3.36 months). HCs were recruited from the local community, and had no history of alcohol or drug abuse. These two groups showed no significant differences in age . ANOVA showed no interaction effect  between group and the decision-making time , and no main between group effect . In addition, high-risk choice proportion  had a significant group effect , indicating more high-risk choices in AHAs compared with HCs. Besides, there was a significant interaction effect between group and risky option proportion . Further simple and simple effect test showed a significant difference in the high-risk condition between the two groups  with a tendency for higher-risk in AHAs compared with HCs .Independent samples F = 5.045, p = 0.031] and a significant interaction effect between group and hemisphere  were observed in the SPN component. Further simple effects test found no significant difference in left hemisphere activity  while right hemisphere activity showed a significant difference between the two groups . Interestingly, the mean amplitude of SPN in the right hemisphere was higher in AHAs than HCs  = 6.806, p = 0.013], indicating that more negative FRN was elicited for AHAs vs. HCs  , suggesting that high risk options  evoked more negative FRN compared with low risk counterparts . However, no significant interaction was found for group risk .Topographic maps for AHAs and HCs depicting voltage differences across the scalp for FRN from 290 to 350 ms after feedback onset are presented in Figure During the FRN time window, in the loss-gain 9 condition, difference of sources were mainly identified in the ACC, while in the loss-gain 99 condition differences were located in the inferior parietal lobule (IPL) and superior parietal lobule (SPL) between the two groups Figure , Table 1F = 11.230, p = 0.002]. Further simple effects test showed a larger P300 in high risk  than low risk  choices for the gain condition, but not for the loss condition .Grand average ERP waveforms at Pz elicited by gain and loss are presented in Figure During the P300 time window, significant voxel clusters of differential activation between the two groups were found in the superior frontal gyrus (SFG) and MFG for the gain 9 condition, in the IPL for the loss 9 condition, in the SFG, MFG, ACC, inferior frontal gyrus (IFG), and orbital gyrus for the gain 99 condition, and in the SPL and IPL for the loss 99 condition Figure , Table 1This study assessed the behavioral and neural characteristics of risky decision-making in AHAs, in a simple gambling task. We expected behavioral and neural response abnormalities of risky decisions in AHAs. Despite no significant differences in final scores and decision-making times between the AHA and HC groups, behavioral results showed that AHAs tend to select more high-risk options compared with HCs in the gambling task, confirming our hypothesis that AHAs are more risk-seeking compared with HCs. This corroborates previous reports that drug addicts have a high rate of risk-taking behaviors  and outcome-evaluation (indexed by FRN and P300) stages (Zheng and Liu, At the later cognitive appraisal stage, P300 was larger for high-risk outcomes and gain trials Figure , indicatAlthough this study revealed the characteristics of different decision-making stages in AHAs compared to HCs from the ERP and source localization perspectives, it remains unclear how different brain regions interact with each other during decision-making. Moreover, different cognitive states during risk decision-making were assessed in this study, but whether a specific brain area plays a key role in the entire decision-making process was not evaluated. We did not measure the level of the stress or anxiety during/after experiment, which largely prevented us from linking the behavioral or neural response with the psychological states of subjects. In addition, low-density EEG montage and the use of volume conduction head model template in this study might limit the spatial resolution and accuracy of source reconstruction (Liu et al., Our findings revealed significant differences in ERPs and corresponding neural sources between AHAs and HCs during the decision-making process; these differences are reflected in the anticipation and outcome-appraisal stages. We assume that such differences are probably caused by an abnormal reflective system due to decreased activation in the right anterior insula and MFG. This study provides electrophysiological evidences for understanding the neural mechanisms of abnormal decision-making behaviors in heroin dependence, and has the potential to offer novel biomarkers for withdrawal treatment in addicts.Study conception and design: BH and QL. Acquisition of data: QZ and HL. Analysis and interpretation of data: HL, BH, HW, and QL. Manuscript writing: All authors.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.