To evaluate the utility of the pharmacokinetic modeling derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in differentiating benign from malignant adnexal masses. A total of 43 patients with 49 complex adnexal masses  underwent preoperative DCE-MRI examinations on a 3 Tesla MRI. Using extended Tofts’ model, quantitative analysis was performed in the solid components of all tumors. Three pharmacokinetic parameters were defined as volume transfer coefficient (Ktrans), the rate constant (Kep), and the plasma volume (Vp). Semi-quantitative analysis was also performed and the values of relative signal intensity (SI rel) wash-in-rate (WIR), the initial area under the curve (iAUC60), time-to-peak (TTP) and wash-out-rate (WOR) were calculated. Receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic characteristics of each DCE-MRI parameter in differentiating borderline/malignant tumors from benign lesions and to provide the optimal cutoff values for these variables.SI rel had the highest diagnostic value . Ktrans had the second highest AUC=0.836 . The other factors found to be acceptable diagnostic parameters for borderline/malignant lesions included WIR , iAUC60 , Vp , SI max , SI peak  and Kep . Quantitative DCE-MRI is a relevant tool for differentiating benign from malignant adnexal masses. Among all the DCE parameters, SI rel and Ktrans are the most accurate discriminators. Adnexal masses are among the leading indications of gynecological surgery . As a result of the recent advances in surgical methods, different treatment approaches are available for adnexal lesions which are selected based on the probability of malignancy . In this regard, ultrasonography (US) has been commonly utilized as an initial imaging modality for estimating the risk of malignancy index (RMI) based on the specific features of malignancy on US (U-score), patient’s menopausal status and serum CA-125 levels . However, in some circumstances the lesion remains indeterminate and requires further assessments with other imaging modalities .Conventional contrast-enhanced magnetic resonance imaging (MRI) is indicated for all indeterminate adnexal masses, which provides an accuracy of 84-93% for differentiating benign and malignant lesions . However, its application is limited due to various reasons, particularly the dependence of its accuracy on the experience of reader .Recent studies have shown that a more accurate characterization of adnexal masses could be made using new objective MR parameters. For instance, dynamic contrast-enhanced MRI (DCE-MRI) is a technique that evaluates the leakage of contrast agent from capillaries into the extravascular extracellular space  and its findings can be analyzed through three different approaches: descriptive, semi-quantitative and quantitative. Descriptive analysis is the most frequently used method while semi-quantitative and quantitative parameters are extracted using appropriate mathematical models .In the semi-quantitative analyses, changes of signal intensity is assessed over time and the individual variabilities such as cardiac output, arterial blood pressure and contrast agent dosage are not taken into account . As previously established, among the semi-quantitative parameters, the maximal slope of the time-intensity curve is the most accurate for differentiating benign from malignant adnexal masses . In order to address individual variabilities, a pharmacokinetic model is used, in which an arterial input function (AIF) is incorporated . Although various studies have reported promising results for using the quantitative approach, but use of different methods has caused them to yield incongruent results .Currently there is not enough data available to determine which methodology and pharmacokinetic model is more accurate. Accordingly, this study was designed to evaluate the feasibility of semi-quantitative analysis along with the extended three-parametric two-compartment Tofts’ model, and to determine which parameter can differentiate between benign and malignant adnexal lesions more accurately.Study Design and Sample PopulationBetween January 2015 and March 2016, patients with a diagnosis of a complex adnexal mass on a trans-vaginal ultrasound, were enrolled in this prospective study. Complex solid cystic lesions with a solid component larger than 2 mm and multiloculated cysts with septae thicker than 2 mm were considered complex, and were included in the study. Patients with contraindications to contrast administration based on their recent serum creatinine levels were excluded. All participants underwent preoperative MRI and DCE-MRI, and were scheduled for surgical resection within 40 days of imaging. Eventually, a total of 47 patients were recruited, of which 4 were excluded from the study; three due to consideration of non-surgical treatment and one due to poor quality imaging. Of the remaining 43 patients, 12 women had cystectomy, 20 women had unilateral salpingo-oophorectomy and 10 subjects had bilateral salpingo-oophorectomy with hysterectomy and omentectomy. For one patient, resection of the tumor was not possible because the tumor was highly vascular and fragile; therefore, only open biopsy was performed and histopathological assessment of the lesion was consistent with ovarian Primitive neuroectodermal tumor (PNET).Six patients had bilateral lesions including two papillary serous carcinomas, one ovarian lymphoma, one ovarian metastasis, one mucinous cyst adenocarcinoma, and one ovarian PNET. In bilateral tumors, each lesion was considered as one individual study case. Thus, a total of 43 patients with 49 adnexal masses were included in our analyses. Imaging techniqueAll MRI examinations were performed on a 3 Tesla MR unit  with a phased-array pelvic coil. The range of scan was between the umbilicus and the pubic symphysis. The patients were fasting for at least 6 hours before the imaging. An antispasmodic agent (1ml of Hyoscine) was given by intramuscular injection immediately before image acquisition to minimize the artifacts caused by bowel peristalsis. An abdominal belt was also used to help reduce respiratory motion-related artifacts. The sequences acquired before the injection were as follows: Sagittal T2-weighted fast spin-echo , axial T2-weighted fast spin-echo  and axial gradient-echo T1-weighted sequences with and without fat suppression .Then, a dose of 0.1 mmol.kg−1 of gadoterate meglumine  was administered with an MR-compatible power injector at a rate of 2 mL.s−1, followed by a bolus of 20 mL saline solution (0.9%).DCE sequences were obtained from the tumor’s solid components  based on the findings of initial non-enhanced sequences, and consisted of axial DCE T1-weighted gradient-echo sequence (3D Turbo FLASH) . DCE images were acquired at 7.6/frame intervals, beginning 10 seconds before the bolus injection for a total of 380s (50 time frames). Finally, delayed enhanced axial and sagittal T1-weighted gradient-echo images with breath-hold were obtained 6 min after injection.Imaging analysisAll images were reviewed by two experienced radiologists blinded to the results of histopathological assessments (M.G with 10 years and S.P with 4 years of experience in gynecologic imaging). Color-coded Ktrans maps were created for all slices of adnexal masses, and regions of interest (ROIs) were manually placed on the hot-spots of the tumor. Three small ROIs were placed on the most enhancing part of the lesion, trying to include only a few pixels so that it does not involve the adjacent tissues.Pharmacokinetic AnalysisPharmacokinetic (PK) image analysis was performed, assuming a low-dose protocol and subsequently, presence of a linear relationship between the changes of signal intensity and concentration of the contrast agent within the ROI. Quantitative DCE parameters including Ktrans (the volume transfer rate), Kep (the rate constant) and vp (the fraction of plasma volume) were computed for the selected ROIs based on extended Tofts PK model :In the above equation, δ is the time lag between the onset of the concentration rise in the tissue and the beginning of arterial input concentration, Cp represents the concentration in the plasma which is calculated assuming a hematocrit level (HCT) of 45% and using (1-HCT) Cb. AIF or Cb was obtained through a population-averaged model proposed by Weinmann. To acquire the best fit, the population-based AIF was calibrated to the AIF obtained from 10 randomly selected patients among our study population, in whom the iliac artery was selected as the region of arterial input. The time lag, δ, was selected automatically by increasing the parameter by 1 unit in a range of values repeatedly and was selected as the value returning the best results for goodness of fit evaluation parameters. All the mentioned quantitative analyses were performed by in-house MATLAB scripts  and 2. Semi-Quantitative AnalysisSeveral DCE-MRI curve descriptors including, maximum signal intensity (SI max), relative signal enhancement (SI rel), time to peak (TTP), wash-in-rate (WIR), wash-out-rate (WOR), and initial area under the time-intensity curve (iAUC60) were calculated as semi-quantitative parameters .Statistical analysisMean values of individual parameters were compared among benign, borderline and malignant adnexal lesions using One-way ANOVA test. The significance of differences between benign and malignant lesions, and benign and borderline/malignant tumors were also assessed using independent two-tailed Student’s t-test for normally distributed data or otherwise by Mann-Whitney U-test, after examining the normality of data for variables based on Shapiro-Wilk test. A p value of less than 0.05 was considered statistically significant. Receiver operating characteristic (ROC) curve analysis was also performed to evaluate diagnostic characteristics of each DCE-MRI parameter in differentiating borderline/malignant tumors from benign lesions and, to provide the optimal cutoff values for these variables. All statistical calculations were performed using SPSS 23.0 for Windows platform . Ethical considerationOur institutional review board of the university hospital approved the study protocol. The objectives and methods of the survey were thoroughly explained for the subjects invited for participation. They were reassured that their inclusion in the study will not affect their treatment, will not pose additional charges to them and their information will be considered confidential and used anonymously, and only the main researchers will have access to them. Eventually, an informed written consent was obtained from all patients willing to participate. The study protocol was in accordance to the guidelines of the Helsinki’s Declaration. Data from a total of 43 patients with 49 adnexal masses were included in our analyses, which comprised of 27 benign (55.1%), 3 borderline (6.1%) and 19 malignant (38.8%) lesions. Considering the invasive potential of borderline tumors, we categorized the three borderline cases in the same group as malignant lesions for analysis purposes. The mean age of the patients was 39.9±10.5 years, ranging from 18 to 59 years. Diagnostic characteristics of evaluated parameters for differentiating borderline/malignant lesions from benign tumors are presented in Our study demonstrates the feasibility of performing PK analysis using the extended three-parametric two-compartment Tofts’ model for differentiating between malignant and benign adnexal masses. Previous studies have also shown the usefulness of PK parameters in evaluating adnexal masses. The fact that PK analysis can be used in this setting owes to the presence of disorganized neoangiogenetic vessels in malignant tumors that lack muscular coating, which allows for the contrast leakage into extravascular extracellular space .In one of the previous studies on the same topic, using a four-parametric two-compartment model on a 1.5 Tesla MRI machine, Thomassin-Naggara et al. found a higher value of the tissue blood flow (FT), a higher blood fraction volume (Vb), a lower interstitial volume (Ve) and a higher relative AUC (rAUC) in malignant adnexal tumors compared to benign lesions . Their results also showed that the borderline ovarian tumors have higher FT and lower lag time (Dt) in comparison with the malignant lesions.Carter et al., (2013) evaluated the PK parameters using extended Tofts’ model on a 3 Tesla MRI scanner. Having placed the ROI on the most solid area of the adnexal masses, the results of their study showed that most DCE parameters were significantly higher in malignant tumors, among which Kep was the most accurate. The present survey was also based on the three-parametric two-compartmental extended Tofts’ model in a 3 Tesla MRI machine. Using a high magnetic field in these two studies provided the advantage of increased signal to noise ratio . Our study was consistent with previous reports in demonstrating the feasibility of PK parameters in differentiating malignant from benign adnexal masses; however, incompatible with the findings of Carter et al., (2013) our results revealed that among the PK parameters the most accurate one was the Ktrans, found to be higher in malignant lesions. In this study we also evaluated the semi-quantitative DCE-MRI parameters in these adnexal lesions. As shown previously by Bernardin et al., (2012) among the semi-quantitative parameters the malignant tumors had higher values of SI max, SI rel and WIR, and the latter was the most accurate predictive parameter. We also found SI max, SI peak, SI rel and WIR to be significantly higher in malignant lesions compared to benign tumors, and we included iAUC60 in our analyses as well, which was similarly higher in malignant masses. Incompatible with their’s results, we showed that the most accurate parameter among these variables for predicting malignant lesions is the SI rel, which was also found to be a better predictive factor than the PK parameters we assessed with an AUC of 0.872. The overall accuracy of SI rel was calculated to be 79.6% with a sensitivity of 95.5% and a specificity of 66.7%, assuming a cut-off value of 121.4. In our study population there were three borderline tumors, two of which had Ktrans values in the malignant range , and one had a low Ktrans value in the range of benign lesions with a histopathological report of papillary serous type tumor. There was also an ovarian PNET in our study population, which is a very rare pelvic tumor of young women treated initially by non-surgical interventions . PK analyses of this tumor showed a Ktrans value of 0.264, which was much higher than other malignant tumors in our study population. This finding can be attributed to the highly fragile vessels formed in this type of malignancy.We included 2 dermoid cysts and 16 cases with endometriomas. Although these lesions generally have no solid components, have thin enhancing walls and are easily diagnosed on conventional MRI images, all the included cases in the current study met the aforementioned inclusion criteria and were eligible for inclusion. The small sample size in the present study was one of the limitations of this survey and so further investigations with larger sample populations should be conducted to confirm or negate our findings. Moreover, in this study we classified borderline tumors in the same group as the malignant lesions. Although these two categories may share some characteristics, but could be distinguished from each other using PK parameters. Therefore, it is suggested that further studies include more patients with borderline tumors so that a more comprehensive comparison could be made between their characteristics and that of malignant lesions. In conclusion, this study demonstrated that PK modeling of DCE-MRI is a relevant tool for estimating the likelihood of malignancy in the adnexal tumors. Our results also showed that SI rel and Ktrans have higher diagnostic values over other quantitative PK and semi-quantitative parameters in differentiating benign and malignant adnexal tumors.


Hirota bilinear form. The residual symmetry and finite symmetry transformation of the nonlocal AB-Boussinesq system are also studied.The generalized Boussinesq equation is a useful model to describe the water wave. In this paper, with the coupled Alice-Bob (AB) systems, the nonlocal Boussinesq system can be obtained via the parity and time reversal symmetry reduction. By introducing an extended Bäcklund transformation, the symmetry breaking rogue wave, symmetry breaking soliton and symmetry breaking breather solutions for a nonlocal Boussinesq system are obtained through the derived  The technical assumption was so-called 3. These solutions possess rich structures, such as single soliton, infinitely many singular soliton, cnoidal wave and the interaction of the different coherent structures. Nonlocal integrable peakon equations were also constructed and shown to have peakon solutions through this principle4. A new elegant form of the N-soliton solutions of the AB-KdV system which derived from the nonlinear inviscid dissipative and barotropic vorticity equation in a β-plane channel was obtained5. This system was applied to the two correlated monopole blocking events which was responsible for the snow disaster in the winter of 2007/2008 happened in Southern China. On the basis of the Lie point symmetry, the nonlocal symmetry of the AB-mKdV system was gained and the explicit solution of the system was presented6. By using the Darboux transformation, some types of shifted parity and time reversal symmetry breaking solutions, including such localized structures as one-soliton, two-soliton, and rogue wave solutions were explicitly depicted for the AB-mKdV system7. This methodology also induced a large number of nonlinear systems, including some integrable ones, such as the NLS,  KdV,  KP and sG systems from nothing  equation with shifted parity and delayed time reversal was derived through the multi-scale expansion method9. A special approximate analytical solution of the original two-vortex interaction model was given to describe two correlated dipole blocking events with a lifetime. Exact solutions of a new general nonlocal modified KdV equation were obtained including periodic waves, kink waves, solitary waves, kink- and/or anti-kink-cnoidal periodic wave interaction solutions, which can be utilized to describe various two-place and time-delayed correlated events10. The two-place nonlocal integrable models were systematically extended to multi-place nonlocal integrable (and nonintegrable) nonlinear models by means of the discrete symmetry reductions of the coupled local systems11. Especially, various two-place and four-place nonlocal NLS systems and KP equations were obtained. An integrable two-place nonlocal real system with three different nonlocal properties was derived which one nonlocality was related to a special nonlocal KdV system, while the other two were associated with the nonlocal Boussinesq systems12. The multisoliton solutions for the nonlocal KdV and Boussinesq systems were investigated and the possible prohibitions on multisoliton solutions were also discussed.Recently, Lou proposed various intrinsic models named the Alice-Bob systems (ABSs) to describe two-place physical problems13. This Boussinesq Eq. , \documentsystem is researched by introducing two auxiliary variables, and the finite symmetry transformation is obtained through solving the initial value problem. In brief, the variable coefficient with shifted parity and delayed time reversal in nonlocal AB- Boussinesq system is discussed, from which abundant of controllable symmetry breaking solutions are illustrated by changing parameters of events A and B. It is valuable to the nonlocal AB symmetric system analysis and the associated applications with mathematical models into more real physical systems for further study.It is believed that there are some really novel phenomena in two-place nonlocal systems compared with local one. In this article, we studied the nonlocal Boussinesq equation coupled with AB systems. Furthermore, After introducing the bilinear variable transformation, the different expressions of function in the Bäcklund transformation are obtained. By choosing special parameters, the abundant solutions, including symmetry breaking rogue wave, soliton and breather solutions of the AB-Boussinesq system, are discussed in detail. At last, with the derived modified Bäcklund transformation, the residual symmetry of the nonlocal Boussinesq


It is well established that repeated social defeat stress can induce negative long-term consequences such as increased anxiety-like behavior and enhances the reinforcing effect of psychostimulants in rodents. In the current study, we evaluated how the immune system may play a role in these long-term effects of stress. A total of 148 OF1 mice were divided into different experimental groups according to stress condition  and pre-treatment  before each social defeat or exploration episode. Three weeks after the last social defeat, anxiety was evaluated using an elevated plus maze paradigm. After this test, conditioned place preference (CPP) was induced by a subthreshold dose of cocaine (1 mg/kg). Biological samples were taken four hours after the first and the fourth social defeat, 3 weeks after the last defeat episode, and after the CPP procedure. Plasma and brain tissue  were used to determine the levels of the pro-inflammatory cytokine interleukin 6 (IL-6). Results showed an increase of peripheral and brain IL-6 levels after the first and fourth social defeat that was reverted three weeks later. Intraperitoneal administration of the anti-inflammatory drug indomethacin before each episode of stress prevented this enhancement of IL-6 levels and also reversed the increase in the rewarding effects of cocaine in defeated mice. Conversely, this protective effect was not observed with respect to the anxiogenic consequences of social stress. Our results confirm the hypothesis of a modulatory proinflammatory contribution to stress-induced vulnerability to drug abuse disorders and highlight anti-inflammatory interventions as a potential therapeutic tool to treat stress-related addiction disorders. Scientific evidence suggests that alterations of inflammatory parameters are linked to a vulnerability to mental illnesses , includiBoth clinical and preclinical studies have shown that psychostimulants such as cocaine or methamphetamine activate the central and peripheral components of the innate immune system , 25, 26.Another cornerstone of our understanding of drug addiction is stress, with emotional stressors representing the main source of stress in humans . Life-thRecent basic research shows that stress induces activation of the immune system, thereby promoting the stimulation of microglia and leukocytes, altering the levels of peripheral and brain cytokines and leading to monocytes trafficking into the brain , 42. MorIn response to this link between immune response, vulnerability to addiction and stress, some researchers have employed anti-inflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs) as a therapeutic approach. For instance, the inflammatory potential of ethanol is well established , 46, 47;Based on the above-mentioned observations, we hypothesize that the long-term sensitization to the rewarding properties of cocaine, constantly reported in experimental animals after social defeat stress, is mediated by a pro-inflammatory state. Likewise, we hypothesize that this inflammatory mechanism is also underlying the long-term anxiety-like behavior displayed by social defeated animals. In order to test these hypotheses, we first determined whether there is an increase in peripheral and brain inflammatory response after social stress by measuring plasmatic and brain levels of interleukin 6 (IL-6). We then determined if anti-inflammatory treatment with indomethacin before each stress episode blocks the inflammatory response induced by social stress, and therefore the enhanced cocaine response and anxiety-like behavior expected after social stress experiences.ad libitum, except during behavioral tests. The experimental protocol has been approved by an Institutional Review Committee for the use of animal subjects . Procedures involving mice and their care were conducted according to national, regional and local laws and regulations, which are in compliance with the Directive 2010/63/EU. All efforts were made to minimize animal suffering and to reduce the number of animals used.A total number of 148 OF1 adult mice  were used in this study. The animals were housed as previously described in detail  in groupThe anti-inflammatory indomethacin  was dissolved in 5% DMSO (dimethyl sulfoxide) and injected intraperitoneally (i.p.) at a dose of 5 or 10 mg/kg 30 minutes before each social defeat. For CPP, a dose of 1 mg/kg of cocaine hydrochloride  was employed. This dose of cocaine was selected on the basis of previous CPP studies showing 1 mg/kg to be a sub-threshold dose , 54, 55.The experimental design is depicted in Biological samples were taken four hours after the first and fourth social defeats, 3 weeks after the last social defeat, and following the CPP procedure. In the case of the control group (EXP), samples were also taken four hours after the first exploration session and after the CPP procedure.The social defeat protocol performed in this study was validated and described in detail in previous research papers from our research group , 36, 44.Place conditioning consisted of three phases and took place during the dark cycle , followiIn the second phase (conditioning), animals underwent two pairings per day. First, they received an injection of physiological saline before being confined to the vehicle-paired compartment for 30 minutes. After a 4-hour interval, they received cocaine immediately before being confined to the drug-paired compartment for 30 minutes. In the third phase or post-conditioning (Post-C), the time spent by the untreated mice in each compartment during a 15-minute observation period was recorded. The difference in seconds between the time spent in the drug-paired compartment in the Post-C test and that spent in the Pre-C test is a measure of the degree of conditioning induced by the drug. If this difference is positive, then the drug is considered to have induced a preference for the drug-paired compartment, whereas the opposite indicates the induction of an aversion.All groups in which a preference for the drug-paired compartment was established underwent an extinction session every 72 hours, which consisted of placing the mice in the apparatus for 15 minutes. This was repeated until the time spent in the drug-paired compartment by each group was similar to that of the Pre-C.The effects of non-contingent administration of a priming dose of cocaine were evaluated 24 hours after the confirmation of extinction. Reinstatement tests were the same as those for the Post-C (free ambulation for 15 minutes), except for the fact that mice were tested 15 minutes after administration of the drug . This procedure was repeated with progressively lower priming doses until a non-effective priming injection was determined.The elevated plus maze (EPM) test was carried out essentially following the procedure previously described by Daza-Losada and coworkers . The mazTo obtain blood and tissue samples, unperfused mice were sacrificed by cervical dislocation and then decapitated. Blood was collected from the neck into a Microvette CB 300 capillary tube . Blood samples were kept on ice, and plasma was separated from whole blood by centrifugation  and transferred to sterile 0.2 ml microcentrifugue tubes. Plasma samples were stored at -80°C until IL-6 concentration determination.3VO4, and 10 mM PMSF). Brain homogenates were kept on ice for 30 minutes and centrifuged at maximum speed for 15 minutes, after which the supernatant was collected and protein levels were determined by the Bradford assay from ThermoFisher (Ref: 23227).Brains were rapidly removed and the prefrontal cortex (PFC), striatum (STR) and hippocampus (HIP) were dissected following the procedure described by Heffner and coworkers  and keptTo determine IL-6 concentration in plasma and tissues, we used a Mouse IL-6 ELISA Kit obtained from Abcam (Ref: ab100712) following the manufacturer’s instructions. To determine absorbance, we employed an iMark microplate reader (Bio-RAD) controlled by Microplate Manager 6.2 software. The optical density was read at 450nm and the final results were calculated using a standard curve following the manufacturer’s instructions, and were expressed as pg/ml for plasma, and pg/mg for tissue samples.For the CPP data, the time spent in the drug-paired compartment during Pre-C and Post-C tests was analyzed with a mixed three-way ANOVA, with two between-subjects variables—Pre-treatment, with three levels , and Stress, with two levels (RSD and EXP)—and a within-subjects variable—Days, with two levels (Pre-C and Post-C). For the EPM data, a two-way ANOVA, with two between-subjects variables—Pre-treatment, with two levels , and Stress, with two levels (RSD and EXP)—was employed. In all cases, post-hoc comparisons were performed with Bonferroni tests. In addition, the groups showing CPP, extinction and reinstatement values were analyzed by a Student’s t-test.Data concerning IL-6 concentration were analyzed using a single factor analysis ANOVA, with a between-subjects variable: Stress, with 4 levels . For the IL-6 levels measure performed after the CPP procedure, we used an ANOVA with a between-subjects variable: Stress, with 3 levels . Data are presented as mean ± SEM. A p-value < 0.05 was considered statistically significant. Analyses were performed using SPSS v22.ANOVA of the CPP data  showed aThe data of the EPM test are presented in Regarding plasmatic circulating IL-6 levels , the ANOs <0.05), in the STR (p <0.001) and the PFC (p <0.05) after the fourth defeat, and only in the hippocampus three weeks after the last social defeat (p <0.05).Similar results were observed in the three brain areas studied. The ANOVA revealed a significant increase in IL-6 protein levels in the STR F = 7.878, p <0.001 ; PFC F3 and hippThe ANOVA F = 2.971, p <0.05 of plasmatic IL-6 levels in animals pretreated with indomethacin before social stress  showed tThe ANOVA F = 19.279, p <0.001 of plasmatic IL-6 levels after cocaine CPP  revealedSimilar results were obtained in the STR, as ANOVA F = 5.644, p <0.01 showed increases in IL-6 protein levels in defeated animals after CPP when compared with stressed mice previously treated with indomethacin (p <0.05).In the current study, we explored the role of the immune system in the long-term effects of social stress on anxiety-like behavior and the conditioned rewarding effects of cocaine in mice. Social defeat induced a long-term increase in anxiety when evaluated with the EPM test and produced a significant increase in the conditioned reinforcing effect of cocaine in the CPP paradigm. With the aim of determining a possible role of the immune response in the genesis of these stress effects, we first verified that social defeat increased levels of the proinflammatory cytokine IL-6. Pre-treatment with the anti-inflammatory drug indomethacin before each stress episode prevented this enhancement of IL-6 levels and reversed the increase in the rewarding effects of cocaine in defeated mice. Conversely, this protective effect was not observed with respect to the anxiogenic consequences of social stress.It has been widely demonstrated, in humans and animal models, that stressful experiences have a modulatory effect on the behavioral and physiological response to drugs . In geneWe hypothesized that these behavioral consequences of social stress are somehow mediated by a neuroinflammatory immune response. To validate this hypothesis, we first determined if social stress could trigger an inflammatory response. We observed increased levels of the cytokine IL-6 in defeated mice four hours after social defeat episodes. Socially defeated animals displayed significantly higher plasmatic and brain  IL-6 levels after the first and fourth social defeat when compared with exploration mice. This is not surprising, as other researchers have also reported increased levels of proinflammatory cytokines in response to social stressors , 68, 72.However, most of these previous reports only dealt with the acute inflammatory consequences of social stress. We have focused on long term-effects in the present study by extending the timeframe of the IL-6 profile and determining its levels three weeks after the stress episode, immediately before performing the behavioral tests. Three weeks after the last social defeat encounter a significant increase in IL-6 levels was registered only in the hippocampus of socially defeated mice, which is considered a central structure in CPP establishment , 74. AftOnce we confirmed the existence of an acute immune reaction triggered by social stress episodes, we aimed to determine if the increased sensitivity to the rewarding properties of cocaine and anxiety-like behavior is somehow modulated by this pro-inflammatory response. Considering that cytokine IL-6 levels were generally similar in stressed and non-stressed mice (with the exception of the hippocampus) when they performed the anxiety and CPP tests, the different behavior of defeated mice can be explained by an initial role of the pro-inflammatory response by which long-term adaptations are promoted. For this reason, we decided to block the development of an inflammatory response by administering the anti-inflammatory indomethacin before each social stress episode. At the highest dose of indomethacin (10 mg/kg), we registered a general assuaging of the increase in IL-6 levels after the first and fourth social defeat compared with levels displayed in non-treated animals, although significantly higher levels of IL-6 in plasma continued to be measured after the first defeat when compared with the exploration group.We also detected increased levels of IL-6 after the CPP procedure. Administration of four daily low doses of cocaine (1mg/kg) induced an enhancement of plasmatic IL-6 cytokine levels in all animals. This enhanced immune signaling was more pronounced in animals under the stress condition. Socially defeated animals presented increased IL-6 levels in plasma, with these levels proving to be statistically higher than in non-stressed animals. Again, pretreatment with indomethacin reversed this enhancement in the effect of stress, and socially defeated animals pretreated with the anti-inflammatory displayed similar IL-6 levels to the exploration group after cocaine CPP. The potential of cocaine as a xenobiotic that can activate proinflammatory central immune signaling is well documented . We have found that the inflammatory potential of cocaine is exacerbated by previous stress experience, whereas an anti-inflammatory pre-treatment before stress can reverse it. It is known that previous stress experiences can sensitize peripheral and central components of the immune system , 69, 70.Once we had demonstrated that indomethacin was capable of reducing the release of cytokine induced by social defeat, we set out to evaluate if this decrease was related to the behavioral consequences of stress. Administration of the higher dose of indomethacin (10 mg/kg) before each social defeat completely reversed the stress-induced increase in the rewarding properties of cocaine, since defeated mice treated with this anti-inflammatory did not develop CPP induced by cocaine. One possible mechanism by which neuroinflammation can enhance the rewarding properties of cocaine is the activation of the hypothalamus hypothalamic-pituitary-adrenal (HPA) axis. It had been demonstrated that IL-6 promotes the activation the HPA axis at the hypothalamic level , 80, leaConversely, the anti-inflammatory treatment failed to prevent anxiety-like behavior in our socially defeated animals. While there is general agreement in the literature about the link between an enhancement of IL-6 levels and the induction of anxiety (See revision on ), the deThe results of this research surpass current basic knowledge and take on a clear translational relevance, since IL-6 levels have been found to be altered in humans under conditions of social stress , and eveS1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.


N-acetyltransferase 2 (NAT2) gene may increase the risk of experiencing such toxicity events.Tuberculosis (TB) patients receiving anti-tuberculosis treatment may experience serious adverse drug reactions (ADRs) such as hepatotoxicity. Variants of the NAT2 variants and anti-tuberculosis drug-related toxicity.To provide a comprehensive evaluation of the evidence base for associations between This was a systematic review and meta-analysis. We searched for studies in Medline, PubMed, EMBASE, BIOSIS and Web of Science. We included data from 41 articles (39 distinct cohorts of patients). We pooled effect estimates for each genotype on each outcome using meta-analyses stratified by country.NAT2 acetylators were statistically significantly more likely to experience hepatotoxicity than rapid acetylators . Heterogeneity was not detected in the overall pooled analysis (I2 = 0%). NAT2 acetylator status was significantly associated with the likelihood of experiencing anti-tuberculosis drug-related hepatotoxicity.We assessed the quality of the included studies, which was variable, with many areas of concern. Slow/intermediate We encountered several challenges in performing robust syntheses of data from pharmacogenetic studies, and we outline recommendations for the future reporting of pharmacogenetic studies to enable high-quality systematic reviews and meta-analyses to be performed. TUBERCULOSIS (TB) is one of the most important challenges in global health. There were an estimated 1.3 million TB deaths in 2016 among human immunodeficiency virus (HIV) negative people and 374 000 deaths among HIV-positive people.1TB patients receiving a combination of these drugs may experience adverse drug reactions (ADRs), the most serious of which is anti-tuberculosis drug-induced hepatotoxicity (ATDH). Reported incidence rates of ATDH among patients treated with standard multidrug treatment vary from 2% to 28%, depending on the regimen given, definition of ATDH and patient characteristics such as age, race and sex.2N-acetyltransferase 2 (NAT2) gene, which codes for the drug-metabolising enzyme, NAT2.NAT2 polymorphisms may affect the activity of the NAT2 enzyme, altering the chemical modification of anti-tuberculosis drugs and their metabolites in the liver, leading to hepatic adverse reactions.The proposed genetic risk factors for ATDH include polymorphisms of the NAT2 acetylator status may be associated with INH-related hepatotoxicity because NAT2 is one of the main enzymes involved in INH metabolism in the liver. There are three phenotypes of acetylator status. Individuals who are slow NAT2 acetylators have higher plasma drug concentrations. This may be beneficial for treatment efficacy, but may also cause an accumulation of toxic metabolites as part of the metabolic activation of acetylhydrazine to harmless diacetylhydrazine. INH suppresses the acetylation of acetylhydrazine to produce more toxic metabolites, which contributes to the increased risk of hepatitis.INH is the anti-tuberculosis drug for which the genetic contribution to ATDH has been most widely studied and is best understood. Specifically, it is thought that *15 haplotype has been reported to be a predictor of RMP-induced liver injury;8RMP and PZA have also been reported to be hepatotoxic;The objective of this systematic review and meta-analysis was to evaluate evidence on the effect of NAT2 on anti-tuberculosis drug-related toxicity in TB patients receiving anti-tuberculosis treatment. Meta-analyses investigating the effect of NAT2 on toxicity outcomes have been published,NAT2 variants. Studies investigating associations between other genetic variants and anti-tuberculosis drug-related toxicity will be reported separately.This review was conducted in line with the methods outlined in our protocol (PROSPERO registration number: CRD42017068448).We included cohort studies, case-control studies and randomised controlled trials (RCTs). We did not include studies on case series because this type of study design would be inappropriate to investigate the effect of genetic variants on anti-tuberculosis drug-related toxicity. We did not require a minimum number of enrolled patients for a study to be included in our review.We included studies that recruited TB patients who were either already established on anti-tuberculosis treatment or commencing treatment , and who were genotyped to investigate the effect of genetic variants on anti-tuberculosis drug-related toxicity. We only included studies where >50% of included patients were TB patients receiving anti-tuberculosis treatment.We included studies that measured any drug-related toxicity outcomes.* and An information specialist (EK) designed the search strategy * and A.2The search results were imported to Covidence.We obtained the full text for each potentially relevant study. One reviewer (MR) assessed eligibility based on the selection criteria. A second author  independently assessed a sample of 10% of studies for eligibility. Disagreements between the two reviewers at the abstract and full-text screening stages were resolved through discussion, and by consulting a third author if necessary.The primary outcome of this review was hepatotoxicity by any definition used by the original investigators. The secondary outcomes were all other toxicity outcomes.We designed and piloted a data extraction form. We collected data on study design, participant characteristics, and treatment regimen and outcomes. One author (MR) extracted data in accordance with the methods outlined in the Cochrane HandbookWe contacted individuals who were listed as authors of multiple included articles to enquire whether there was overlap between articles in terms of the patient cohorts. We examined locations, dates of recruitment and other study characteristics to identify articles that reported outcomes for the same patient cohort. If an author confirmed that multiple articles reported outcomes for the same patient cohort, or if we suspected this based on reported study characteristics, we assigned a group identifier (GI) to these articles, and ensured that no data for the same patient cohort were included more than once in any meta-analysis.One author applied criteria for the quality assessment of pharmacogenetic studiesNAT2 and any anti-tuberculosis drug-related toxicity outcome that were investigated by at least two studies. The effects of both NAT2 acetylator status (as predicted using genotyping methods) and individual NAT2 single-nucleotide polymorphisms (SNPs) were investigated.We performed meta-analyses for associations between The primary analysis compared risk of hepatotoxicity for slow/intermediate acetylators in comparison with rapid acetylators. Data were pooled from studies that reported data for each acetylator group separately together with data from studies that combined slow and intermediate acetylator groups.Two sensitivity analyses were conducted. The first was pairwise comparisons of slow vs. rapid acetylator status, and intermediate vs. rapid acetylator status. Here, it was only possible to include data from studies that reported on each acetylator group separately. The second was comparison of slow vs. rapid/intermediate acetylator status. Here, data were pooled from studies that combined data for intermediate and rapid acetylator groups, and from studies that reported data for each acetylator group separately.NAT2 SNPs. For each SNP, two pairwise comparisons were undertaken: heterozygous genotype vs. homozygous wild-type (wt), and homozygous mutant-type vs. homozygous wt. For SNPs investigated by one study only, odds ratios (ORs) comparing genotype groups were calculated and summarised in a table, together with the pooled estimates from the meta-analyses. There were insufficient data to perform meta-analyses for an association between NAT2  and other toxicity outcomes; ORs and 95% confidence intervals (CIs) for each pairwise comparison were calculated and reported in a table.The secondary analysis compared the risk of hepatotoxicity between genotype groups for Meta-analyses were performed using Stata v 14 (metan package) ;The HuGENet HuGE Review Handbook recommends that meta-analyses of genetic association studies be stratified by ethnicity, and that meta-analyses should only be performed if effect estimates for different ethnic groups appear sufficiently similar.2I statistic. If substantial heterogeneity had been observed (>50%),We assessed heterogeneity by visually examining forest plots, and by referring to the We assessed the possibility of selective reporting as part of the quality assessment. Potential sources of selective reporting considered were genetic variants, outcomes and modes of inheritance.20We produced a funnel plot for the primary analysis to assess the risk of publication bias.www.prisma-statement.org).A Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart showing the selection of studies during the literature search is provided in NAT2 variants and anti-tuberculosis drug-related toxicity; from these articles, 40 distinct patient cohorts were identified. In this review, we include data from 40 articles (39 distinct patient cohorts).Forty-six articles reported data for the association between Twenty-seven articles reported the reasons for choosing all genes and SNPs investigated. For the 13 articlesThe median sample size was 170 (interquartile range 108.5–285). Only two articlesEleven articles described case-control studies, 27 articles described prospective cohorts, one article described a retrospective cohort and one article described an RCT. For one case-control study,Only three articlesFor most articles (29/40), on comparison of the number of participants included in the analyses with the study sample size, it was apparent there were no missing genotype data. For the remaining 11 articles,One article mentioned undertaking tests for population stratification;Twenty-three articlesNineteen articles made a specific assumption regarding the underlying mode of inheritance.There was large variation in the definition of hepatotoxicity . Of the Nine articles did not provide justification for the choice of outcomes, but outcomes were in line with the main study aim as conveyed in the Introduction section of the article.Six articles32I2 = 0%).A forest plot displaying the results of the primary analysis is given in I2 = 60.0%), but there were no significant differences between intermediate and rapid acetylators . The sensitivity analysis that compared slow acetylators with rapid/intermediate acetylators suggested that slow acetylator status significantly increased the risk of hepatotoxicity .Results of the sensitivity analyses are provided in Moderate heterogeneity was observed in the sensitivity analyses of slow vs. rapid acetylator status, and slow vs. rapid/intermediate acetylator status. Such moderate heterogeneity may have been due to the variable distribution of genotypes in different geographic areas.The funnel plot for the primary analysis  providedNAT2 SNPs. A summary of all data for the association between NAT2 SNPs and hepatotoxicity is provided in I2 =0%; AA vs. GG, OR 2.05, 95%CI 1.24–3.40, I2 =47.7%; 857G-A: GA vs. GG, OR 1.30, 95%CI 1.03–1.64, I2 = 0.9%; AA vs. GG, OR 1.99, 95%CI 1.02–3.91, I2 = 11.3%).1 For 590G-A and 857G-A, both heterozygous genotype and homozygous mutant-type significantly increased hepatotoxicity risk compared with homozygous wt ; however, no significant difference was observed for heterozygous genotype compared with homozygous wt .2 For 282C-T, homozygous mutant-type significantly increased hepatotoxicity risk compared with homozygous wt ; however, no significant difference was observed for homozygous mutant-type compared with homozygous wt . The lack of statistical significance for the latter comparison may have been caused by the relatively small number of homozygous mutant-type patients (n = 162) among the patients contributing data to this analysis (n = 3604)3 For 481C-T, heterozygous genotype significantly increased hepatotoxicity risk compared with homozygous wt .4 For 341T-C and 803A-G, no significant differences were observed for either pairwise comparison . This moderate heterogeneity may have been due to the variable distribution of genotypes in different geographic areas  is provided in A summary of all data for the association between For peripheral neuropathy, no significant associations were reported for either of the pairwise comparisons conducted for acetylator status, 191G-A or 341T-C. Similarly, for skin rash and eosinophilia, the pairwise comparisons for acetylator status demonstrated no significant effects. None of the SNPs investigated by Kim et al. had a significant effect on anti-tuberculosis drug-induced maculopapular eruption.NAT2 variants and anti-tuberculosis drug-related toxicity outcomes, as previously identified and as our systematic review confirmed. However, we established that performing robust synthesis of this evidence is challenging due to the variability between studies in terms of how participants are classified according to genotype; choice and definition of outcomes and variants to investigate; ethnicity of participants; and methodological quality. In conducting our review, we carefully considered these challenges, stratifying meta-analyses by genetic variants, genotype contrasts and outcomes. We also stratified further by the country where the study was conducted as a proxy for ethnicity, which has not been widely reported. We supplemented our data synthesis with a rigorous assessment of the methodological quality of included studies.There is substantial evidence for the association between Where possible, meta-analyses were undertaken to improve the power to estimate genetic effects. We found that slow/intermediate acetylators were significantly more likely to experience hepatotoxicity than rapid acetylators. This result is consistent with the findings of several meta-analyses,NAT2 gene have not been published, so our results add to the existing knowledge of the association between NAT2 variants and hepatotoxicity.Meta-analyses on individual SNPs of the INH remains an essential drug in the treatment of active TB and is the mainstay of chemoprophylaxis in latent tuberculous infection (LTBI), an intervention that is being rapidly expanded in recent strategies to eliminate TB as a public health problem. The global use of INH will therefore greatly increase worldwide in the coming decade. While transaminase testing is a readily available biomarker of possible ATDH, baseline values have modest predictive value and routine monitoring is not generally recommended. Where slow acetylator status is common, pharmacogenetic testing could make a clinically useful contribution to risk stratification for ATDH. However, the need for testing of a relatively large panel of SNPs and the current lack of a clear substitute to INH for LTBI chemoprophylaxis mean that such a strategy may not be cost-effective or feasible. Studies investigating the cost-effectiveness and/or feasibility of such a strategy would be beneficial. Nevertheless, based on the nearly three-fold increased risk of ATDH in slow acetylators observed in this review, pharmacogenetic epidemiology should certainly be a factor in national policymaking on the need for transaminase monitoring during treatment of active TB and LTBI locally.The quality of included studies varied, with some areas of concern. Most studies were significantly smaller than typically required to provide sufficient power,20As the quality assessment was qualitative rather than quantitative, it was not possible to exclude studies from meta-analyses based on a single summary score. Although we identified issues of concern relating to some of the quality criteria, we did not identify any studies that were thought to be of particularly poor quality overall, so we did not deem it necessary to exclude any single study in sensitivity analyses.Most included studies did not report the ethnic background of participants. We therefore performed analyses stratified by the country in which the study was conducted as a proxy variable for ethnicity. It is clear that this approach is not ideal as the population of any given country is often ethnically diverse. However, stratifying by country was deemed the most suitable approach in the absence of definitive information on ethnicity.An additional challenge was identifying distinct patient cohorts from the included articles. If multiple articles report data for the same patient cohort, data for this patient cohort must only be included in meta-analysis once, otherwise a unit-of-analysis error occurs.There was considerable variability in the definitions of hepatotoxicity in the included studies, which introduced heterogeneity into the meta-analyses. Jorgensen et al.NAT2 diversity across Africa,Finally, an important limitation of the systematic review was a lack of evidence from studies conducted in Africa. There is a great deal of 1 Report the number of patients in each genotype group;2 Report outcomes for each genotype group separately ;rs number of each genotyped SNP;3 Report the 4 Report the ethnicity of included patients;5 If a study includes more than one ethnic group, provide the summary data specified in 1) and 2) per ethnic group;6 Provide the reference to the published protocol;7 Provide information on patient cohort overlap;8 Report full details of all variants and outcomes investigated, and of all analyses undertaken;9 Consensus should be reached between experts in specific areas of research on the definitions of outcomes that are commonly reported in pharmacogenetic studies of a particular treatment.We made several recommendations regarding the reporting of future pharmacogenetic studies to facilitate the conduct of high-quality systematic reviews and meta-analyses, and thus improve the power to detect genetic associations.We also recommend that articles adhere to the criteria of the quality assessment tool74This review showed that slow/intermediate acetylators were significantly more likely to experience hepatotoxicity than rapid acetylators. Therefore, pharmacogenetic testing may be useful in clinical practice in terms of risk stratification for ATDH during treatment of TB. However, more studies are needed to overcome the reported methodological limitations and to assess if this strategy might be feasible and cost-effective.


Is the availability of buprenorphine-waivered physicians associated with buprenorphine treatment use and prescription opioid use among Medicaid enrollees?In this economic evaluation study using the all-capture Medicaid prescription data between 2011 and 2016, a 10% increase in the number of buprenorphine-waivered physicians was associated with an approximately 10% increase in the Medicaid-covered buprenorphine prescribing rate and a 1.2% reduction in the opioid prescribing rate.Expanding capacity for buprenorphine treatment holds the potential to improve access to opioid addiction treatment, which may further reduce prescription opioid use and slow the ongoing opioid epidemic in the United States. This economic evaluation study uses Medicaid prescription data from 2011 to 2016 to analyze the association of number of physicians permitted to prescribe buprenorphine for opioid addiction treatment with use of buprenorphine treatment and prescription opioid use among Medicaid enrollees. Expanding treatment for opioid addiction has been recognized as an essential component of a comprehensive national response to the opioid epidemic. The Drug Addiction Treatment Act and its amendments attempted to improve access to treatment by involving office-based physicians in the provision of buprenorphine treatment.To estimate the association of availability of buprenorphine-waivered physicians with buprenorphine treatment use and, secondarily, with prescription opioid use among Medicaid enrollees.This economic evaluation study used state-level panel data analysis to estimate the association between the number of buprenorphine-waivered physicians and the Medicaid-covered buprenorphine prescribing rate and opioid prescribing rate among Medicaid fee-for-service and managed care enrollees throughout the United States between January 1, 2011, and December 31, 2016.Buprenorphine prescribing rate and opioid prescribing rate, measured as the number of buprenorphine prescriptions and opioid prescriptions covered by Medicaid on a quarterly basis per 1000 enrollees.The sample included 1059 quarterly observations. Two additional 100-patient-waivered physicians per 1 000 000 residents (approximately a 10% increase) were associated with an increase in the quarterly number of Medicaid-covered buprenorphine prescriptions of 0.46  per 1000 enrollees and a reduction in the quarterly number of opioid prescriptions of 1.01  per 1000 enrollees. Furthermore, 5 additional 30-patient-waivered physicians per 1 000 000 residents (approximately a 10% increase) were associated with an increase in the quarterly number of Medicaid-covered buprenorphine prescriptions of 0.37  per 1000 enrollees and a reduction in the quarterly number of opioid prescriptions of 0.96  per 1000 enrollees. A 10% increase in the number of buprenorphine-waivered physicians was associated with an approximately 10% increase in the Medicaid-covered buprenorphine prescribing rate and a 1.2% reduction in the opioid prescribing rate.Expanding capacity for buprenorphine treatment holds the potential to improve access to opioid addiction treatment, which may further reduce prescription opioid use and slow the ongoing opioid epidemic in the United States. These programs required patients to take daily medications administrated onsite under the direct observation of program staff.19The Drug Addiction Treatment Act of 2000 (DATA 2000) expanded the provision channels of buprenorphine treatment from stand-alone opioid treatment programs to general medical care. Under the DATA 2000, office-based physicians who have a board certification in addiction medicine or psychiatry or complete an 8-hour course of buprenorphine prescribing training are qualified to provide buprenorphine treatment through a waiver.30-patient-waivered physicians). Subsequently, the Office of National Drug Control Policy Reauthorization Act of 2006 amended the DATA 2000 by raising the patient limit from 30 to 100. Allowing qualified physicians to provide buprenorphine treatment for up to 100 patients (hereafter referred to as 100-patient-waivered physicians) further helped absorb the unmet treatment need of people with opioid addiction.20 If the expansion of buprenorphine treatment capacity can effectively increase buprenorphine treatment use, it may hold the potential to reduce the prevalence of opioid addiction, which may further help reduce the number of opioid prescriptions being misused.Although the DATA 2000 waiver mechanism made the first legislative attempt to involve office-based physicians in the provision of buprenorphine treatment, it only allowed a buprenorphine-waivered physician to treat up to 30 patients at any given time  reporting guideline and the 24-item CHEERS checklist.23Our study used deidentified data from publicly available sources and was deemed an exempt human research study by the University of Kentucky institutional review board. We followed the recommendations of the Consolidated Health Economic Evaluation Reporting Standards  guidelines in 2009 and 2010 for appropriate opioid prescribing in chronic pain management, as well as the surgeon general’s warning letter about the opioid crisis in 2016, raised the awareness among physicians and patients of the risks associated with prescription opioid use.30 Moreover, the Centers for Disease Control and Prevention Guideline for Prescribing Opioids for Chronic Pain,31 released in 2016, set the stage for a wave of state laws that recommend or require limits on opioid prescribing duration aligned with the guideline. Although our fixed-effects study design accounted for the nationwide common shocks, states may have responded differently to them. As such, we chose our study window to avoid conflating these policies with the effects of buprenorphine-waivered physicians.We used quarterly, state-level Medicaid fee-for-service and managed care data between the first quarter of 2011 and the second quarter of 2016 (2011 is the first year in which state reporting of Medicaid managed care data became mandatory and nearly complete under the Affordable Care Act data collection requirements). The managed care data capture many high-risk, low-income adult enrollees who recently gained Medicaid coverage under the expansion provisions of the Affordable Care Act or the Section 1115 waiver. Using a study period of 2011 onward not only allows for the inclusion of Medicaid managed care prescription data, but also minimizes the influence of some major common shocks before 2011 or after 2016 that may cause disruptions to buprenorphine use and opioid use. For instance, following the 2010 OxyContin reformulation, there was an initial decline in OxyContin misuse and a shift away from OxyContin to other opioids such as heroin, which then leveled off as the individuals who continued to use these substances changed their preferred route of administration or managed to defeat the abuse-deterrent formulationsThe first outcome variable of our study, buprenorphine treatment use, was measured by Medicaid prescriptions for, and spending on, buprenorphine treatment on a quarterly basis per 1000 Medicaid enrollees. Each drug product in the State Drug Utilization Data can be identified by a unique 11-digit, 3-segment National Drug Code number. We excluded a few formulations of buprenorphine generally prescribed for pain management rather than for opioid addiction treatment . eAppendix 1 in the 32 Effective October 2014, hydrocodone combination products have been rescheduled from Schedule III to Schedule II.5 Accordingly, we classified all hydrocodone combination products as Schedule II opioids throughout the entire study period. Within the category of Schedule II opioids, we further created 6 major subcategories of commonly used, highly addictive opioids: oxycodone, hydrocodone, fentanyl, morphine, oxymorphone, and hydromorphone. In addition to the contemporaneous effect, we also estimated 1-, 2-, 3-, and 4-quarter lagged effects of buprenorphine treatment use on prescription opioid use.The second study outcome was prescription opioid use. In parallel with buprenorphine treatment use, prescription opioid use was measured by quarterly opioid prescriptions and spending per 1000 enrollees. We excluded buprenorphine products classified as medications for opioid addiction treatment from our measurement of prescription opioids. We classified opioid products into 2 categories based on the Controlled Substance Act scheduling: Schedule II opioids and Schedule III-V opioids. The Schedule II opioids are generally considered to have a higher addiction and overdose liability.The independent variable of interest was the availability of buprenorphine-waivered physicians. We counted the total number of buprenorphine-waivered physicians per 1 000 000 state residents; then we counted the numbers of 100-patient-waivered physicians and 30-patient-waivered physicians separately. eAppendices 1 and 2 in the 33 . The secondary analyses used a 2-stage least-squares instrumental variable model, which is analogous to a mediation analysis,35 to provide further evidence that buprenorphine treatment use served as one of the key pathways from expanding the availability of buprenorphine-waivered physicians to reducing prescription opioid use. eAppendix 3 in the The main analyses estimated the effects of availability of buprenorphine-waivered physicians on buprenorphine treatment use and the effect on prescription opioid use. We estimated linear weighted least-squares regression models with state and quarter 2-way fixed effects to account for unobserved time-invariant state heterogeneity as well as national secular trends and common shock related to the study outcomes36 The clustered standard errors allow for arbitrary within-state correlation in error terms but assume independence across the states.37 Statistical significance was assessed at P < .05 using 2-tailed tests. The main analyses were adjusted for overall physician supply, general economic conditions, and concurrent policies that may be correlated with prescription opioid use and buprenorphine treatment use by Medicaid enrollees . Sensitivity analyses also included group-specific linear trends to account for the unobserved groupwide confounding factors such as trajectory pattern and public sentiment that evolve over time at a constant rate. eAppendix 2 in the All analyses used sample weights (weighted by the state populations) and standard errors were clustered at the state level using Stata/SE statistical software version 15 (StataCorp LLC).We found that 2 additional 100-patient-waivered physicians per 1 000 000 residents (approximately a 10% increase) were associated with an increase in the quarterly number of Medicaid-covered buprenorphine prescriptions of 0.46  per 1000 enrollees, equivalent to a 5.6% increase. Two additional 100-patient-waivered physicians per 1 000 000 residents were further associated with a reduction in the quarterly number of opioid prescriptions of 1.01  per 1000 enrollees, a relative 0.6% reduction .We also found that 5 additional 30-patient-waivered physicians per 1 000 000 residents (approximately a 10% increase) were associated with an increase in the quarterly number of Medicaid-covered buprenorphine prescriptions of 0.37  per 1000 enrollees or 4.5%, as well as a further reduction in the quarterly number of opioid prescriptions of 0.96  per 1000 enrollees, or 0.6% .In addition to the effects on buprenorphine and opioid prescribing rates, expanding the availability of buprenorphine-waivered physicians was also found to be associated with an increase in Medicaid spending on buprenorphine and a reduction in opioid spending. The estimated spending effects on were comparable in size to the prescribing effects .The estimated reductions in prescription opioid use were largely concentrated in Schedule II opioids, especially in oxycodone, hydrocodone, and oxymorphone. These 3 subcategories of Schedule II opioids were commonly prescribed for noncancer pain and have a high potential for addiction and overdose . CollectCombining the effects of 100- and 30-patient-waivered physician availability and translating the absolute change into relative percentage change, we estimated that a 10% increase in the number of buprenorphine-waivered physicians  was associated with a 10% increase in the Medicaid-covered buprenorphine prescribing rate and a 1.2% reduction in the opioid prescribing rate. In addition to the prescribing effects, a 10% increase in the number of buprenorphine-waivered physicians was also associated with a 9.4% increase in buprenorphine spending and a 3.2% reduction in opioid spending .The mediation analyses provide exploratory evidence that increasing buprenorphine treatment use may have been one of the key pathways from expanding buprenorphine-waivered physician availability to reducing prescription opioid use . We foun38 On July 22, 2016, the Comprehensive Addiction and Recovery Act was signed into law, including a provision that further extended the DATA waiver privilege to nurse practitioners and physician assistants.39 On March 29, 2017, the Trump administration established a bipartisan Commission on Combating Drug Addiction and the Opioid Crisis and announced new actions to address the epidemic. The commission’s strategy prominently features proposals to improve access to opioid addiction treatment by expanding the capacity for buprenorphine treatment, especially for underserved populations and areas.40Our study has direct policy relevance to the ongoing opioid epidemic. On July 6, 2016, the Department of Health and Human Services released a final rule, effective on August 8, 2016, that allows eligible office-based physicians to request approval to treat up to 275 patients. The final rule also includes requirements to ensure that patients treated by these buprenorphine-waivered physicians receive a full array of evidence-based services that minimize the risk of misuse and diversion.43 Our findings suggest that expanding capacity for buprenorphine treatment may help address this challenge: the 2-way fixed-effects estimates indicate that a 10% increase in the number of buprenorphine-waivered physicians was associated with a 10% increase in the Medicaid-covered buprenorphine prescribing rate and a 1.2% reduction in the opioid prescribing rate. We also provided exploratory evidence that increasing buprenorphine treatment use may have been 1 of the key pathways from expanding buprenorphine-waivered physician availability to reducing prescription opioid use. The sustained effect of buprenorphine treatment use on prescription opioid use further suggests that buprenorphine treatment may have helped patients to achieve longer-term reduction in opioid use and recovery from opioid addiction, rather than simply acting as a temporary substitute for prescription opioids. As such, the investment in buprenorphine treatment may generate further savings in health care expenditures, criminal justice costs, and labor productivity.An estimated one-third of opioid prescriptions and 40% of opioid spending between 2011 and 2015 were misused, of which Medicaid shared a disproportionately large burden.45 Furthermore, the uncertain status of the essential health benefits provision of the Affordable Care Act and the lack of explicit inclusion of opioid agonist medication-assisted treatment benefits in the essential health benefits may expose buprenorphine treatment to more stringent future restrictions.46Despite the potential of expanding buprenorphine treatment capacity to reduce prescription opioid use, financial and logistic barriers to buprenorphine treatment still remain. Many states have imposed prior authorization, dosage or duration limits, and extensive counseling documentation requirements on Medicaid coverage for buprenorphine treatment, which may discourage Medicaid patients and physicians from considering such treatment.19 our findings imply 6 to 14 additional Medicaid enrollees being prescribed buprenorphine per 100-patient-waivered physician and 2 to 5 additional Medicaid enrollees per 30-patient-waivered physician. To put our estimates into context, existing literature showed that the average caseload of a buprenorphine-waivered physician was about 45% to two-thirds of the approved patient limit.48 Considering the overrepresentation of Medicaid enrollees among opioid addiction patients, our Medicaid-focused estimates seem to be consistent with and on the lower side of the existing statistics. One may speculate that some Medicaid enrollees may have faced difficulties in access to buprenorphine-waivered physicians and treatment, or paid out-of-pocket for buprenorphine treatment to circumvent the Medicaid restrictions and requirements.Our study found that 2 additional 100-patient-waivered physicians and 5 additional 30-patient-waivered physician per 1 000 000 residents were associated with an increase in the quarterly number of Medicaid-covered buprenorphine prescriptions by 0.46  and 0.37  per 1000 enrollees, respectively. Given the total population size, Medicaid enrollment, and the guideline-recommended 14- to 30-day refill interval,50 Furthermore, future research may investigate whether new buprenorphine-waivered physicians have obtained waivers as a result of a training program requirement or as a recipient of free continuing medical education, as opposed to those who have obtained waivers as a result of specialization in psychiatry and addiction medicine. The new 275-patient-waivered physicians would be another group to examine in future studies.In light of the remaining financial and logistic barriers and policy uncertainty surrounding access to buprenorphine treatment, active physician participation and a supportive state policy environment are needed to ensure that buprenorphine treatment achieves its full potential in addressing opioid addiction and the ongoing epidemic.Our study is subject to the following limitations. First, we only studied opioid prescriptions covered by Medicaid. Furthermore, our aggregate data do not allow us to determine whether and to what extent an estimated change in opioid prescriptions reflects inappropriate use. We expect a significant proportion of the effect of buprenorphine treatment use on prescription opioid use to be on the reduction of misuse, rather than a reduction in appropriate pain management. Nonetheless, our data do not allow us to directly scrutinize this expectation.51Second, although we included only the buprenorphine formulations that are FDA approved for opioid agonist medication-assisted treatment, we cannot rule out the off-label, unapproved use of buprenorphine. Concerns have been voiced over the emerging phenomenon of the misuse of buprenorphine.Third, our measures of prescription counts did not account for variations in formulation, strength, and dosage. The data lack the information to convert prescription counts to more standardized values such as morphine milligram equivalents. Nonetheless, we provided separate estimates for major subcategories of opioids. The estimates suggest that the reductions in opioid prescriptions were largely concentrated in Schedule II opioids, which, to some extent, helps alleviate the concern about potential substitution of prescriptions with higher morphine milligram equivalents  for those with lower ones that would otherwise undermine the study findings.Fourth, as with any observational study, we cannot establish a causal chain from buprenorphine-waivered physician availability, through buprenorphine treatment use, to prescription opioid use. Buprenorphine treatment use constitutes one of the potential pathways from buprenorphine-waivered physician availability to prescription opioid use. There may be other pathways in this causal chain. For instance, once they have received the buprenorphine prescribing training, buprenorphine-waivered physicians may become more aware of the opioid crisis and the problems with overprescribing opioids, thus reducing the use of prescription opioids in pain management. Nonetheless, the consistent results from the main and sensitivity analyses, as well as the supporting evidence from the secondary instrumental variable analyses, strongly suggested that expanding the availability of buprenorphine-waivered physicians has the potential to increase buprenorphine treatment use by Medicaid enrollees and reduce their prescription opioid use.Our study uses timely, comprehensive Medicaid administrative data and provides empirical evidence that an increase in the number of physicians qualified to provide buprenorphine treatment was associated with a significant increase in buprenorphine prescriptions and spending covered by Medicaid, which may have induced economically meaningful reductions in Medicaid-covered opioid prescriptions and spending. Our findings suggest that expanding treatment capacity for buprenorphine holds the potential to improve access to opioid addiction treatment, which may further reduce prescription opioid use and help slow the ongoing opioid epidemic in the United States.


The purpose of this review is to explore the role of monocyte chemoattractant protein-1 (MCP-1 or CCL2) in the processes that underpin bone remodelling, particularly the action of osteoblasts and osteoclasts, and its role in the development and metastasis of cancers that target the bone.MCP-1 is a key mediator of osteoclastogenesis, being the highest induced gene during intermittent treatment with parathyroid hormone (iPTH), but also regulates catabolic effects of continuous PTH on bone including monocyte and macrophage recruitment, osteoclast formation and bone resorption. In concert with PTH-related protein (PTHrP), MCP-1 mediates the interaction between tumour-derived factors and host-derived chemokines to promote skeletal metastasis. In breast and prostate cancers, an osteolytic cascade is driven by tumour cell–derived PTHrP that upregulates MCP-1 in osteoblastic cells. This relationship between PTHrP and osteoblastic expression of MCP-1 may drive the colonisation of disseminated breast cancer cells in the bone.There is mounting evidence to suggest a pivotal role of MCP-1 in many diseases and an important role in the establishment of comorbidities. Coupled with its role in bone remodelling and the regulation of bone turnover, there is the potential for pathological relationships between bone disorders and bone-related cancers driven by MCP-1. MCP-1’s role in bone remodelling and bone-related cancers highlights its potential as a novel anti-resorptive and anti-metastatic target. Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC-motif chemokine family (as CCL2); a large group of cell signalling molecules and cognate receptors. MCP-1 was the first discovered human chemokine and is well-known as a potent chemotactic factor for monocytes . It is pA further characteristic of chemokine biology is the proteolytic processing of chemokines , which cThe roots of disease can be found in the physiological mechanisms that underpin an organ's normal action. Physiological bone remodelling is a critical contributor to overall health, having roles in growth, structural preservation, repair and mineral homeostasis –10. BoneOsteoblasts are specialised bone-forming cells that arise from pluripotent mesenchymal stem cells –14. OsteBone cancer presents in two unfavourable forms: primary and secondary . PrimaryMetastases require intricate communication between cancerous cells and endogenous cells at the metastatic site. This interaction facilitates the dissemination of cancer cells to other locations within the body and adds a complex dimension to cancer treatment. The formation of bone metastases is controlled, in part, by the pre-metastatic and metastatic niches, in which the homing and colonisation of cancerous cells to a related tissue location occurs through the action of endogenous factors . The rolThe relationship of MCP-1 to bone remodelling is manifested through its involvement in osteoclast differentiation and maturation. Given a long-established role in monocyte responses to infection and injury, it was somewhat unexpected that MCP-1 had a role in osteoclast biology and, in particular, its stimulation of osteoclast formation. Since MCP-1 stimulates chemotaxis of monocytes –26.Osteoclasts differentiate from cells of the monocyte lineage; however, osteoclast differentiation requires the presence of M-CSF and RANKL: M-CSF is required for precursor cell survival while differentiation itself is controlled by RANKL . MonocytThe interaction between osteoblasts and osteoclasts with respect to RANKL and M-CSF is well known; the interaction between osteoblasts and osteoclast progenitors is not. Early work showed that recruitment of monocytes to the bone surface is mediated by MCP-1 . Li et aMCP-1 is rapidly induced in human osteoclast progenitors when differentiating into osteoclasts. This induction of MCP-1 precedes a similar but weaker induction of chemokine (C-C) motif ligand-3 (CCL3) •. Like MThe literature shows clear involvement of MCP-1 in bone remodelling, specifically in osteoclast differentiation; however, its exact role in this process remains to be determined. As mentioned, the primary receptor for MCP-1 is CCR2, which is expressed in human osteoclast precursors  and exisBinder et al. (2009) reported that CCR2-/- mice had a small but significant increase in tibial trabecular bone volume and a reduced number of osteoclasts in the bone at 10 weeks of age. Similarly, Sul et al. (2009) found a small but statistically significant increase in trabecular bone volume at the distal femur at 14 weeks of age. In vitro osteoclast formation assays showed that MCP-1-/- mice produced fewer osteoclasts that were about one-half as effective at bone resorption as wild type osteoclasts. Using mouse cells, Sul et al. (2012) confirmed earlier observations seen in human cells ••; that In addition to having a role in the cells that underpin bone function, MCP-1 also has a role in the hormonal regulation of bone. Decreased serum calcium activates the parathyroid gland to secrete PTH, increasing osteoclast activity to release calcium from bone and normalise serum calcium levels . ExcessiRemarkably, MCP-1 is the most iPTH-responsive gene with > 200-fold induction in bone after 14 days of iPTH treatment ••. Both It now appears, though, that MCP-1 is involved in regulating both the anabolic and catabolic actions of PTH via osteoclast formation . An osteDiseases of the bone characteristically include some type of interruption, interference or inhibition of the processes associated with bone remodelling. Bone loss induced by inflammatory diseases is believed to occur as a result of direct or indirect effects of inflammatory cytokines or inflammatory cytokine networks . RheumatPatients suffering from RA have significantly higher serum concentration levels of MCP-1 compared to control patients, with this increase in serum concentration levels also positively correlated with the number of osteoclasts cultured from their peripheral blood monocytes (PBMCs) . RecentlOne commonly used treatment for late-stage arthritis is the total joint replacement (TJR) . In caseUndoubtedly, one of the most readily associated diseases with bone is osteoporosis. Osteoporosis is a condition characterised by the disruption to the critical balance between bone resorption and bone formation, in which a favouring of bone resorption leaves the skeleton in a state of disharmonic predilection to bone loss •, 51. ThMCP-1 is implicated in a number of cancers and, coupled with its involvement in bone remodelling, places it in a central position to link these roles with bone metastasis . This alThe formation of bone metastases is controlled, in part, by the pre-metastatic and metastatic niches . It has Since the early 90s, PTHrP derived from cancer cells has been recognised as a key signal driving metastatic osteolysis •. PTHrP,With similar importance to osteoclast recruitment is tumour-associated macrophage (TAM) recruitment. The role of TAMs in tumour progression is varied and occurs at different levels; from the promotion of genetic instability to the suppression of protective adaptive immunity. TAMs have important roles across all stages of tumourigenesis, encompassing cancer stem cell nurturing and metastasis promotion . While tMCP-1 expression by tumours is correlated to the number of TAMs. Tewari et al.  reportedMCP-1, along with a manner of other inflammatory proteins, has been noted in many papers as an important component of cancer progression. Table These studies present a convincing case for the involvement of MCP-1 in skeletal metastasis. In a model of breast cancer bone metastasis, Ottewell et al. (2014) introduced the probability of a causal relationship between post-menopausal osteoporosis and the colonisation of disseminated breast tumour cells in bone, noting a significant difference in bone tumour occurrence in post-menopausal and pre-menopausal animals, where 83% of ovariectomised animals exhibited cancerous bone lesions compared to 17% of sham animals ••.Wright et al. (2017) expanded on this concept, investigating the potentially negative effects that some pharmaceuticals may have through activation of bone resorption. They specifically examined aromatase inhibitors (AI), a popular anti-cancer drug used in the treatment of breast cancers in post-menopausal women. They observed that prevention of AI-induced bone resorption mitigated the formation of bone metastases; subsequently implicating this treatment-induced bone loss in metastatic breast cancer progression .Breast cancer frequently eventuates in bone metastases . The rolThe formation of metastases is only possible because of the intricate communication between disseminated cancer cells and the metastatic organ of choice. Cancer cells communicate best with organs that have similar microenvironmental properties as the organ in which they originated. MCP-1 is ever-present within the breast tumour microenvironment as it drives breast cancer progression; similarly, MCP-1 is ever-present within the bone as a mediator of bone remodelling, and even more so within osteoporotic bone, given the increased osteoclastic expression of the chemokine. These similarities in MCP-1 expression may facilitate the communication between breast cancer cells and the tumour microenvironment, presenting MCP-1 as a driving factor of breast cancer bone metastasis.Advances in cancer biology have highlighted the inherent need for, and the great advantage of, targeted therapies. There is mounting evidence to suggest a pivotal role of MCP-1 in the mechanisms that underpin bone remodelling and skeletal metastasis. Evidence suggests a strong tumourigenic role for MCP-1 in cancer through its effects on monocyte recruitment, activation of tumour-associated macrophages, induction of angiogenesis, metastasis promotion and osteoclastogenesis. In breast and prostate cancers, an osteolytic cascade is driven by tumour cell–derived PTHrP that upregulates MCP-1 in osteoblastic cells. This accelerates tumour growth via enhanced osteoclastic and endothelial cell activity in bone marrow, supported by increased angiogenesis. Presently, MCP-1’s specific role in breast cancer bone metastasis, as to whether it promotes bone metastases or protects the bone from breast cancer cell invasion, remains inconclusive; however, its pivotal role in bone remodelling and breast cancer progression highlights the need for further investigation. Development of successful treatments for metastatic cancers proves challenging, especially for skeletal metastases where surgery to excise tumours is often impossible. Targeting MCP-1 has promise as a therapeutic option with increasing evidence for its role as a primary contributor to the development of several types of metastatic lesions; however, the reported metastatic rebound following anti-MCP-1 therapy in experimental animals suggests this pursuit must be navigated thoroughly and cautiously.