The evidence that exposure to ozone air pollution causes acute cardiovascular effects is mixed. We postulated that exposure to ambient levels of ozone would increase blood markers of systemic inflammation, prothrombotic state, oxidative stress, and vascular dysfunction in healthy older subjects, and that absence of the glutathione S-transferase Mu 1 (GSTM1) gene would confer increased susceptibility. This double-blind, randomized, crossover study of 87 healthy volunteers 55–70 years of age was conducted at three sites using a common protocol. Subjects were exposed for 3 h in random order to 0 parts per billion (ppb) (filtered air), 70 ppb, and 120 ppb ozone, alternating 15 min of moderate exercise and rest. Blood was obtained the day before, approximately 4 h after, and approximately 22 h after each exposure. Linear mixed effect and logistic regression models evaluated the impact of exposure to ozone on pre-specified primary and secondary outcomes. The definition of statistical significance was p<0.01. There were no effects of ozone on the three primary markers of systemic inflammation and a prothrombotic state: C-reactive protein, monocyte-platelet conjugates, and microparticle-associated tissue factor activity. However, among the secondary endpoints, endothelin-1, a potent vasoconstrictor, increased from pre- to post-exposure with ozone concentration . Nitrotyrosine, a marker of oxidative and nitrosative stress, decreased with increasing ozone concentrations, with marginal significance . GSTM1 status did not modify the effect of ozone exposure on any of the outcomes. These findings from healthy older adults fail to identify any mechanistic basis for the epidemiologically described cardiovascular effects of exposure to ozone. The findings, however, may not be applicable to adults with cardiovascular disease. Exposure to air pollution is a well-established risk factor for cardiovascular morbidity and mortality. Most of the evidence supporting an association between air pollution and adverse cardiovascular effects involves exposure to particulate matter . TropospMeta-analyses have concluded that increased ambient ozone concentrations are associated with an increased risk/rate of stroke , but notControlled exposure studies at pollutant concentrations relevant to ambient concentrations can explore the mechanistic basis for the associations observed in epidemiological studies between short-term exposures to ozone and acute cardiovascular disease outcomes, and identify markers of effect that would be useful in future observational  studies. Recently, two controlled human exposure studies , 28 in hWe postulated that initial reactions of inhaled ozone with lipid substrates on airways surfaces generate a variety of reactive chemicals that activate multiple airway cells. Mediators released from these cells enter the circulation and may cause activation of blood elements, notably platelets, and increase the production of acute phase reactants such as C-reactive protein (CRP). Activated endothelial and blood cells also generate and release membrane-enclosed vesicles (microparticles) that contain a variety of biologically active intracellular molecules. Such vesicles may serve an endocrine-like function by adhering to other cells and transferring their contents . These systemic changes could result in prothrombotic as well as other cardiovascular effects.Given that ozone exposure generates oxidative stress, individuals with impaired anti-oxidant defenses may be at increased risk for acute health effects. Soluble cytosolic GSH transferases (GST) belong to a supergene family with multiple classes which might be involved in detoxification of post-exposure inflammation-related oxidants or in the resolution of inflammation. The GST Mu 1 (GSTM1) gene   is partiThe Multicenter Ozone Study in oldEr Subjects (MOSES) was a controlled human exposure study in which we exposed a relatively large number of healthy older adults, who may be more susceptible to air pollution-induced cardiovascular health effects than younger adults , to filtwww.healtheffects.org. Briefly, MOSES was a controlled exposure study of the acute cardiovascular effects of ozone inhalation in healthy non-smoking adults (ages 55 to 70 years). The study was conducted in three clinical centers: the University of Rochester Medical Center (URMC), the University of North Carolina (UNC), and the University of California, San Francisco (UCSF); exposures were conducted from 2012 to 2015. The subjects underwent 3 randomized exposure sessions, each consisting of 3 consecutive days. At approximately noon on the first day of each session phlebotomy was performed; the second day involved randomized exposures to clean air (0 ppb), 70 ppb, and 120 ppb ozone for 3 h while alternately exercising and resting for 15 min, with phlebotomy 4 h after exposure; phlebotomy was performed again on the third day, 22 h after exposure. Exposure sessions were separated by a minimum of 2 weeks. Blood was collected from the antecubital vein of the arm not used for other procedures, into tubes containing sodium citrate. In situ platelet and endothelial activation were minimized by a short tourniquet time, minimizing trauma at needle entry, and discarding the first 5 mL of blood. Subjects with difficult venous access were excluded. The subjects, investigators, and study personnel, with the exception of the individual controlling the exposure chambers, were unaware of the nature of the exposures. The total duration of subject participation, from the screening visit to the final visit, typically varied between 3 and 6 months, but could be as long as 12 months. The design for MOSES has been previously described in detail . The stuWritten informed consent was obtained from all subjects at each clinical center. The study was approved by the institutional review board at each center and by the US EPA Human Subjects Research Review Official. A Certificate of Confidentiality was obtained from the US Department of Health and Human Services. The study was registered with ClinicalTrials.gov, Identifier NCT01487005.1) and forced vital capacity (FVC) ≥75% of NHANES predicted values and FEV1/FVC ≥0.65); ability to complete the training exercise regimen chosen to induce an inspired ventilation rate of 15–17 L body temperature and pressure, saturated (BTPS) /min/m2 body surface area (BSA), without exceeding 80% of predicted maximal heart rate; normal baseline 12-lead resting ECG, and absence of significant ST depression while performing the 15-min required level of exercise targeted for the exposure period; ability to avoid a specific list of medications and supplements.Subjects were recruited through advertisements at the three clinical centers. Inclusion criteria were as follows: nonsmoking males and females of all ethnic backgrounds, ≥55 and ≤70 years of age; normal spirometry ; plasma cotinine level >3 ng/mL; body mass index (BMI) >35 and <18; blood pressure >140 systolic or >90 diastolic or on anti-hypertension medications other than diuretics; pregnancy or nursing (breastfeeding); on the following medications: prednisone, statins, beta-blockers, anticoagulants, current systemic estrogen therapy, tamoxifen; and current occupational exposures to high levels of vapors, dust, gases, or fumes.Subjects were not studied within 6 weeks of a respiratory infection and were asked to abstain from caffeinated and alcoholic beverages starting with lunch on the day before the exposure through the post-exposure day.The exposures took place in ventilated, climate-controlled chambers at each of the clinical centers. The chamber characteristics and specific protocols for generating and measuring ozone were site-specific and are described elsewhere .All three centers generated ozone by silent electric-arc discharge in breathing-quality (USP grade) oxygen from a gas cylinder. The dilution air entering the exposure chamber was purified by passing it through Purafil, charcoal, and HEPA filters. The effluent from the ozone generator was mixed with the filtered air immediately before entering the chamber. The ozone output was controlled by varying the voltage of the generator and adjusting the flow rate. Temperature and relative humidity were targeted at 22°C and 40%, respectively. The concentration of ozone breathed by the subject was monitored continuously during the exposure by calibrated and externally certified analyzers.bold below) and several secondary outcomes, based on either previous evidence in the ozone health effects literature or clinical relevance.The MOSES outcomes were selected based on hypothesized cardiovascular mechanisms of action of ozone. For each potential mechanistic pathway we identified one or more primary outcomes , and a prothrombotic state , fibrinogen, and flow-cytometric indices of platelet activation, including monocyte-platelet conjugate count, activated platelet [CD62P+] count, platelet-derived microparticle [CD42b+] count, activated platelet-derived microparticle [CD42b+/62P+] count, tissue factor expressing microparticle [CD142+] count, and CD40 ligand-expressing microparticle [CD154+] count).In this paper, we report effects on peripheral blood biomarkers of systemic inflammation, oxidative stress, and endothelial function (++ activates added factor X (FX) (Enzyme Research Lab). The resulting FXa was then assayed colorimetrically (absorbance at 405 nm) in the MP pellet suspension along with suitable controls and standards in a 96-well plate with Pefachrome FXa 8595  [Plasma samples for the MP-TFA assay were analyzed as previously described , 38, at zerland) . An esseWe measured blood platelet activation and circulating microparticles using immunofluorescence and flow cytometry (FC), with modifications of methods previously reported . The FC The list mode data collected at each Center were analyzed using FloJo software . Platelets and monocyte-platelet conjugates were analyzed using a method adapted from Li et al. . MicropaCRP, IL-6, 8-isoprostane, nitrotyrosine, fibrinogen, ET-1, and vWF were measured in blood plasma by the AssayGate commercial laboratory . The specific assays used are described below. The laboratory utilized custom-designed antibody kits that are proprietary. The limit of detection (LOD) of each assay was defined as the value calculated from the standard curve at the point lying 2 standard deviations above the mean background media fluorescence intensity for 10 replicates.Luminex bead-based multiplex immunoassays were used to measure CRP, fibrinogen, vWF, IL-6, and P-selectin . Dilution was performed for samples falling outside the range of the standard curves. Samples were tested in duplicate. Positive controls with known analyte concentrations and negative controls on each bead plate allowed for assay quality assurance. Sandwich enzyme-linked immunosorbent assays (ELISA) were used to measure ET-1 and nitrotyrosine. A competitive ELISA was used to measure 8-isoprostane. A 4-parameter standard curve was prepared relating color intensity to the concentration of the target on each plate. Positive and negative controls on each plate allowed for assay quality assurance.https://www.healtheffects.org/system/files/MOSES-Pt1-AddtlMaterials-5.pdf.Power calculations for selected outcomes were performed prior to study initiation and again during the study. Details of these analyses can be found at Outcomes were calculated by subtracting the pre-exposure value from each post-exposure value. Any of these outcome variables that were not normally distributed, via the Kolmogorov-Smirnov test, were transformed using the natural logarithm . This resulted in more normal distributions of these outcomes. Outcome variables that were normally distributed were summarized using means with SDs, and those that were skewed, and transformed as described above, were summarized using both means with SDs and medians with inter-quartile ranges. Age was treated as a continuous value and centered by subtracting the mean age for the cohort.Linear mixed effect models, with a random subject effect and a ‘variance components’ covariance structure, were used to evaluate the impact of exposure to ozone on the pre-specified primary and secondary outcomes. Ozone was treated as a categorical (3-level) variable, with clinical center and time of measurement  included in all models. When there was a statistically significant effect of ozone on an outcome, the ozone by time interaction was then assessed to determine whether the ozone effect was consistent across the individual post-exposure times. Since this study involved a large number of comparisons, we pre-specified a limited number of outcomes as primary (three of these primary variables are reported here), and used α = 0.01 as the threshold for statistical significance, with α = 0.05 as the threshold for marginal significance. Results were then interpreted within the context of coherence among related variables, and plausibility. Significant changes in secondary variables were to be considered hypothesis-generating but not definitive, unless there were changes consistent with effects on the primary outcome variable(s) for that response pathway. In that case, the secondary variable was considered supportive of the findings for the primary variable(s). Analyses were performed using SAS, Version 9.3 or later  and R version 3.2.2 or later .A total of 1,062 individuals responded to the advertisements of the three clinical centers, and 594 of these underwent telephone screening . Eighty-The ozone concentrations in the chambers of all three centers were close to the target values. The mean (SD) temperature was 22.3 (0.7)°C, compared to the target of 22°C, and the mean (SD) relative humidity was 41.4 (3.0) %, compared to the target of 40%.Ozone caused no change in plasma CRP, IL-6, 8-isoprostane, or P-selectin. Descriptive statistics are shown in Nitrotyrosine decreased after 120 ppb ozone. In linear regression analysis, ozone effects on nitrotyrosine were marginally statistically significant (p = 0.017). As shown in ET-1 increased after 120 ppb, but not after 70 ppb ozone. In linear regression analysis, ozone effects on ET-1 were statistically significant . There were no significant ozone effects on MP. MP expressing CD40L showed a significant ozone-sex interaction . We observed a marginally significant ozone-age interaction  and increased with 120 ppb ozone (317 ug/mL) relative to 0 ppb ozone exposure, but this did not reach statistical significance (p = 0.048). There were no significant age, sex or GSTM1 interactions -dependent response . We hypoEndothelin-1 (ET-1) is a potent vasoconstrictor peptide originally isolated from endothelial cells that is now known to be produced by multiple cell types when stimulated, and has been linked to air pollution exposure –53. To oIncreased propensity for vascular thrombosis has been posited as one of the pathways by which exposure to air pollution leads to adverse cardiovascular events. We found no convincing evidence for ozone effects on platelet activation, circulating MP, or MP-TFA. We did find a significant ozone-sex interaction for CD40L+ MP, which decreased in females and increased in males following ozone, compared with air exposure, suggesting this response to ozone exposure may differ by sex. However, none of the other thrombosis markers supported an ozone effect or sex interaction, so we conclude there is no effect on prothrombotic vascular status at these ozone exposure levels in this population.A few other controlled human exposure studies have examined ozone effects on markers of coagulation or thrombosis, with variable results. Frampton et al.  exposed Arjomandi et al.  found noA recent controlled human exposure study examined the effect of varying temperature on the effects of ozone exposure . HealthyA few panel studies have examined the effects of ozone exposure on propensity for thrombosis, but the findings are also mixed. Strak et al.  obtainedA weight-of-evidence review  describeThis multi-center study has a number of strengths. It is one of the largest controlled human exposure studies of ozone to be conducted to date, providing greater statistical power than previous studies. The study was designed to comprehensively evaluate the mechanistic pathways by which air pollutants may contribute to acute cardiovascular toxicity.The lack of exposure to a higher inhaled dose of ozone limits comparability to the previous controlled exposure studies described above. We cannot therefore generalize the absence of blood biomarker effects to populations exposed to higher concentrations. Also, since we studied acute responses to short-term ozone exposures, we cannot exclude the possibility of effects of chronic or repeated exposures, or effects delayed longer than 22 h after exposure.The chamber ozone exposure levels of 120 ppb and 70 ppb used in MOSES were only slightly higher than peak ambient ozone levels occurring at the study centers on some days during the study periods. Thus, it is possible that ambient ozone and other pollutant exposures experienced by the study subjects before and during the study may have independently impacted the study biomarkers, and/or modified, blunted, or lessened any biomarker responses to the controlled ozone exposures. These questions are being examined and will be published separately.By design, we restricted the participants to older, healthy subjects, who were physically fit enough to complete the exercise regimen, and the findings may not be representative of people in other age groups. People with pre-existing cardiovascular disease may differ in their responses to ozone exposure. The choice to study older subjects was based on the hypothesis that these individuals would be at increased risk for acute cardiovascular effects of ozone, but younger individuals are known to be more responsive to the effects of ozone on lung function, and could also be more responsive to cardiovascular effects.Subject to the same limitations, other data from the MOSES study also failed to reveal significant acute effects on cardiovascular function , althougIn this multicenter clinical study of older healthy subjects, ozone exposure caused no effects on systemic inflammation. Blood levels of nitrotyrosine decreased with ozone exposure with marginal statistical significance, the opposite of our hypothesis. Blood levels of the potent vasoconstrictor, ET-1, increased with ozone exposure. The results of our study do not support acute effects of low-level ozone exposure in healthy older subjects on systemic inflammation, oxidative stress, or a pro-thrombotic state. We cannot exclude the possibility of effects with higher ozone exposure concentrations or more prolonged exposure, or the possibility that subjects with underlying vascular disease, such as hypertension or diabetes, would show effects under these conditions.S1 Appendix(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.S2 Checklist(DOCX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


This study reports a medium-term follow-up of a randomised, double-blind, placebo-controlled trial of omega-3 polyunsaturated fatty acids (PUFA) in ultra-high risk for psychosis (UHR) patients. Primary outcomes of interest were transition to psychosis and symptomatic and functional outcome. A secondary aim was to investigate clinical predictors of medium-term outcome. Three hundred four UHR participants were recruited across 10 specialised early psychosis services in Australia, Asia, and Europe. The intervention consisted of 1.4 g/daily of omega-3 PUFA or placebo, plus up to 20 sessions of cognitive-behavioural case management (CBCM), over the 6-month study period, with participants receiving further CBCM sessions on basis of need between months 6–12. Mean time to follow-up was 3.4  years. There was a modest increase in transitions between 12-month and medium-term follow-up (11–13%) and substantial improvement in symptoms and functioning between baseline and follow-up, with no differences between the treatment groups. Most improvement had been achieved by end of the intervention. 55% of the sample received mental health treatment between end of intervention and follow-up. Omega-3 PUFA did not provide additional benefits to good quality psychosocial intervention over the medium term. Although most improvement had been achieved by end of intervention the substantial rates of post-intervention mental health service use indicate longer-term clinical need in UHR patients. The post-intervention phase treatment or the longer-term effect of CBCM, or a combination of the two, may have contributed to maintaining the gains achieved during the intervention phase and prevented significant deterioration after this time. Omega-3 fatty acids, when given alongside traditional therapies, provide no extra benefits to patients at high risk of developing psychosis. In attempts to validate a previous clinical trial wherein omega-3 reduced the risk of ultra-high-risk patients transitioning to psychosis, Patrick McGorry, of the National Centre of Excellence in Youth Mental Health, Australia, and his team looked to replicate the findings in a larger cohort. Despite improvements in symptoms seen in a medium-term follow-up of 304 patients after ~3.4 years, no extra benefit or reduced transitioning was seen in those additionally receiving omega-3 fatty acids. The bulk of treatment benefit was seen between 0–12 months, but the data indicate a clear need for ongoing patient care. This study also revealed psychopathology score level, anxiety, and societal functioning to be strong indicators of psychosis transition risk. In line with the clinical staging model,12 during the earliest stage of illness, safer interventions, such as long-chain omega-3 polyunsaturated fatty acids (PUFA) and cognitive behavioural therapy (CBT), are regarded as the preferred option for first-line treatment. CBT has been found to be effective in many, though not all, of the published trials.19 The most striking result from the trials to date was the finding that omega-3 PUFA were greatly superior to placebo in reducing transition risk, and psychiatric morbidity in general, not only during the treatment period, but also for a prolonged period (median 6.7 years) subsequently.21 Omega-3 PUFA are safe, provide broad-spectrum health benefits, and represent a simple and relatively inexpensive treatment strategy.Twelve trials assessing psychosocial and pharmacological interventions, alone or in combination, have been conducted in UHR cohorts. Meta-analyses have shown that these interventions are effective, with an overall risk reduction of transition to psychosis of 54% at 12 months, with a number needed to treat (NNT) of 8.23 At the 12-month follow-up, we observed no difference in transition rates between the two groups (p = .76), with both groups improving on clinical and functioning measures.22 The failure to replicate the original study may have been due the lack of efficacy of omega-3 PUFA in this patient population. However, other possible reasons are that the lower than expected transition rate (~11% across groups) prevented a test of the efficacy of this treatment and that the other treatments received in both groups  and antidepressant medication) may have introduced a ceiling effect beyond which omega-3 PUFA, even if effective, could not be shown to confer additional benefit.22 CBCM was not provided in the original study and the rate of antidepressant prescription was lower in that study (10% compared to 62% in the current study.) In the current paper, we report a medium-term follow-up of the NEURAPRO study sample in order to determine if there was an increase in the rate of transition to psychotic disorder after cessation of treatment. Clinical and functional outcomes in the two treatment groups, as well as baseline predictors of outcome, were also of interest.We therefore attempted to replicate this finding from a single site trial in a larger cohort of 304 UHR participants across 10 sites internationally (the “NEURAPRO” study).24 Morrison and colleagues14 reported that cognitive therapy was superior to monitoring over 1 year. However, at 3-year follow-up, cognitive therapy was no longer superior in preventing transition,15 although it was found to be associated with a reduced likelihood of being prescribed antipsychotic medication. A study of olanzapine versus placebo showed trend-level benefits for reducing transition rate in favour of olanzapine after 12 months, although the difference did not reach statistical significance.25 At 2-year follow-up transition rate did not differ between the two groups.26 Bechdolf and colleagues27 reported that an integrated psychological intervention was superior to supportive counselling in high-risk patients at 24-month follow-up. Ising and colleagues18 reported benefits in favour of CBT compared to treatment as usual at 4-year follow-up, both in terms of transition rate and remission from UHR status. When pooled together, the medium-term outcome studies (24–48 months) have shown benefits in favour of specific interventions compared to comparison conditions, with the transition risk being reduced by 36% and a NNT of 12.5Previous medium-term outcome studies of UHR intervention trials have varied in their findings. The first medium-term follow-up found that the benefits observed at 6 months of combined risperidone and CBT compared to “needs based intervention” did not persist at 3–4-year follow-up.The aim of the current study was to investigate the medium-term outcome  of the NEURAPRO cohort. While the equally positive outcomes of the two treatment groups  achieved at 12-month follow-up may have been due to the CBCM and/or antidepressant medication received in both groups rather than due to the trial intervention (omega-3 PUFA), it was of interest to investigate whether these benefits were maintained over the medium term or whether there was deterioration in clinical outcomes after cessation of treatment. Secondary aims were to investigate baseline clinical predictors of medium-term outcome and the relationship between clinical measures at the end of the intervention period and symptoms/functioning at medium-term follow-up.22 Medium-term follow-up data were collected for 270 cases . Baseline characteristics are presented in McGorry et al.n = 98, 41%), hospital inpatient admissions , UHR services , community health services , drug and alcohol services , forensic services , and other services . Information on pharmaceutical treatment was available for 240 cases (eTable One hundred thirty-three participants (55%) had received mental health treatment between end of the study period and medium-term follow-up. These consisted of: general mental health services  were known to have transitioned to psychosis. Nineteen of these transitioned cases were from the omega-3 PUFA-treated group (12%) and 21 from the placebo-treated group (14%). Of the 40 transitioned cases, 31 (77.5%) were determined using the CAARMS, 6 (15%) were clinician-determined, and 3 (7.5%) were determined using hospital records. eTable n = 106, Vienna: n = 72, and other sites: n = 126). The baseline MADRS score was divided into two levels in randomisation:<21 and ≥21. The stratified log-rank test indicated that there was no significant difference between the two treatment groups in transition rates (p = 0.77). Cox regression was also used to compare the transition rates with site and MADRS score as stratifying variables. The hazard ratio of omega-3 PUFA vs. placebo was 0.91 (95% CI = 0.41–1.70), p = 0.77.The survival curves comparing transition rates of the two treatment groups are shown in Fig. General linear model analysis was used to compare the two treatments on symptom and functioning measures at medium-term follow-up with the corresponding baseline score, site and baseline MADRS score as covariates. As the follow-up time had a substantial range, its effect was accounted for as a factor of three levels . In order to examine whether the difference between the two treatments varied between the different levels of follow-up time, the interaction between follow-up time and treatment was also analysed.p-values for the interaction term  of all the measures were non-significant, indicating that the difference between the two treatments did not vary between different follow-up times. The p-values for treatment  were also non-significant for all measures, indicating that the two treatments did not differ in their effects. Approximately 50% of cases for whom data were available (n = 166) had remitted from UHR status, with no difference in remission rates between the treatment groups .All symptom and functioning measures showed improvement between baseline and medium-term follow-up Table . The p-vMost improvement on the symptom and functioning measures appeared to have been achieved by end of the intervention period (12 months), with only minimal further improvement between 12-month and medium-term follow-up. For example, the mean BPRS score across both treatment groups reduced by 7.8 (SD = 8.9) points by 12 months and by a further 1.5 by points by medium-term follow-up; mean SANS score reduced by 6.9 (SD = 11.7) points by 12 months and the same at medium-term follow-up; mean MADRS score reduced by 9.3 points (SD = 9.5) by 12 months and by a further 0.4 points at medium-term follow-up; mean SOFAS score increased by 14.5 points (SD = 18) by 12 months and by a further 0.6 points by medium-term follow-up.The list of potential predictors included demographic variables, symptom and functioning measures, and recruiting site. As in the other analyses, recruiting site was treated as a factor of three levels  with Melbourne used as the reference level. The association between each variable and transition risk was tested individually using Cox regression Table .Table 2Cp = 0.0000002), ethnicity (p = 0.002), and migrant status (p = 0.033) being selected as independent predictors of transition, with CAARMS Disorganised Speech, BPRS Psychotic sub-scale, BPRS Anxiety, SANS total, and SANS sub-scales, Global Functioning  score no longer remaining significant predictive variables. The finding of ethnicity and migrant status as predictive seemed to be driven by higher transition risk among non-Caucasian, non-migrants. This group showed a higher hazard ratio for transition to psychosis than Caucasian non-migrants , Caucasian migrants , and non-Caucasian migrants . This finding seems to be accounted for by the fact that the sites most highly represented by non-Caucasian non-migrants (Hong Kong and Singapore sites) had a somewhat higher transition rate (16%) than the other sites over the medium term. However, the strength and precision of this finding is limited by the small sample sizes of these sites, as indicated by the wide 95% CIs.A stepwise cox regression was then performed to analyse the predictive value of the variables after adjusting for each other. This analysis resulted in total BPRS score  at follow-up  score, SANS Attention subscale and site eTable , with thThe correlations of clinical measures between medium-term follow-up and baseline and end of treatment (12 month) are presented in eTable This paper reports a medium-term follow-up of an intervention trial of omega-3 PUFA plus CBCM vs. placebo plus CBCM in a UHR sample. The findings indicated a non-trivial but modest increase in proportion of known transitions between 12-month and medium-term follow-up (11–13%), with no significant difference in transition rate between the two treatment groups. On average, participants improved in their clinical symptomatology/functioning between baseline and medium-term follow-up, regardless of treatment condition, and approximately half remitted from UHR status. In fact, the group-level improvement was substantial, with clinical ratings indicating improvement from moderate/serious symptomatology and functional impairment at entry to mild symptoms and functional impairment at follow-up. On the whole, this improvement appeared to have been achieved by the end of the intervention period (12 months), with only minor further improvement between 12-month and medium-term follow-up.The strongest independent baseline predictor of transition to psychosis was general psychopathology levels . Participants of a non-Caucasian non-migrant background had a somewhat higher transition risk, which seemed to be a site effect. Baseline anxiety, negative symptoms (particularly attention disturbances), and poor role functioning independently predicted poor functional outcome. A further site effect was also apparent, with participants from the Melbourne site showing substantially poorer functioning at medium-term follow-up.27 However, it is inconsistent with other studies26 which reported a substantial escalation in transition rate after end of treatment and concluded that the benefits of specific intervention were only apparent during the treatment phase. There are a number of possible reasons for the only modest increase observed in the current study. All participants received CBCM during the intervention phase. The CBT aspect of this intervention was specifically directed toward cognitive restructuring, building resilience against stress, and stress management.23 These aspects of the psychotherapy may have been protective over the longer term, rather than just dealing with immediate mental health and contextual difficulties during treatment. This is consistent with the higher medium-term transition rate in the Hong Kong and Singapore sites compared to the other sites, because these sites provided a lower number of CBCM sessions (mean of 7.2 sessions compared to 10.6 sessions among the other sites). The possibility of the effect of CBCM, with omega-3 PUFAs not conferring additional benefits, is indicated by the lack of difference in transition rate between the treatment groups at 12-month and medium-term follow-up, as well as by the substantial clinical improvements achieved in both groups by the end of the intervention phase.The modest increase in transition rate after cessation of specific targeted intervention is consistent with some previous medium-term follow-up studies.28) and conferred benefit over the longer term . The use of antidepressants in approximately 20% of participants over the follow-up period may also have contributed to preventing the transition rate from escalating more substantially over this period, as well as possibly also contributing to the ongoing improvement in psychopathology and functioning. Additionally, approximately 10% of the sample used antipsychotic medication over the follow-up period . Finally, the use of omega-3 supplementation or change in dietary habits was not controlled after the study intervention period, which may have contributed to lack of difference between the treatments groups at medium-term outcome and the modest overall transition rate, if indeed omega-3 PUFAs do have a protective effect in high-risk patients. Although most clinical improvement seemed to have been achieved by the end of the intervention period (12 months), the high proportion of the sample who received  mental health support after the intervention period indicates ongoing clinical need in this population after 1 year of care.Approximately half (55%) of the sample for whom data were available received ongoing mental health treatment after the end of the study intervention period, which may have had the effect of maintaining the gains achieved during the intervention phase as well as preventing deterioration. A substantial proportion of the sample (62%) received antidepressants during the intervention phase, which may had both an immediate effect on lowering the transition rate  vs. medium-term (>2 year) outcome in this sample. Baseline depression was a strong predictor of transition by 12 months,22 with a MADRS cut-off score of 14 distinguishing between a 16.5 vs. 0% 12-month transition rate, whereas depression did not appear as a significant predictor over the medium term, with general psychopathology scores playing a stronger predictive role. This suggests, from a clinical point of view, that inferring level of risk based on clinical characteristics of new patients should not be based solely on research into predictors of short-term outcome, but also on variables that predict the longer-term outcome of patients. For example, if results from the current cohort were to be used to form a clinical risk calculator,40 entry depression scores might have identified those at short-term risk but missed those at medium-term risk. The results also show, as one would expect, that clinical state over the medium term appears to be better predicted by clinical state at the end of 12 months of treatment than by baseline symptom and functioning scores.The prediction analysis conducted in this report reaffirms43 While anxiety is known to be associated with compromised functioning in adult populations,45 its role may have been somewhat neglected in psychosis risk research and youth mental health more broadly. The predictive role of anxiety, general psychopathology, depression (over the short term) and negative symptoms for poor outcome (psychosis onset and poor functioning) reinforces the value of a broad, formulation-based target of intervention for the UHR group.46 Although attenuated positive psychotic symptoms are a common feature of the population , a range of general psychopathological characteristics play a role in predicting poor outcome and should therefore be a focus of intervention.Baseline anxiety, poor role functioning, and negative symptoms (particularly attention disturbances) predicted poor functional outcome. The predictive role of negative symptoms is consistent with previous findings in UHR cohorts.Functioning levels at medium-term outcome were found to vary by recruitment site, with the Melbourne site showing poorer functioning than other sites. This may have been due to socio-economic factors, given that a large portion of the Melbourne site’s catchment area is of deprived socio-economic status compared to the other recruiting sites. This differential outcome by site does not seem to have been due to differences in treatment received over the follow-up period, with 55–60% of participants at each recruitment site receiving treatment over this period.https://clinicaltrials.gov/ct2/show/NCT01429454; https://clinicaltrials.gov/ct2/show/record/NCT02597439). The efficacy of omega-3 PUFAs in sub-groups of patients should also be investigated—for example, in those with aberrant membrane fatty acid levels or inflammatory markers. Predictive modelling in this patient population should also incorporate time-dependent or dynamic characteristics,48 rather than relying solely on baseline clinical variables, and examine predictors of persistent or incident non-psychotic outcomes.50 Group-level prediction analysis, as we have conducted in the current report, may mask important sub-group  differences, which will be the focus of subsequent reports. Finally, although a high follow-up rate (89%) was achieved on the main outcome of interest (transition to psychosis), availability of follow-up data on other outcomes (59% for functioning and 41–46% for symptom measures) were more modest. This may have introduced an attrition bias toward more favourable outcome data, given that previous research51 indicates difficulty recontacting members of adolescent and young adult psychiatric cohorts is associated with increased presence of disorder at follow-up.Given the negative findings of this trial, future work needs to further investigate whether omega-3 PUFA may play a role in treatment of this patient population. Other trials are currently underway to this end , although high rates of post-intervention mental health service use indicate ongoing clinical need after this time. This post-intervention phase intervention or the longer-term effect of CBCM, or a combination of the two, may have contributed to maintaining the gains achieved during the intervention phase and prevented significant deterioration after this time.52 on the basis of need between months 6 and 12. See Markulev et al.23 for full details of study methodology, inclusion/exclusion criteria and interventions, and McGorry et al.22 for the 6 and 12-month results.This was a randomised, double-blind, placebo-controlled treatment trial of omega-3 PUFA plus CBCM or placebo plus CBCM. Treatment was provided for 6 months, with participants receiving further CBCM53 The National Health and Medical Research Council of Australia (NHMRC) National Statement on Human Research was adhered to and appropriate ethical approval was obtained by each site . Written informed consent was obtained for those younger than 17 years, parental or guardian consent was sought. The trial was registered at the Australia and New Zealand Clinical Trials Registry (ID 12608000475347).The study was performed in accordance with the Declaration of Helsinki.Medium-term follow-up of the sample was conducted in 2015–2016. The procedure consisted of the following steps to locate and recontact participants: (1) accessing the National Death Index to determine whether any participant had died since last contact, (2) research files, (3) public mental health service record systems, (4) National Electoral Roll, (5) telephone directory, (6) previous contacts, and (7) internet-based searching. Re-contacted participants were invited to a comprehensive face-to-face interview. If individuals did not consent to face-to-face assessment, they were asked for a brief telephone assessment, enabling collection of a minimum dataset. The brief assessment consisted of a sub-set of measures, including determination of transition status and functioning levels. When participants could not be contacted, hospital records were consulted to collect details of public health service contact, date of contact and diagnosis.42 Diagnoses were determined with the Structured Clinical Interview for DSM-IV-TR Axis I Disorders. If CAARMS data were not available, the public mental health records were accessed to determine if the participant had developed a psychotic disorder. Secondary outcome measures included the Brief Psychiatric Rating Scale (BPRS),54 the Scale for the Assessment of Negative Symptoms (SANS),55 the MADRS,56 the Young Mania Rating Scale (YMRS),57 the SOFAS,58 and the Global Functioning: Social59 and Role60 scales.The main outcome of interest was transition to psychosis, with transition defined on the basis of operationalized criteria and assessed with the Comprehensive Assessment of the At-Risk Mental State (CAARMS).56 score (<21 and ≥21) as stratifying factors. General linear modelling was used to compare the secondary outcomes between the two treatment groups. Baseline clinical predictors of outcome were examined using stepwise cox regression. The relationship between clinical measures at the end of the treatment phase and medium-term follow-up was examined using Pearson correlation.The primary analysis used the intention-to-treat approach, comparing the difference in transition rates between the treatment groups using survival analysis with the stratified log-rank test and Cox regression with recruitment site and baseline Montgomery-Asberg Depression Rating Scale (MADRS)The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary material


Self-harm is a major risk factor for suicide, with older adults (older than 65 years) having reportedly greater suicidal intent than any other age group. With the aging population rising and paucity of research focus in this age group, the extent of the problem of self-harm needs to be established. In a primary care cohort of older adults we aimed to investigate the incidence of self-harm, subsequent clinical management, prevalence of mental and physical diagnoses, and unnatural-cause mortality risk, including suicide.The UK Clinical Practice Research Datalink contains anonymised patient records from general practice that routinely capture clinical information pertaining to both primary and secondary care services. We identified 4124 adults aged 65 years and older with a self-harm episode ascertained from Read codes recorded during 2001–14. We calculated standardised incidence and in 2854 adults with at least 12 months follow-up examined the frequency of psychiatric referrals and prescription of psychotropic medication after self-harm. We estimated prevalence of mental and physical illness diagnoses before and after self-harm and, using Cox regression in a matched cohort, we examined cause-specific mortality risks.Overall incidence of self-harm in older adults aged 65 years and older was 4·1 per 10 000 person-years with stable gender-specific rates observed over the 13-year period. After self-harm, 335 (11·7%) of 2854 adults were referred to mental health services, 1692 (59·3%) were prescribed an antidepressant, and 336 (11·8%) were prescribed a tricyclic antidepressant (TCA). Having a diagnosed previous mental illness was twice as prevalent in the self-harm cohort as in the comparison cohort  and with a previous physical health condition prevalence was 20% higher in the self-harm cohort compared to the comparison cohort (1·20 [1·17–1·23]). Adults from the self-harm cohort (n=2454) died from unnatural causes an estimated 20 times more frequently than the comparison cohort (n=48 921) during the first year. A markedly elevated risk of suicide (hazard ratio 145·4 [95% CI 53·9–392·3]) was observed in the self-harm cohort.Within primary care, we have identified a group of older adults at high risk from unnatural death, particularly within the first year of self-harm. We have highlighted a high frequency of prescription of TCAs, known to be potentially fatally toxic in overdose. We emphasise the need for early intervention, careful alternative prescribing, and increased support when older adults consult after an episode of self-harm and with other health conditions.National Institute for Health Research Greater Manchester Patient Safety Translational Research Centre. Suicide is a major public health issue worldwide. With consideration of socioeconomic differences between regions, suicide risk rises with increasing age.Non-fatal self-harm is the strongest risk factor for subsequent suicide.8Evidence before this studyWe searched Embase and PubMed with initial title terms related to self-harm  and an older population . We included further combination terms related to epidemiology of self-harm, including “mortality”, “epidemiol*”, “prevalence”, “incidence”. We checked publications covering all ages for adult populations to ensure older age bands were included in results sections. Further combination searches for clinical management, including referrals and treatment prescribing, and mental and physical diagnoses, were also included. We searched for articles published from Jan 1, 2000, to Dec 31, 2017, and set no restriction on language, method, or quality of publication. We also checked citations of relevant publications. No publications were found on self-harm specific to older age populations at primary care level. Most studies relating to self-harm among adults of an older age were based on hospital emergency department attendance, mortality statistics, or register-based studies of adult populations of all ages. These reported on risk factors for self-harm repetition or suicide or described incidence, self-harm method, and suicide as a primary outcome. Clinical management in older adults after self-harm was rarely reported. One multicentre, hospital-based study in England reported self-harm across all adult age ranges and provided some data on referrals after hospital admission. We did not identify any publications on prescribing patterns in older adults after self-harm. One US study, investigating specific self-poisonings, detailed medication use at the time of self-harm. Reports on mental and physical illness diagnoses specific to self-harm populations were scarce. Most presented illness diagnoses as risk factors to suicide outcome and were from small psychological autopsy studies. Mortality risk literature was based only in hospital settings.Added value of this studyThis study is a novel primary-care-based study with linkage to hospital and mortality records to establish the largest primary care dataset of self-harm in older adults of its kind. We have identified a group of older adults who registered with a general practice as being at high risk of premature unnatural death, particularly within the first year of a self-harm episode. Further, we have identified low referral rates contrary to National Institute for Health and Care Excellence (NICE) guidelines, which suggest that older adults should be seen by a mental health professional after an episode of self-harm. Additionally, we have identified a high rate of prescription of tricyclic antidepressants (TCAs); NICE guidelines clearly recommend their avoidance after self-harm. We have also shown an increased prevalence of psychiatric and physical diagnoses before self-harm, previously shown to be important risk factors for self-harm.Implications of all the available evidence:Our findings suggest that health-care professionals in primary care have the opportunity to consider self-harm risk when older adults consult with complex mental or physical health-care needs. Additionally, after self-harm in an older adult, improvement of referral rates and consideration of possible alternative medication, with particular avoidance of TCAs, might reduce the risk of escalating self-harm behaviour and associated mortality risk.A 3% annual increase has been seen in the proportion of adults aged 60 years and older in the global population.In England, the National Institute for Health and Care Excellence (NICE) clinical guidance recommends that older adults are assessed by an old-age psychiatrist or mental health specialist after a self-harm episode because of raised levels of suicidal intent at older age.Electronic primary care patient records, containing routinely collected information on symptoms, diagnoses, prescribed medication, and referrals to secondary care services, were extracted from the Clinical Practice Research Datalink (CPRD). This source contains over 4·4 million active patient records from 674 registered general practices, covering 6·9% of the UK population. It is broadly representative of the national population in terms of age, gender, and ethnicity.The study cohort consisted of adults aged 65 years and older with a self-harm episode recorded during calendar years from 2001 to 2014. Self-harm episodes were identified by Read codes from patients' electronic primary care health records, which capture health-care interaction from multiple sources, including information captured during a GP consultation and notification of an episode to the patient's GP after hospital attendance. Self-harm was defined, as in NICE Clinical Guideline CG16,In the first analytical phase, we calculated annual incidence from the number of older adults with a record of self-harm during each calendar year in relation to the total number of older adults registered with a GP who were at risk during the same year. Stratified on age band, IMD, and region of residence, we applied strata-specific rates to calculate directly standardised incidence of self-harm as the outcome. The IMD is an ecological deprivation measure derived as a composite of several socioeconomic indicators for small areas based on practice location or patient's residential postcode.In the second analytical phase, we assessed clinical management as an outcome according to referrals to specialist mental health services and prescription of psychotropic medication by a GP during the year after the index self-harm episode. We captured psychiatric referrals using the Family Health Services Authority Psychiatry code, National Health Service specialty fields, and further Read codes identified as indicating a psychiatric referral. Psychotropic medication included prescriptions issued for antidepressants, antipsychotics, and hypnotics or anxiolytics. We stratified clinical management variables by gender, age group, and IMD quintile on the basis of general practice location.For the third analytical phase, we implemented a matched cohort study design to describe mental and physical illness prevalence as an outcome in both a self-harm and comparison cohort and to investigate mortality risk as an outcome after self-harm. We restricted the cohort to patients registered and up-to-standard for research purposes at general practices for at least a year before cohort entry, within England, and participating in the CPRD-HES-ONS linkage scheme. We included incident self-harm episodes recorded in the CPRD between Jan 1, 2001, and March 31, 2014, in which no previous self-harm episode was identified from both the primary care record and via linkage to HES records from ICD-10 codes X60–X84.9. Each incident episode was matched by age, gender, and registered practice with up to 20 comparison individuals with no record of self-harm on the index self-harm date of its corresponding matched case. We opted to sample this many patients in the comparison cohort to maximise statistical power and precision for examination of unnatural death as a rare outcome.We identified psychiatric and physical illness diagnoses recorded before index date entry and separately identified new diagnoses after the index date from Read and ICD-10 codes recorded in CPRD or linked HES records in both the self-harm and comparison cohorts. The psychiatric diagnostic categories included depression, anxiety disorders, personality disorders, bipolar disorder, schizophrenia-spectrum disorders, and dementia. Physical health diagnoses, based on the Charlson comorbidity index, included cancer, cerebrovascular disease, chronic pulmonary disease, congestive heart disease, diabetes with and without complications, hemiplegia, metastatic tumour, mild and moderate liver disease, myocardial infarction, peptic ulcer disease, peripheral vascular disease, renal disease, and rheumatological disease. We generated dichotomous indicator variables for any psychiatric or physical illness. Additionally, we calculated the Charlson comorbidity index scoresWe defined classification of cause-specific mortality using the underlying cause of death code from ICD-10 and included all natural deaths , all unnatural deaths (V01–Y98), and suicides . To estimate the risk of all-cause and cause-specific mortality, we used Cox regression modelling, producing both unadjusted hazard ratios [HRs] and HRs adjusted by IMD quintile based on a patient's postcode. Adjustments by mental illness diagnoses were not appropriate in this instance because these were suspected to be on the causal pathway. We did, however, report prevalence of diagnosis for serious mental illness before and after the index date. The index date of the self-harm cohort was defined as the occurrence of the first ever recorded self-harm event. Follow up started from the index date in both the self-harm cohort members and their matched comparators. Records were right-censored at the date of the first occurrence of either the end of the follow-up period , the practice last data collection date, date of transfer out of the CPRD , or death. We evaluated the proportional hazards assumption formally using Schöenfeld's residuals.All code lists applied in this study are published online and we used Stata/SE 14.2 for all analyses.The funders of the study had no role in study design, data collection, analysis, or interpretation. The corresponding author had full access to all the study data and final responsibility for publication submission.During the 2001–14 observation period, 4124 adults aged 65 years and older were identified as having an incident self-harm episode. Of these, 3327 (80·7%) involved ingestion of drugs, 234 (5·7%) involved self-cutting, a further 118 (2·9%) were by some other means, and 435 (10·6%) had no method specified. The overall incidence was 4·1 per 10 000 person-years and stable gender-specific incidences were observed across the entire 13-year period . Across vs 9·7%; χ2=7·84; p=0·005; vs 66·2%; χ2=5·49, 1 df; p=0·01). Women were more often prescribed psychotropic medication than men across all psychotropic medication categories  of 2854 older adults were referred to mental health services, although more women were referred than men . A diagnosed previous mental illness was more than twice as prevalent in members of the self-harm cohort than the comparison cohort , as was a subsequent diagnosis of mental illness  deaths occurred in the self-harm cohort compared with 12 683 (25·9%) deaths in the comparison cohort, of which 54 (5·9%) were unnatural deaths and 36 (4·0%) were suicides in the self-harm cohort versus 275 (2·2%) unnatural deaths and 12 (<0·1%) suicides in the comparison cohort . MortaliThis cohort study used CPRD, one of the world's largest electronic primary health-care datasets, and found that referral rates to mental health services were low, with women more likely to be referred than men, and likelihood of referral from general practices in more socially deprived localities was lower than from less deprived areas. Prescription rate was high for psychotropics within 12 months of the self-harm episode. 12% of the self-harm cohort were prescribed TCAs. Mental illness diagnosis was more than twice as prevalent with a higher level of multimorbidity among the self-harm cohort compared with the comparison cohort. We found that older adults (65 years and older) after self-harm were at increased risk of dying unnaturally, particularly of suicide, compared with individuals without a history of self-harm.This is the first study to focus on self-harm exclusively among an older aged population from a primary care perspective. We report for the first time the clinical management in primary care during the first year after self-harm and found a low rate of referral to psychiatric services. NICE guidelines for short-term management of adults after self-harmThe NICE guidance for the long-term management of self-harm (CG133)We report a high proportion of TCA prescribing after self-harm; TCAs are known to be fatally toxic in overdose and some TCAs more harmful than others.Few studies report mental health diagnoses before and after self-harm in older adults, with most based on psychological autopsy and suicide or investigating risk factors. In a retrospective cohort% had a repeat episode after deliberate self-harm.Within 12 months of initial self-harm, 14·4% of older adults had a further self-harm episode, which is similar to findings reported from a hospital-based studyThe higher rate of repetition found in our cohort and the markedly increased risk of suicide and unnatural death, particularly within 12 months of a known self-harm episode, identify a high-risk group of older individuals within primary care. Additionally, with older aged adults showing greater suicidal intent inferred from previous studies,The low referral rates we report and the high rates of referral back to primary care after hospital admission from previous hospital-based studies, emphasise the importance of a GP's role to ensure individuals are assessed and monitored effectively after self-harming behaviour. Additionally, the inequity in referral across differing socioeconomic status highlights an important target for improvement across the health-care system.As highlighted by a systematic reviewThe present study did, however, have some limitations. First, the coding recorded within the CPRD and HES datasets is reliant on the clinicians who assign and enter the codes. Second, some hospital-treated cases of self-harm will not have been reported to an individual's GP and, therefore, will not have been captured in CPRD.This novel primary-care-based study with linkage to hospital admissions and mortality records furthers understanding of self-harm among older aged adults. We have identified a group at high risk of premature unnatural death and identified areas of improvement for clinical management of older-aged adults in primary care. Health-care professionals should take the opportunity to consider the risk of self-harm when an older person consults with other health problems, especially when major physical illnesses and psychopathology are both present, to reduce the risk of an escalation in self-harming behaviour and associated mortality.code lists see www.clinicalcodes.orgFor more on the Clinical Practice Research Datalink see www.crpd.com/researcherFor the UK Health and Social Care Information Centre see www.hscic.gov.uk/hesdataFor the UK Office for National Statistics see www.ons.giv.uk/ons/indexFor the


There are a wide range of therapies for metastatic colorectal cancer (CRC) available, but outcomes remain suboptimal. Learning the role of the immune system in cancer development and progression led to advances in the treatment over the last decade. While the field is rapidly evolving, PD-1, and PD-L1 inhibitors have a leading role amongst immunomodulatory agents. They act against pathways involved in adaptive immune suppression resulting in immune checkpoint blockade. Immunotherapy has been slow to impact the management of this patient population due to disappointing results, mainly when used broadly. Nevertheless, some patients with microsatellite-instability-high (MSI-H) or mismatch repair-deficient (dMMR) CRC appear to be susceptible to checkpoint inhibitors with objective and sustained clinical responses, providing a new therapeutic option for patients with advanced disease. This article provides a comprehensive review of the early and late phase trials with the updated data of PD-1/PD-L1 inhibitors alone or in combination with other therapies . While data is still limited, many ongoing trials are underway, testing the efficacy of these agents in CRC. Current and future challenges of PD-1 and PD-L1 inhibitors are also discussed. Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. The incidence across the globe is different in various countries, but about 55% of the cases occur in more developed regions. Despite this fact, geographic patterns are very similar in men and women . EstimatRecently immunotherapy changed the treatment paradigm and its respective outcomes in many tumor types like melanoma, renal, bladder and lung cancer.Nobel Prize in Physiology or Medicine of 2018 was awarded for the discovery of cancer therapy by the inhibition of negative immune regulation. Research lead by Prof. Honjo identified PD-1 (Programmed cell death protein-1) as a component of inhibition of the immune system, noting that disruption of PD-1 in pre-clinical models resulted in increased immune system activity. This key information helped initiate the development of anti-PD-1/PD-L1 (Programmed death ligand-1) checkpoint inhibitors that are now a standard of care treatments for several tumor types . PD-1 isFindings of previous studies with PD-1 inhibitors in mCRC were not notably encouraging. Responses were only rarely seen . FurtherBesides dMMR/MSI-H patients, other subjects who respond to PD-1/PD-L1 inhibitors have tumors with an even higher TMB known as hypermutated tumors, e.g., tumors harboring polymerase proofreading domain mutations (POLE), which are thought to also be susceptible to immunotherapy , 16.dMMR CRCs are found in 15–20% of stage II and III CRCs and are associated with a better prognosis than proficient mismatch repair (pMMR) tumors. In the metastatic setting, dMMR CRCs represent only around 5% and are associated with a worse prognosis .Researches showed that most CRC patients are not responsive to immunotherapy. Several factors may be involved in this lack of sensitivity. Lack of T-cell infiltration, type 1 T-helper cell activity and low immune cytotoxicity in tumor microenvironment are thought to be involved , 19. ManAn article search was performed in PubMed and Cochrane databases to identify clinical and randomized controlled trials published until August 2018. Multiple algorithms that included the following terms were used: colorectal cancer, colon cancer, immunotherapy, PD-1, PD-L1, MSI-H, dMMR, and microsatellite instability. Inclusion criteria used were published full articles, clinical trials, retrospective series, meta-analyses, and English language. Since immunotherapy is a very active research field, we also explored abstracts from known oncology conferences during 2014–2018. Our systematic review focused on published clinical trials according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines . The floThe first study to show the clinical efficacy of PD-1 blockade in the microsatellite instability subset of colorectal cancer patients was with Pembrolizumab, an Anti-PD1 inhibitor. The trial examined the treatment of tumors with MMR deficiency following a recurrence of disease after standard treatments in a wide range of malignancies. Clinical activity of pembrolizumab 10 mg/kg was evaluated in 3 patient groups: those with dMMR mCRC, pMMR and a third group that included patients with dMMR in other malignancies (non-CRC) . OverallA second phase Ib trial, the KEYNOTE-028, evaluated Pembrolizumab in PD-L1–positive advanced solid tumors. The results for the CRC cohort were published . PatientKEYNOTE-164 was another study with Pembrolizumab. The cohort included patients previously treated with more than one line of therapy with mCRC MSI-H . PatientNivolumab was initially studied in phase I trial on various solid tumors. Only 14 patients enrolled had mCRC. A durable complete response was reported on a single patient. Nivolumab was further evaluated in a phase II trial on 296 patients but no objective responses were achieved among those with mCRC. As the only responder was found to have a deficient MMR (dMMR) tumor, this status was presumed to be a predictor of efficacy .A phase 2 trial, CheckMate 142, provided further evidence for the use of Nivolumab in dMMR/MSI-H mCRC . SeventyThe large population of mCRC patients are not dMMR/MSI-H which make up to 95% of patients. Based on previously reported low efficacy of immunotherapy in unselected patients, several combination regimens with local ablation, chemotherapy or molecularly targeted drugs have been already evaluated  and a laThe combination of immune checkpoint inhibitors with Nivolumab and Ipilimumab (anti-CTLA4) in dMMR/MSI-H mCRC patients were studied in a cohort with 119 patients of the CheckMate 142. Published outcomes demonstrated a consistent clinical effect with an ORR of 55% and a 12-weeks disease control rate-rate 80% . ResponsOther studies combining Pembrolizumab with chemotherapy were published. Pembrolizumab plus Azacytidine was evaluated in a phase 2 trial to assess anti-tumor activity and safety in patients with previously treated mCRC without standard treatment options. The combination showed a tolerable safety profile but had a little anti-tumor effect for MSS mCRC . PembrolThe combination of Atezolizumab an anti–PD-L1 monoclonal antibody with FOLFOX and Bevacizumab was studied in a multi tumor phase I trial. Preliminary data suggests that the combination can promote immune-related activity resulting in enhanced efficacy. However, more robust data is need .Atezolizumab in association with Cobimetinib a MEK1/MEK2 inhibitor in the MAPK pathway was also studied in patients with MSS/MSI-L mCRC in the IMblaze370 trial. The results of primary analysis were presented recently. The study did not however meet its primary endpoint which was OS with a median of 8.9 month with combination vs. 8.5 month with regorafenib. In this study, almost all patients included (91.7%) had tumors with MSS/MSI-L status .Lastly, PD-L2 Fc fusion protein that binds to PD-1, known as AMP-224, was tested in combination with stereotactic body radiation therapy (SBRT) plus cyclophosphamide in mCRC with hepatic metastasis. This combination appeared safe and feasible, but preliminarily data showed no objective responses. Expected in a near future are clinical and correlative data including post-therapeutic radiated and non-radiated tumor biopsies .Many of the published studies are early phase clinical trials, with limited number of mCRC patients recruited. At least six trials with favorable objective response and improved progression-free survival in patients with MSI-H CRCs were observed. These results are encouraging. However, the population of dMMR/MSI-H is very low, representing only 5% of patients in the metastatic setting. ORR in CheckMate142 and KEYNOTE-164 was around 30% with a substantially higher rate 52% reported in KEYNOTE-016; 1-year overall survival rates were very high which may indicate durable responses. .The clinical trials with PD-1/PD-L1 inhibitors also revealed different results according to the patient population studied. Outcomes in MSS/pMMR population including a phase III study with Atezolizumab plus Cobimetinib were negative, not providing any evidence of its use in this context.In non-metastatic CRC, dMMR accounts for approximately 15–20% which can be found more frequently. An adjuvant trial with an anti-PD-L1 is ongoing, but it remains unknown its effectiveness in this situation. Adjuvant ipilimumab or nivolumab have shown to extend survival in stage III or IV (resected) but mechanisms of resistance in CRCs in this setting are not necessarily the overexpression of immune checkpoints .Particular patients may present primary or secondary resistance to immune checkpoint inhibitors. It will be important in the future to study mechanisms of primary and acquired resistance to PD-1 blockade. Furthermore, there are many rationales for the study of combination therapies with PD-1 inhibitors and compounds targeting other immune regulators . They might prove together more effective or have other properties after certain resistances. The highlighted any many other strategies involving targeted therapy, chemotherapy and radiotherapy are ongoing with the aim to enhance response to immunotherapy , 3.Current predictive biomarkers for the efficacy of anti PD1 are dMMR status and POLE mutations. POLE mutations are associated with an ultramutated phenotype and are reported in 1–2% of colorectal cancers . Still aThe challenges pointed out, and many others are ahead and will include other clinical and feasibility issues. Questions like how to increase or reinforce the efficacy of immunotherapy, the optimal duration and combinations of treatments  and indiThe outcome of CRC patients has improved considerably over the past two decades. The efficacy of systemic therapies and biomarker-based treatments has been predominant in this positive change. A better understanding of the interaction between a tumor and the immune system in the last decade led to the development of new agents, in particular, the PD-1/PD-L1 inhibitors.Even though PD-1 inhibitors have shown efficacy in dMMR CRCs, there are still many questions which need to be answered, e.g. how could we conceive a response on pMMR CRCs and how to find predictive biomarkers of efficacy. dMMR/MSI-H tumors only account for a small 5% of mCRC. There is no doubt that extending the benefit of immunotherapy to a broader, microsatellite stable population would be the next but nevertheless difficult step.Many trials are already in progress exploring combinations strategies. Other important topics that in the future will be important to address are the role of immunotherapy and anti-PD1 therapy in the prevention of CRC, conversion therapy of potentially resectable liver metastases, and adjuvant or neoadjuvant treatment. Trials already published have been received by gastrointestinal oncology community with great enthusiasm, and future studies with PD-1 inhibitors mCRC will further help us to decipher the many pieces of a big puzzle with immunotherapy.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 , carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this  Vaccinium macrocarpon, Ericaceae), a native plant of North America, is among the top selling dietary supplements around the world. The juice as well as dietary supplements derived from this berry exert various beneficial effects on human health, including prevention and treatment of urinary tract infections, anti-cancer and antioxidant activities [Cranberry  crosomes . Moreovecrosomes . Indeed,crosomes . In contcrosomes . Long-tecrosomes . Besidescrosomes . Individin vitro .etc.), and dosage schemes . Moreover, most of the studies have focused only on selected enzymes in one organ (mostly liver) and a comprehensive view has been lacking.The abovementioned data show that information about the effect of cranberry extract/juice on drug-metabolizing enzymes has been inconsistent and insufficient. The inconsistence of the results from various studies is mainly due to the use of different experimental conditions. There are differences not only in the model systems, but also in the administered substances  on the activity and expression of a panel of drug-metabolizing enzymes  in rats. In addition to the hepatic enzymes most studied in the literature, intestinal enzymes were also included in this project to obtain more complete information. With the aim to increase reproducibility and repeatability of our experiments, CystiCran®, a patented and standardized cranberry extract, was used in this study.Therefore, the present ®  contains 1.6 mg of anthocyanidins and 36 mg of proanthocyanidins in one tablet, mainly those comprising A-type linkages, which have been associated with preventing adhesion of uropathogenic bacteria to uroepithelial cells [p.o. administration of CC (by gastric gavage) was chosen. The two dosage schemes employed represented two possible situations: regular long-term consumption of dietary supplement as a prevention of urinary tract infection  and short-term overdose by dietary supplement . In order to obtain more comprehensive information about the impact of cranberry extract on drug-metabolizing enzymes, their activities and expressions were studied not only in liver, but also in small intestine. Moreover, anthocyanins and proanthocyanidins are polyphenols with low absorption [Cranberry standardized extract CystiCranal cells . In the sorption , and thuin vivo, proanthocyanidin oligomers with a degree of polymerization lower than 5 are absorbable [in vivo [Proanthocyanidins are stable during gastric transit. While degradation of polymeric proanthocyanidins to the corresponding monomers is negligible in the gastrointestinal tract sorbable . Upon absorbable . Unlike sorbable , phase Isorbable . Neverth[in vivo .2 group) resulting in new chemical entities with different physico-chemical and biological properties. Reactions carried out by phase I enzymes usually transform active drugd into their inactive metabolite(s), but in certain cases, phase I biotransformation causes bioactivation of xenobiotics. Enzymes responsible for phase I biotransformation of drugs and other xenobiotics are abundantly present in the liver, gastrointestinal tract, lungs and kidneys. Many of them are inducible by various xenobiotics. Induction of drug metabolism may arise as a consequence of increased synthesis, decreased degradation, activation of enzymes or a combination of these three processes, although it should be emphasized that the majority of enzymes are induced at the transcriptional activation level. Thus, enzyme induction takes place only after its prolonged exposure to the xenobiotic [The phase I of drug metabolism includes oxidation, reduction or hydrolytic reactions, which lead to introduction or uncovering of functional groups  were assessed in microsomal fractions of rat liver  and anthocyanidins  have been recognized as  of CBR1 ,25. To oion used ,27.In phase II, xenobiotics or their phase I metabolites undergo conjugation reactions with endogenous compounds such as glutathione or UDP-glucuronic acid. Conjugation usually introduces hydrophilic ionizable functional groups onto the molecule of a xenobiotic, thus making it more polar and facilitating its renal excretion. Almost all phase II biotransformation reactions lead to detoxification/deactivation of the parent xenobiotic or metabolites formed in phase I. Glucuronidation, catalyzed by UGT, is the most common conjugation pathway in humans. Typical UGT substrates are xenobiotic or eobiotic alcohols, phenols or carboxylic acids. On the other hand, electrophilic compounds are mostly conjugated with glutathione through the action of GSTs .p-nitrophenol as a substrate. In the liver, UGT activity was significantly increased in CC-treated rats compared to the control group . GST activity was elevated by 13% in the liver of both CC-treated groups compared to the control group, while an insignificant decrease in GST activity was observed in the small intestine of rats treated with CC B.i.e., about 50 µg per 0.5 mg cranberry supplement capsule) [et al. [p-nitrophenol glucuronidation was found in rat liver and small intestine after 14-day intake of quercetin. Another constituent capable of UGT and GST activities induction is the stilbene resveratrol. This compound caused 1.8-fold and 1.3-fold increases of UGT and GST activities in the liver of male rats upon 28-day intake of resveratrol, respectively [Several constituents found in cranberry may be responsible for increased GST and UGT activities in rat liver. Consumption of various fruits and plants, which are rich sources of anthocyanidins as well as proanthocyanidins, was shown to elevate UGT and GST activities in rat liver ,30. Obsecapsule) . This as [et al. , in whicectively . Moreoveectively . Inductiectively .in vitro need not to be found also in vivo because the concentrations of active compounds in organism is influenced by their bioavailability. While high concentrations of bioactive compounds directly influence the activities of the studied enzymes in the in vitro experiments, the activity as well as expression of these enzymes are affected by low concentrations of these compounds in the in vivo study.Certainly, it is necessary to take into consideration that the observed effects of cranberry extract may not be ascribed to only one chemical constituent as it is a complex mixture of various phytonutrients. Individual components of this mixture may interact with each other and the resulting biological activity of the cranberry extract is therefore dependent on their synergistic/antagonistic behavior. Moreover, biological effects of cranberry juice/extract are concentration-dependent. Thus, its inhibitory activity observed In our study, the activities of drug-metabolizing enzymes were assessed primarily as the activities which are more important than protein and mRNA levels from a pharmacological/toxicological point of view. When elevated activity of a certain enzyme was found, the corresponding protein was quantified using an immunoblotting technique. The amounts of liver CYP1A1, CYP3A4, and UGT1A proteins were detected in the microsomal fractions of control as well as CC-treated rats, while levels of hepatic CBR1 and GSTP proteins were quantified in the cytosolic fractions. Calnexin and β-actin were used as loading controls in microsomal and cytosolic fraction, respectively. Protein quantification did not revealed any significant changes in the proteins amount after CC treatment .etc.) [Although the protein quantification was not in full agreement with the activities of biotransformation enzymes, similar trends were found in several studied enzymes . Some discrepancies may be explained by differences in enzyme activity assessment and performance of immunoblotting. In the case of UGT and GST, enzyme activity was assessed using universal substrates covering most of the isoforms, while immunoblotting was performed using antibody specific for one isoform  or one enzyme family (UGT1A). Therefore, observed changes in enzyme activity may be caused by different isoform(s) . Moreover, modulation of various biotransformation enzymes by cranberry extract may occur at several levels  .Benzyloxyresorufin, 7-ethoxyresorufin, 7-methoxyresorufin, 7-pentoxyresorufin, menadione, 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrophenol (NP), reduced glutathione (GSH), UDP-glucuronic acid, NADPH, and chemicals used for realization of electrophoresis were products of Sigma-Aldrich . Precision Plus molecular weight standard and non-fat dry milk were purchased from Bio-Rad . For immunoblotting, rabbit polyclonal anti-UGT antibody , rabbit polyclonal anti-GST alpha antibody, rabbit monoclonal anti-CBR1 antibody, rabbit polyclonal anti-calnexin antibody, rabbit polyclonal anti-beta actin antibody , rabbit polyclonal anti-CYP1A1 antibody , rabbit polyclonal anti-CYP 3A4 antibody (Sigma-Aldrich), bovine anti-rabbit IgG antibody conjugated with horseradish peroxidase and chemiluminescence kit  were used. All other chemicals used were of HPLC or analytical grade.ad libitum. The animal protocols used in this work were evaluated and approved by the Ethic Committee of the Ministry of Education, Youth and Sports (Protocol 20363/2011-30). They are in accordance with the Guide for the Care and Use of Laboratory Animals . At 12 weeks of age, rats were randomly divided into three groups of four. Rats of the first group (CC-14L) were orally administered with therapeutic dose of CystiCran® once daily for a period of 2 weeks; three times higher dose of CystiCran® was administered at once by gastric gavage to the second group of rats , and the third group represents untreated controls. At the end of experiment, animals were sacrificed by decapitation. Livers and small intestines were removed immediately, the intestinal contents were washed out with cold 0.9% saline solution and mucosa was scraped. Both tissues were stored at −80 °C until preparation of subcellular fractions.Male Wistar rats were obtained from Meditox . They were housed in air-conditioned animal quarters with a 12 h light/dark cycle. Food (a standard rat chow diet) and water were provided Frozen liver or mucosa from small intestine were thawed at room temperature up to 15 min and processed to microsomal and cytosolic fractions. The subcellular fractions were isolated by differential centrifugation of the tissue homogenate  and storEnzyme activities were assayed in the cytosolic and microsomal fractions from homogenates of liver and small intestinal mucosa of control and treated rats. The enzyme assays (each performed in 4–8 replicates) were repeated three times. The amount of organic solvents in the final reaction mixtures did not exceed 1% (v/v).O-dealkylase , 7-methoxyresorufin O-dealkylase , 7-pentoxyresorufin O-dealkylase  and 7-benzyloxyresorufin O-dearylase  were measured at 37 °C using fluorimetric determination of arising resorufin [The activities of several CYP isoforms were assessed. The activities of 7-ethoxyresorufin esorufin . Each suCarbonyl reductase 1 activity was measured in the cytosolic fractions using menadione as a substrate . ConsumpS-glutathione was detected at 340 nm by Tecan Infinite M200 multimode microplate reader .The cytosolic glutathione S-transferase activities were assessed by standard colorimetric assay using CDNB as an electrophilic substrate , which wp-nitrophenol were assayed as described by Mizuma et al. [p-nitrophenol was measured at 405 nm by the Tecan Infinite M200.The microsomal UDP-glucuronosyltransferase activities towards a et al. . Absorba® TurboTM Transfer System . Protein concentrations were determined using the BCA protein assay (Sigma-Aldrich). The membranes were blocked in 5% non-fat dry milk/TBS-Tween-20 for 2 h. Immunodetection of biotransformation enzymes was performed using corresponding primary antibodies . The bands were visualized with respective horseradish peroxidase-conjugated secondary antibodies using the chemiluminescence kit according to manufacturer's instructions. Calnexin and β-actin served as the loading controls for microsomal and cytosolic fraction, respectively. Intensity of bands was evaluated using a C-DiGitTM Blot Scanner .Microsomal proteins of rat liver were separated by SDS-PAGE (10% stacking gel)  and subsp < 0.05.All calculations were done using Microsoft Excel and GraphPad Prism 6. All values were expressed as mean ± SD. One-way Anova was used for the statistical evaluation of differences between control and treated groups, and differences were considered as significant when in vivo administration of standardized cranberry extract in both studied dosage schemes caused only mild changes of some activities of drug-metabolizing enzymes in rat liver, while those in small intestine were not affected. Interestingly, long-term consumption of regular dose has more pronounced effects on drug-metabolizing enzymes’ activities than short-term overdose by cranberry extract. Nevertheless, consumption of cranberry juice/extract in reasonable amounts seems to be safe and serious supplement–drug interactions do not seem probable.In conclusion,