Rift Valley fever virus (RVFV) is a lethal threat to humans and livestock in many parts of Africa, the Arabian Peninsula, and the Indian Ocean. This systematic review’s objective was to consolidate understanding of RVFV epidemiology during 1999–2021 and highlight knowledge gaps relevant to plans for human vaccine trials.The review is registered with PROSPERO (CRD42020221622). Reports of RVFV infection or exposure among humans, animals, and/or vectors in Africa, the Arabian Peninsula, and the Indian Ocean during the period January 1999 to June 2021 were eligible for inclusion. Online databases were searched for publications, and supplemental materials were recovered from official reports and research colleagues. Exposures were classified into five groups: 1) acute human RVF cases, 2) acute animal cases, 3) human RVFV sero-surveys, 4) animal sero-surveys, and 5) arthropod infections. Human risk factors, circulating RVFV lineages, and surveillance methods were also tabulated. In meta-analysis of risks, summary odds ratios were computed using random-effects modeling. 1104 unique human or animal RVFV transmission events were reported in 39 countries during 1999–2021. Outbreaks among humans or animals occurred at rates of 5.8/year and 12.4/year, respectively, with Mauritania, Madagascar, Kenya, South Africa, and Sudan having the most human outbreak years. Men had greater odds of RVFV infection than women, and animal contact, butchering, milking, and handling aborted material were significantly associated with greater odds of exposure. Animal infection risk was linked to location, proximity to water, and exposure to other herds or wildlife. RVFV was detected in a variety of mosquito vectors during interepidemic periods, confirming ongoing transmission.With broad variability in surveillance, case finding, survey design, and RVFV case confirmation, combined with uncertainty about populations-at-risk, there were inconsistent results from location to location. However, it was evident that RVFV transmission is expanding its range and frequency. Gaps assessment indicated the need to harmonize human and animal surveillance and improve diagnostics and genotyping. Given the frequency of RVFV outbreaks, human vaccination has strong potential to mitigate the impact of this now widely endemic disease. Rift Valley fever virus (RVFV) is a globally important mosquito-transmitted zoonosis that is also directly transmissible via aerosolization of body fluids from infected animals. RVFV outbreaks cause mass mortality of young livestock and pregnancy losses in both humans and animals. Severe human cases also result in hemorrhagic fever, encephalitis, and death. Loss of livestock additionally threatens the livelihood of people who depend on animals for income and food. In endemic areas, initiation of RVFV outbreaks is connected to weather events that cause excess rainfall, leading to flooding and subsequent mosquito blooms. However, the natural cycle of RVFV transmission is complex, requiring congregation of susceptible mammalian hosts and mosquito vectors in suitable environments. Several human vaccine candidates are in different stages of development, but none are yet licensed for use in human populations. In this systematic review, we assessed the 1999–2021 frequency and distribution of RVFV outbreaks among humans, animals, and vectors to identify potential locations and population targets for a human RVFV vaccine efficacy trial. It focuses on current understanding of RVFV epidemiology and the identification of gaps that pose critical barriers to controlling expansion of RVFV and implementing new protective measures including human vaccination. Phlebovirus genus, with three main lineages, with East African, West African, and Southern African groupings. In the absence of efficacious human drugs and/or vaccines against RVF, the virus has the potential to trigger significant public health emergencies in affected areas.Rift Valley fever virus (RVFV) remains an important emerging arboviral pathogen due to its recent geographic spread and its combined disease and financial impacts on vulnerable human populations . SpecifiAedes spp. are important in maintenance during interepidemic periods, whereas other Diptera serve as secondary vectors during outbreaks in sub-tropical and temperate regions of the world  = 19%) and touching an aborted animal fetus  were strongly linked to risk of severe (rather than mild) human RVF disease. Older age and death of a family member were also linked to more severe human disease [Out of the 135 included papers assessing human RVFV exposure and disease, 12 presented data on human risk factors associated with more severe RVF disease presentation . Persons disease . In Suda disease . Environ disease ,128.Severity of human RVF was associated with the presence of concomitant co-infections. A fatal outcome in a travel related case was attributed to the presence of concurrent hepatitis A virus infection . Additio95% = 1.07, 12.64, PAR% = 47%) [Four studies performed in three countries , examined the risk factors linked to death from RVF. In Kenya in 2007, consuming and handling products from sick animals and village and district location were linked to risk of death  ,130. In % = 47%) . One stu% = 47%) .Because human risk of RVFV exposure is closely associated with the local presence of infected livestock, we reviewed factors related to animals’ risk of RVFV infection.Herd Immunity Levels. Our systematic review included results from 174 animal studies, in which 144 tested domestic livestock and 26 tested wild animals for RVFV exposure and/or acute infection. Of all livestock studies included, 32 were conducted during an active epizootic, 10 were conducted just after an outbreak in the post-epidemic phase, and 97 were conducted during interepidemic years. For wildlife surveys, 5 were carried out during an active epizootic, 5 were done in the post-epidemic phase, and 14 were performed during interepidemic periods. Of all animal risk factors, sex and age were the most studied, and older animals and those that had lived through outbreaks were more likely to have been exposed. A wide range of anti-RVFV IgG seroprevalence was observed among livestock in endemic countries  and ratFor herd level risks, the observed effects of herd size has varied across studies. A 2013–2014 study in a high risk area of Kenya showed that medium sized herds of 50–100 animals had a significantly higher seroprevalence compared to small herds  or very large herds  , though The differing levels of RVFV susceptibility across livestock species  further For individual-level animal risk, studies examining cattle had the most significant findings. Abortion was the most recognized clinical sign associated with RVFV infection and recent abortion was significantly associated with RVFV exposure . This exRVFV has been found in a variety of wild mammals . Their rAmong the included papers in this review, we identified 17 studies in six countries that included vaccinated animals. Countries that had vaccinated cattle, sheep, or goats for RVFV were Kenya, Tanzania, South Africa, Egypt, and Saudi Arabia ,141–145.The range of RVFV-endemic areas is expanding, and the virus appears to travel well ,149,150.In animal, humans or vectors are summarized in When implementing surveillance for detecting RVFV transmission, one major constraint is the limited availability of accurate diagnostics in places where RVF disease occurs. For both human and animals, reliance on a clinical presentation to prioritize RVFV testing is complicated by the significant clinical diagnostic overlap with other febrile infection syndromes.Although detection of circulating RVFV RNA by RT-PCR can be a definitive diagnosis of active infection, because the human and livestock viremic period is typically only 4–6 days (up to 14 days if hemorrhagic human disease), serologic testing is more extensively used to investigate the epidemiology of recent and past infection. We extracted data from 207 included review reports on the type of assays recently employed for diagnosis of acute RVF. No studies tested livestock and wild animals together for acute infection. The assays used to detect acute RVFV infections are summarized in During RVF epidemics, assessment of incidence, prevalence, and associated risk factors for infection are often based on case series of suspected patients, rather than well-designed, population-based surveys that enumerate laboratory-confirmed cases vs. non-cases. Epidemic and epizootic outbreak surveys are further hampered by limited access to the affected rural communities, which are often experiencing severe flooding caused by excessive rainfall, and very limited access to diagnostic testing. In addition, survey incidence/prevalence calculations may be imprecise because of lack of accurate census information. As a result of these factors and the irregular frequency of disease outbreaks, the epidemiology of RVFV transmission and the risk factors for RVFV-related human disease are not well defined. In some endemic areas, post-epidemic serosurveys have shown high community exposure to RVFV ,153–158,Aedes spp. mosquitoes [Our review indicated that during the last two decades, new patterns of RVF epidemiology have emerged. The virus continues to expand its range across Africa countries and into new regions within endemic countries ,159. It squitoes  indicatesquitoes –169, andsquitoes . Thus, tThe review identified 9 actionable critical knowledge gaps for vaccine development and testing. Until this knowledge is improved, clinical trial planning and therefore vaccine development will continue to face practical implementation challenges that can’t easily be overcome:Gap 1: Defining the likely interval between significant RVF outbreaks is challengingGap 2: The unpredictability of livestock herd immunityGap 3: Differences in seasonality across endemic/enzootic locations.Gap 4: Data challenges related to bias based on limited study designs and inconsistent reporting.Gap 5: Delineating human risk factors.Gap 6: Need for consistent naming of RVFV lineages and strains.Gap 7: Need for consistent RVFV testing frameworks.Gap 8: Evolving better approaches to diagnostics.Gap 9: The need for One Health approaches for RVFV detection and control.The limited understanding of transmission persistence during interepidemic periods is a major gap affecting our ability to control continued expansion of RVFV, both within and between countries. Prospective studies during interepidemic periods are needed to provide a deeper understanding of how RVFV survives then subsequently thrives in an endemic area. Study findings could then suggest how mitigation efforts, such as vaccination of high-risk humans, can be implemented even before existing surveillance systems detect an outbreak.Defining periodicity of outbreaks is challenging because, without active case finding, passive detection is an imperfect, trailing indicator of transmission, as it requires a large enough case number of RVF clinical syndromes  to identify events. Over the last two decades, increased availability and sensitivity of RVFV diagnostic assays, including genotyping, have contributed to increased recognition of viral transmission. However, without baseline interepidemic surveys in both high and low risk areas it is not possible to determine whether RVF outbreak waves are due to persistence or to re-introduction of RVFV transmission. As the outbreak intervals for RVFV remain undefined, the lack of studies during interepidemic periods limit our ability to understand viral maintenance and make it difficult to qualify endemicity in support of mitigation efforts such as vaccination. More useful data would include concomitant active surveillance of vectors, livestock, and humans, and information on rainfall, temperature, mosquito larval sources, and the frequency of risk-related human behaviors. Because RVF livestock outbreaks are up to five times more likely to occur where outbreaks have previously occurred, and human outbreaks are more likely to occur in the same locations as livestock outbreaks ,171, areKenya, South Africa, Madagascar, and Mauritania have had recurrent human and domestic animal RVF outbreaks in the last two decades and are thus most likely to experience additional outbreaks in the near future, and thus could be most suitable for a human vaccination trial. Notably, areas with recurrent outbreaks tend to have more robust surveillance which may account for the higher frequency of RVF detection in those locations . This could impact the predictability of outbreaks, and strongly affect the validity of vaccine trial endpoints. Based on findings identified in this systematic review, RVF can be more likely in a given location, compared to a similar nearby location due to many factors, including the total number of susceptible hosts (influenced by herd immunity), the presence of wildlife reservoirs , the watCurrent prevention and readiness efforts for RVFV outbreaks rely mostly on early warning prediction and a rapid livestock vaccination response, both of which occur with variable reliability. Recovery from infection and vaccination with live-attenuated vaccines are thought to result in lifelong neutralizing antibodies in livestock  and indeMost livestock animal studies have focused on cattle, sheep, and goat seroprevalence. Although some surveys have used power analysis to determine how many animals to sample from each species, many surveys have still opted for convenience sampling, focusing either on sheep and goats, which are easier to handle, or on just cattle alone. The interpretation of such livestock prevalence surveys should be viewed with caution. Animal age is also likely to influence the results—cattle and dairy animals tend to live longer than sheep or goats, so it can appear that their cumulative burden of exposure is higher. However, evidence of infection among younger animals does provide evidence for recent RVFV transmission activity, and increased seroprevalence between years can indicate an unrecognized outbreak. When multiple species are surveyed, rates should be calculated based on animal-years at risk and better attempts should be made to age animals based on their dentition. Factors that confound RVFV exposure risk should be captured, such as the wealth and knowledge of the farmer , area liAs the previous systematic review by Clark, et al.  has highIt is understood that livestock viral amplification is an important predecessor of human outbreaks. The greater number of acute animal events (N = 273), compared to human events (N = 132) identified in this review could be due to our increasing ability to detect smaller animal outbreaks or could represent our inability to reliably detect acute human cases associated with smaller animal outbreaks ,177. AftAs climate change modifies seasonal weather patterns, differences in outbreak incidence are becoming more prominent among at-risk locations, but these changes may make epidemics harder to predict. Eighty-nine (31%) of our included studies specifically mention flooding at the outbreak site. A defined “rainy season” and “dry season” in tropical locations can guide risk assessment, but it is not just rain that makes an outbreak more likely. Congregation of many susceptible hosts and large volume slaughtering of animals Aedes vertical transmission cannot be ruled out [Pastoralist herds tend to have higher RVFV exposure, and this is influenced by the types of ecozones where the herds circulate. Pastoralists follow water and pasture availability as they become available in a seasonal cycle, which may involve movement through high-risk zones. As animal protein demand increases, mostly in urban centers, more livestock will be passing through areas that have previously experienced RVF cases, and in the presence of competent mosquito vectors. Recent infections are more common in nomadic herds compared to agro-pastoralist herds  and in huled out .In addition to climate, the variety of suitable hosts present in those areas will also vary from location to location. All potential wildlife reservoir species should be considered when comparing incidences between two geographically distinct areas because wild animals’ behaviors and range is intimately tied to topography and resource availability. In Botswana, wildlife hunters’ seroprevalence was 27% compared to a 3% community level exposure , showingA systematic review by Bron and colleagues  has summHospital-based surveys for hemorrhagic fever surveillance are an attractive tool because they do not require household field visits and blood samples are typically more available for testing ,189,190.Limitations in our knowledge of detailed human risk factors undermines ability to use targeted strategies, such as human anti-RVFV vaccination, for prevention. For vaccine development, the predefined geographic site(s) in which clinical trials will be conducted must contain individuals who: 1) are likely to be at a high infection risk during the trial; 2) are likely to exhibit the primary endpoint upon infection; and 3) represent populations in which the vaccine will likely be distributed if found efficacious. Our meta-analysis of human risk factors across multiple 1999–2021 studies confirms the increased RVFV infection risk associated with male gender, general livestock care, contact with animal abortus, butchering animals, and milking risks identified in previous reviews and meta-analysis ,191. NotIn most places where RVFV outbreaks occur, lack of health care infrastructure impedes detailed description of human disease signs and performance of laboratory studies , which might be predictive of severe disease or death. It is possible that other health comorbidities, such as concurrent HIV or hepatitis, can influence the presentation of RVFV disease, but these associations have not yet been widely studied. Further descriptions of the natural history of clinical and laboratory findings in human RVF are needed, and efforts should be made to fully assess the relative risks from direct (non-vector) animal exposures vs. mosquito vector-borne exposure. Frontline healthcare workers need to recognize when testing for RVFV infection is appropriate, based on patients’ significant risk factors, and they should communicate frequently with local veterinary colleagues to identify high-risk transmission periods.Most risk factor analyses rely on participant recall, risking recall bias, and it has been difficult to discriminate the risk of living in the presence of susceptible livestock from risk due to direct participation in husbandry activities or from risk due to local mosquito exposure (including variably zoophilic and anthropophilic mosquito species). Aside from animal rearing activities, the association of human risk with consumption of infected animal products is concerning, as this is practiced by a majority of human populations and the true infectivity by consumption is still not well understood. For example, consumption of raw milk is a now well recognized as an RVFV exposure risk factor ,191, butRVF has been perceived as a disease of poverty, but this is likely due to its greater incidence recognized in rural areas. It is not known whether RVFV has an urban transmission cycle, and so far, the risk of RVF related to socioeconomic status  is poorly defined. Choice of occupation is intimately connected to socioeconomic status and this systematic review confirmed that the highest-risk occupations are associated with livestock exposure. Even within occupational categories, differences in assigned tasks can carry differential risks . We combA possible explanation for variation in RVF attack rates and disease severity, noted between locations and among different hosts, could be differences in RVFV strain virulence. Laboratory studies suggest some strain-specific differences in mortality in experimental animals . HoweverAs highlighted earlier, identification of RVFV outbreaks and determination of the endemic state of a country require consideration of the efficacy of surveillance efforts in place. Well organized and implemented surveillance systems provide the basis for understanding the epidemiology of RVFV and provide an opportunity for warning and an opportunity to fill the gaps in current knowledge.As RVFV diagnostics become more widely available, physicians in hyperendemic regions will be able to consider co-infection with RVFV in their differential diagnoses, especially for pregnant mothers who are at risk for abortion . AdditioThere are concerted efforts to create a pan-viral hemorrhagic fever diagnostic assay. The Uganda Virus Research Institute (UVRI) in Entebbe has been implementing laboratory-based surveillance for viral hemorrhagic fevers (VHF) in Uganda since 2010, which includes testing for RVFV . While tDevelopment of robust RVFV surveillance systems is complex, because both animals and humans display variably detectable disease outcomes when they become infected. Currently, most RVFV surveillance systems are engaged only in response to climate early warning systems and are focused on periods with high-risk for large scale outbreaks. We found that recent re-structuring of the FAO Global Animal Disease Information System (EMPRESi) has provided an open-source, easy to navigate platform for tracking reports of RVFV cases in member countries. Since 2004, the system has recorded 1,186 animal cases of RVFV, of which 1,175 were confirmed. This reporting system relies on mandatory reporting of acute cases by national authorities, the OIE, WHO, FAO field officers, and published reports such as those included in this review. However, this database excludes longitudinal seroconversions, and therefore, excludes evidence of sub-clinical transmission during interepidemic periods and so does not capture the full extent of RVFV burden. Future modeling approaches using only these data should consider this key missing piece in their assumptions. The lack of consistent and systematic open-source data capture currently impedes the design of both animal and human vaccine trials. Although RVFV is a reportable disease in many of the affected countries, a system that relies on passive reporting is likely to miss areas of new emergence when viral activity is below the threshold to detect excess numbers of patients with the more obvious clinical signs of RVF. It should be noted that the partnership of a strong national veterinary service with close ties to a fully equipped national laboratory is crucial to any successful surveillance effort. If farmer-based cellphone reporting is to be successful at identifying RVF cases, farmers need to be sensitized to the greater goals of the system and incentivized to report. Overall, the ideal surveillance system for RVFV would utilize aspects of each of the discussed surveillance types , focusinPost-epidemic performance analyses have found that the lead time given by early warning systems may not be sufficiently long to prepare and vaccinate enough livestock to contain an outbreak. Just as reliance on a passive surveillance system for RVFV is likely to not be enough to detect outbreaks, sole reliance on a remotely sensed early warning systems (EWS) should also be avoided. One major limitation of climatic EWS is that they do not account for the effect of existing herd immunity obtained either through natural infection or prior livestock vaccination. Livestock seroprevalences as high as 60% have been captured in high risk areas  and up tBecause arboviral infections such as RVF often result in self-limited febrile syndromes with non-specific signs and symptoms, isolated cases of infection are frequently misdiagnosed as malaria in sub-Saharan Africa. This highlights the critical need for affordable and accurate point-of-care diagnostics for RVFV –209. In Among the 95 studies that reported human serology, the methods for human anti-RVFV antibody detection exhibited significant variability, with most studies relying on tertiary reference laboratories for final diagnosis. Future efforts should focus on increasing the number and capacity of local laboratories so that the burden of shipping time and costs can be alleviated. The Covid-19 pandemic response has led to expansion of molecular diagnostic capacity in many regions. In the aftermath of the pandemic, these important logistical and laboratory resources should be leveraged to include other viral diseases such as RVF.Serology serves as the basic approach for investigating individual and community level risk factors as it can monitor changes in prevalence over time. Both enzyme-linked immunosorbent assays (ELISA) and viral neutralization tests (VNT) have advantages and disadvantages. Identifying IgG-positive livestock by ELISA has often been the first step in recognizing new areas of emergence ,211. HowExpanding RVFV surveys to include human community populations at-large will identify risk associated with socioeconomic standing, while simultaneously detecting patients with incident infections but without presently known risk factors. To fully understand the ecology of local transmission such broad-based human studies need to be conducted concurrently with robust animal surveys. Recent reviews and policy papers in the human and animal health RVFV literature call for a greater emphasis on a One Health management approach, both in research and control efforts ,212,213.The gaps presented here are based on a collated estimates of recent RVFV transmission. The available case counts and serosurveys in this systematic review contribute to a body of evidence that can guide the way forward to clinical testing of novel preventive and curative interventions.The identified gaps will all affect development of human vaccines for RVF, acting as potential impediments to planning and preparing clinical field trials. Trial feasibility will depend on understanding incidence with sufficient granularity and identification of suitable target populations. Site selection faces challenges in forecasting—timing, duration and location of outbreaks, and study population selection will depend on existing seroprevalence and the duration of protection after prior natural exposure and infection. Definition of clinical endpoints will depend on good characterization of clinical presentations, and availability of diagnostic tests for differentiating non-specific febrile syndromes.RVFV continues to transmit across Africa and nearby Indian Ocean islands at a high rate, but at an irregular frequency. As previously noted, because of its typical mosquito-borne re-emergent transmission, RVFV outbreaks often follow periods of excess rainfall and local flooding, particularly in semi-arid regions. During the 1999–2021 period studied in this review, human outbreaks of RVF occurred at an average rate of 5–6 events per year across the region. The range of transmission has expanded, with human cases documented for the first time in Saudi Arabia, Yemen, Comoros, Mayotte, Burundi, Niger, and Mali, and with re-emergence in Uganda after multiple decades of no activity. Reported human RVF events were most common in Mauritania, Kenya, Sudan, Madagascar, and South Africa, and each of these countries experienced multiple epidemics since 1999. In districts or counties where human cases were reported, the median attack rate was 2.5 per 100,000 (range 0.1 to 91), and median case fatality was 14.3% of severe cases, highlighting the significant population health burden of RVF.Experience with veterinary vaccines indicates that pre-event vaccination can mitigate the spread of RVFV infection. Now that human vaccines are under development, their clinical testing and implementation should be guided by particular focus on the high-risk subgroups (based on location and exposures) identified in this review. Effective vaccine trials will depend on achieving accurate outbreak predictions combined with efficient animal and human case surveillance systems.The feasibility of such trials can be assessed using the present summary of recent epidemiology, while approaches to trial design will be strengthened by simultaneously addressing the gaps we have discussed here. Recommendations for vaccine trial study design will be addressed in greater detail in a subsequent paper by the CEPI RVF study group.S1 Text(DOCX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 TableOutbreaks are indicated by year, country, region, and geographic location, with estimated population size, incidence, and case fatality rates.(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.


In the last years, remarkable progress has been achieved in our understanding of the biology and management of multiple myeloma (MM). Some of these advancements and possible future directions are addressed below and summarized in Table Whole-genome sequencing revealed two types of MGUS: A progressive myeloma precursor condition defined as a clonal entity in which myeloma-defining genomic events have already been acquired at the time of diagnosis and which is associated with a high risk of progression to MM; and an MGUS with a stable myeloma precursor condition, in which myeloma-defining genomic events are rare and which follows an indolent clinical course with a low risk of progression . In thosneg, and C. multiple myeloma—CRABpos.These findings may inaugurate a new era of a genomic definition of monoclonal gammopathies, abandoning the arbitrarily defined categories MGUS, SMM, and MM, which depend on the disease burden but not on the underlying biology. This may be replaced by a genetic definition that may distinguish between three types: A. stable precursor condition of multiple myeloma, B. multiple myeloma—CRABAs indicated above, the presently applied parameters of high-risk SMM do not reflect the genomic structure of individual patients and thus preclude optimal patient selection for early treatment. Further studies will path the way for a biology-based definition or a combination of these factors with conventional parameters of those patients that have already acquired the genetic machinery for malignant progression and thus would benefit most from early treatment initiation. As many experts consider myeloma still as being incurable, the question of optimal patient selection and timing for treatment initiation is highly pertinent. In the future, this question will become less relevant, because of the better differentiation between risk groups and further improvements in treatment outcome.neg either by NGF or MS, and a similar prognostic accuracy of both techniques for PFS. In addition, they identified a small group of either MSneg−MRDpos, and MSpos−MRDneg patients. Whether MS will be used in addition to MRD testing, or whether MS may even replace conventional MRD testing needs to be explored. In case of similar sensitivity and predictability, MS could even become the preferred MRD test because it enables frequent serial testing, which may inform on both reaching and sustaining MSneg, as well as losing MSneg status as an early indication of relapse.Mass spectrometry (MS) shows higher sensitivity for the detection of monoclonal proteins (MP) compared to the conventional electrophoretic techniques  and willneg patients.Present imaging guidelines in MM recommend low dose CT, PET/CT, and whole-body MRI (WB MRI) irrespective of their individual advantages and limitations. This may change if results of a recent prospective randomized study  showing Autologous stem cell transplantation (ASCT) has greatly improved treatment outcome before the introduction of new agents and continues to be a valuable choice in the era of novel agents. ASCT during induction therapy improved PFS over conventional chemotherapy in practically all trials, but OS was found to be similar in most studies . DiscussDaratumumab is a game-changer for MM therapy, active as single-agent therapy in heavily pretreated patients, synergistic with several chemotherapy backbones in later lines of therapy, and finally a valuable combination partner for first-line treatment regimens with an already documented survival advantage in patients not eligible for transplantation (ALCYONE and MAIA) . Studiesneg at a threshold level of 10−6 show prolonged survival compared to MRDpos patients [neg at least once is already associated with a survival advantage, but sustained MRDneg is the ultimate goal. Remarkably, patients with HR cytogenetics achieving MRDneg seem to fair equally well as MRDneg standard-risk patients. A comprehensive meta-analysis has confirmed the close association between MRDneg and survival in practically all treatment scenarios, including younger and older patients, first or later lines of therapy, and different risk groups [neg.New technologies, in particular next-generation sequencing (NGS) and next-generation flow (NGF), have successfully been adapted to enable the detection of one myeloma cell within one million nucleated bone marrow cells. Patients achieving MRDpatients . Achievik groups . This rek groups . Overallneg rates compared to the lenalidomide arm. Presently, new drugs such as CELMoDs and BiTEs are evaluated as maintenance treatments.Lenalidomide maintenance after ASCT is still the gold standard, but trials with proteasome inhibitors and anti-CD38 antibodies, either as single-agent or in combination with lenalidomide, are ongoing. Carfilzomib added to lenalidomide resulted in improved PFS in all cytogenetic risk groups, with the exception of patients with ampl1q in the FORTE study  indicateBelantamab Mafodotin shows remarkable single-agent activity in RRMM patients , which hneg disease and a PFS rate of 77% after one year has been reported in the CARTITUDE-1 study [With the development of CAR-T cell therapy, an old dream of hematologists became true. Harnessing the patient’s own immune system can induce marked tumor responses in far advanced, heavily pretreated patients. A response rate of 97% with high rates of MRD-1 study , but the


Malignant Pleural Mesothelioma (MPM) is a rare and aggressive neoplasm of the pleural mesothelium, mainly associated with asbestos exposure and still lacking effective therapies. Modern targeted biological strategies that have revolutionized the therapy of other solid tumors have not had success so far in the MPM. Combination immunotherapy might achieve better results over chemotherapy alone, but there is still a need for more effective therapeutic approaches. Based on the peculiar disease features of MPM, several strategies for local therapeutic delivery have been developed over the past years. The common rationale of these approaches is: (i) to reduce the risk of drug inactivation before reaching the target tumor cells; (ii) to increase the concentration of active drugs in the tumor micro-environment and their bioavailability; (iii) to reduce toxic effects on normal, non-transformed cells, because of much lower drug doses than those used for systemic chemotherapy. The complex interactions between drugs and the local immune-inflammatory micro-environment modulate the subsequent clinical response. In this perspective, the main interest is currently addressed to the development of local drug delivery platforms, both cell therapy and engineered nanotools. We here propose a review aimed at deep investigation of the biologic effects of the current local therapies for MPM, including cell therapies, and the mechanisms of interaction with the tumor micro-environment. Malignant pleural mesothelioma (MPM) is a fatal asbestos-related malignancy which originates from the mesothelial layer which coat the pleural space. Despite asbestos bans, MPM incidence in Europe and Japan is still rising and it is almost reaching its peak, predicted in 2020 and 2025, respectively. In early disease stages, multimodal therapeutic approaches, encompassing surgery, radiation therapy and chemotherapy can be available at least in patients featuring good performance status. In advanced MPM cases the only option is palliative treatment. Several chemotherapeutics have already been evaluated, in absence of clear clinical efficiency . Modern The poor therapeutic results that impact MPM patient outcomes could be reasonably ascribed as both due to drug inability to reach the site of disease and to the difficulty in reaching adequate intracellular drug concentrations, due to excessive systemic side effects. MPM is characterized by rapid and diffuse local growth, whereas distant metastases rarely appear and arise in advanced phases of disease development. Thus, the pleural space, despite the physiological draining capacity of the membrane that covers it, represents an ideal space for the local chemotherapy treatment in case of neoplastic transformation. Moreover, growing evidence suggests that asbestos-induced inflammation is at the basis of the neoplastic transformation of mesothelial cells and that this unique tumor micro-environment (TME) is involved in the induction of resistance to the applied therapies ,11. AmonBAP1, NF2 and CDKN2A genes and leads to a significant enrichment of tumor suppressor genes. The MPM clonal evolution shapes the MPM micro-environment and modulates immunosurveillance, since neoantigens derived during clonal evolutionary trajectory can modulate the composition of the micro-environment by regulating the infiltration levels of Tregs, CD8+, the HLA system [POS lymphocytes and high CD8POS and high PD-L1 expression associated with poor patient survival [Growing evidence demonstrates that MPM is characterized by specific features of the micro-environment that hamper its pharmacological targeting. Indeed, MPM is not made merely of transformed cells, but a dynamic cross-talk with surrounding stroma is responsible for tumor onset and progression. Every tumor  is made with malignant cells plus stroma: the main and specific issue is related to the role played by asbestos (rather than activation of specific oncogenes) in both driving malignant transformation and modulating tumors surrounding stroma ,12. AlthA system . NotablyA system , being tsurvival ,27,28. Asurvival . Moreovesurvival . HIF1α esurvival . In concsurvival . This obThe specific pattern of growth of MPM could become an Achilles heel. An overview on the strategies that have been developed and/or are under investigation for local therapy in MPM is summarized in Essentially the tools in clinical use to locally vehicle anticancer drugs are cellular-derived or biologically compatible synthetic compounds such as polymer drug conjugates, nanoparticle and liposomal systems, and transdermal drug delivery patches. These novel technologies have made possible the development and use of new anticancer agents as well as a more a safer and more efficient profile of standard chemotherapy. Furthermore, local administration of anticancer therapies combing immune, chemo, radiotherapy and small agents can assure synergistic antiproliferative effects. In this perspective, the heterogeneity of MPM surrounding stroma could be exploited to obtain synergistic effects with conventional treatments.It should be also noted that a tumor micro-environment is also modulated by physical forces. In a context of local drug delivery, they could play a crucial role and should be kept under consideration. They control trans-vascular and interstitial drug transport as well as intercellular cross-talk by modulating the interaction between invading cancer cells and their 3D micro-environments. Interestingly—with respect to MPM—it has been reported that Caveolin 1 (CAV1) acts as a multifunctional scaffolding protein which is involved in cancer growth and progression, modulating tissue responses through architectural regulation of the micro-environment. Caveolae and their components act as modulators of biomechanics and ECM–cell interactions ,45.Images are more than pictures: they are data . A decadOverall, the complex interaction between drugs and the local immune-inflammatory micro-environment which modulates the subsequent clinical response had already been explored with radiomics in several different scenarios, in particular for immune  markers . This apThe application of nanotechnology in medicine opens incredible opportunities in cancer treatment. Given the chemical and physical properties of NPs, they allow: (1) to target specific cell population exploiting both passive targeting by enhanced permeability and retention effects, and active targeting by modifying the surface of nanoparticles with moieties specific only for cancer cells; (2) to increase drugs concentration in tumor bulk reducing side effects; (3) to allow new route of administration for those drugs already used in clinics, increasing their bioavailability. Usually, in cancer treatment the preferentially route of NPs administration is the intravenous injection. However, regarding MPM or other lung diseases, the best approach to administer therapies is the local treatment through aerosol or pleural injection.The most used type of NPs for MPM treatment are liposomes. This is because these kinds of nanovehicles are composed of a mixture of lipids and/or cholesterol, giving the property to be highly biocompatible. Moreover, liposomes give the opportunity to deliver both hydrophobic and hydrophilic compounds, but also genetic material. In MPM treatment, liposomes are exploited to deliver chemotherapeutics, such as doxorubicin ,55, or pNot only liposomes but also other types of nanoparticles are being studying for MPM treatment, such as gold nanoparticles  or pH-reAs mentioned above, the use of NPs allows the targeting of specific cell populations with specific moieties. MPM researchers have exerted the high expression by MPM cells of CD146  and CD44In conclusion, despite the relative novelty of this field, nano-based approaches could be a very promising option for MPM patients, as adjuvant therapy after surgical resection to avoid the relapse of cancer. However, it is very important to study the fate of NPs after pleural injections for every type of material or modification done to NPs, since Marziaroti et al. demonstrated that changing the formulation of liposomes or size could also change the biodistribution and clearance of liposomes .Adoptive transfer of ex vivo cultured and expanded Tumor Infiltrating Lymphocytes (TILs) isolated from a resected tumor specimen has been used from 1988 for the treatment of different tumor types. The treatment with TILs demonstrated a significant disease regression only in melanomas , even ifn = 230 patients to examine the OS in patients treated with DCs loaded with this allogeneic tumor cell lysate, as a maintenance treatment after chemotherapy [In MPM, initial approaches used autologous tumor lysate loaded DCs and have shown unexpected and persistent clinical responses with significantly increased OS (with a maximum of 66 months) after the treatment. Although these are relevant data, this approach is impaired by two important disadvantages: it is time consuming and may not assure the achievement of quality standards for DCs therapy. Allogenic tumor lysates may be of help in bypassing this obstacle, as recently showed in a Phase I clinical trial MesoCancerVa (NCT02395679). In this trial, no dose-limiting toxicities were registered, and radiographic responses were observed. The median progression free survival (PFS) was 8.8 months and the median OS was not reached at a median follow-up of 22.8 months. In a follow up analysis of the peripheral blood T cell receptor β (TCRβ) chain repertoire of nine MPM patients before and five weeks after the start of DCs-based immunotherapy, it was found that clinical responses to DCs-mediated immunotherapy was related to both the pre-existing TCRβ repertoire of total CD3 + T cells and to therapy-induced changes, mainly expanding PD1+CD8+-T cell clones. Thus, the TCRβ profiling could potentially allow to identify and select those subsets of MPM patients that could really benefit from DCs-based immunotherapy. These promising results will be further assessed by the Phase II/III DENIM trial (NCT03610360) which aims to recruit otherapy .Another adoptive cell therapy approach in MPM treatment involves the use of T cells genetically engineered to express a chimeric antigen receptor (CAR) able to recognize a cell-surface antigen and kill cancer cells (CAR-T) .The potential to use CAR-T therapy in MPM has been extensively evaluated, and pre-clinical models using various targets, among which are mesothelin (MSLN), Fibroblast Activation Protein (FAP), Met Proto-oncogene (cMET), pan-ErbB and others, have been tested. The biggest potential limitation of the use of CAR-T therapies in solid tumors could be T-cell exhaustion. However, recent data have pointed out that exposure to checkpoint inhibitors may improve the potency of CAR-T cell therapies, and other treatments such as co-stimulation induction and cytokine-based approaches may also have a role ,76. NeveAnother potential therapeutic strategy is the use of Mesenchymal Stromal Cells (MSCs). MSCs are characterized by their ability to self-renew and differentiate into tissue-specific specialized cells. According to the International Society for Cellular Therapies (ISCT), minimal criteria to define multipotent MSCs are as follows: (1) plastic adhesion capacity in standard culture conditions, (2) surface expression of CD73, CD90, CD105, CD166, CD44 and CD29 markers and absence of CD14, CD34, CD45 and CD31 (3) differentiation capacity in vitro into adipocyte, osteoblast and chondroblast lineages . After eMany different methods of drug delivery have been described in the last decade, among which are immunoconjugates for targeting tumor-specific antigens, nanotools and genetically modified stem cells. Nevertheless, non-modified MSCs are probably the best option for anticancer drug local delivery, as they can rapidly adapt themselves to culture conditions and become home to pathological tissues when injected in vivo in addition to their own antineoplastic activity. MSCs, as well as extracellular vesicles (EVs) obtained from MSCs (MSCs-EV), can release active soluble factors and play an effective immunomodulatory role ,79,80,81MSCs-EV may also represent a potential new Drug Delivery System (DDS) of therapeutic molecules. The main features which identify EVs as promising therapeutic candidates as DDS tools are essentially their high editability and low immunogenicity. The possibility of tumor growth blockades by exploiting cancer messengers is not only intriguing from a scientific perspective, but is also of clinical relevance since it allows a complete bioavailability of therapeutic compounds. Moreover, unlike other drug delivery platforms specifically targeting the cell surface, e.g., , EVs assure a much more precise and complex vehiculation system. However, it should be remarked that a careful evaluation of the TME, the choice of an ad hoc delivery tool as well as the electric charge of the nanocarrier have to be taken into account to obtain the most efficient results ,83,84.Tissue bioengineering has profoundly revolutionized recent therapeutic offers, mainly for local approaches to cancer progression. It involves interdisciplinary research, including biomaterial design and processing, surface characterization, and functionalization for improved cell-material interactions and imaging. Implanted scaffolds and injected hydrogels are two typical biomaterials that provide mechanical structures to tissue constructions, whether cells are suspended within or adhered to a three-dimension hydrogel framework . Cells aIn addition to being a rare disease, MPM is classified as an orphan disease, of little interest to the pharmaceutical industry that does not invest sufficiently in this pathology.This implies that the current therapeutic approaches against this tumor are non-specific and therefore poorly effective, resulting in a very limited life expectancy for the patient. There is, thus, an urgent need to find novel therapeutic strategies. Although few data are, till now, available on MPM, the specific disease features make it potentially susceptible to local therapeutic approaches, taking advantage of progressions in cellular manipulation and bioengineering. Innovative methodologies are landing the clinical scenario and the main results will derive from the pharmacological interaction with the active micro-environment, which surrounds neoplastic masses in the pleural space. The identification of patients likely to respond to specific therapeutic approaches  needs clinical validation studies and requires a constant and productive interaction among the professionals with a complementary multidisciplinary background. Results from future trials could help the fight against MPM  by proposing innovative local therapeutic approaches based on the design of drugs able to treat these still incurable tumors.


Opioid receptors are widely expressed in the human brain. A number of features commonly associated with drug use disorder, such as difficulties in emotional learning, emotion regulation and anhedonia, have been linked to endogenous opioid signalling. Whereas chronic substance use and misuse are thought to alter the function of the mu-opioid system, the specific mechanisms are not well understood. We argue that understanding exogenous and endogenous opioid effects in the healthy human brain is an essential foundation for bridging preclinical and clinical findings related to opioid misuse. Here, we will examine psychopharmacological evidence to outline the role of the mu-opioid receptor (MOR) system in the processing of threat and reward, and discuss how disruption of these processes by chronic opioid use might alter emotional learning and reward responsiveness.In healthy people, studies using opioid antagonist drugs indicate that the brain’s endogenous opioids downregulate fear reactivity and upregulate learning from safety. At the same time, endogenous opioids increase the liking of and motivation to engage with high reward value cues. Studies of acute opioid agonist effects indicate that with non-sedative doses, drugs such as morphine and buprenorphine can mimic endogenous opioid effects on liking and wanting. Disruption of endogenous opioid signalling due to prolonged opioid exposure is associated with some degree of anhedonia to non-drug rewards; however, new results leave open the possibility that this is not directly opioid-mediated.The available human psychopharmacological evidence indicates that the healthy mu-opioid system contributes to the regulation of reward and threat processing. Overall, endogenous opioids can subtly increase liking and wanting responses to a wide variety of rewards, from sweet tastes to feelings of being connected to close others. For threat-related processing, human evidence suggests that endogenous opioids inhibit fear conditioning and reduce the sensitivity to aversive stimuli, although inconsistencies remain. The size of effects reported in healthy humans are however modest, clearly indicating that MORs play out their role in close concert with other neurotransmitter systems. Relevant candidate systems for future research include dopamine, serotonin and endocannabinoid signalling. Nevertheless, it is possible that endogenous opioid fine-tuning of reward and threat processing, when unbalanced by e.g. opioid misuse, could over time develop into symptoms associated with opioid use disorder, such as anhedonia and depression/anxiety. Despite increasing awareness of the risks of problematic opioid use, opioid analgesics are still commonly used to treat acute and chronic pain , 60, 136In the literature, the addictive potential of opioids is strongly linked to their ability to induce pleasure and euphoria. Indeed, one of the more robust indicators used to assess the addictive potential of opioid drugs is drug liking . InteresMolecular imaging studies have reported changes in binding potential after chronic exposure to opioid drugs, consistent with altered endogenous opioid function in opioid users , 132. ItThis narrative review will give an overview of the current state of knowledge on endogenous opioid function in humans, based on behavioural evidence from opioid drug studies. A growing literature of preclinical and human experimental research in healthy subjects has investigated the effects of acute pharmacological administration targeting the mu-opioid receptors. Here we review studies that used drugs to block and/or stimulate the MOR system. Most opioid agonist drugs target mu-receptors . In non-sedative doses, these drugs are thought to inform us about putative functions of endogenous mu-activity. Opioid antagonist drugs  bind to opioid receptors, thereby preventing endogenous ligands (i.e. endorphins) from binding to and stimulating the receptors see Fig. a. All stSpecifically, the review will focus on causal evidence on opioid drug effects on reward, stress and threat in healthy people at the behavioural level. Correlational neuroimaging data from PET or fMRI are also discussed where they complement behavioural effects. Each section considers how the human psychopharmacology findings align with results from studies of opioid function in non-human animals. Finally, we also link the knowledge on acute drug effects with findings in chronic opioid exposed groups. For the sake of brevity, opioid regulation of pain falls outside the scope of the present review, which is also limited to the mu-opioid receptor system. Most of the drugs used as antagonists and some of the agonists are non-specific and also bind to kappa and delta receptors; however, doses are optimised for mu-opioid binding. The limited emerging evidence on kappa-specific activity is exciting, see e.g. Darcq & Kieffer ; KrystalBoth receiving and anticipating rewards can evoke feelings of pleasure in humans. In evolutionary terms, the pleasures of play, food or sex are believed to motivate the individual to engage in these behaviours, securing the individual’s survival and reproductive fitness. Reward processing is often parsed into several components, including liking (the hedonic experience when a reward is anticipated or obtained), wanting  and reward-related learning (e.g. ).Mu-opioid signalling is tightly linked to liking of palatable foods. Groundbreaking rodent research on the role of the MOR system in reward has identified the areas in the rostrodorsal shell of the nucleus accumbens (NAc), the caudal ventral pallidum (VP), the parabrachial (PB) nucleus as well as the anterior orbitofrontal cortex (OFC) and the posterior insula as hotspots that can enhance ‘liking’ responses to food rewards , 19, 92.Does mu-opioid activation also enhance wanting of food rewards? Rodent studies using striatal microstimulation or systemic drugs indicate a clear role for mu-opioids in food wanting. When rats ate chocolate, striatal enkephalin levels surged , and micRodent studies also indicate an important role for the MOR system in encoding liking and wanting of non-food rewards such as social play . Social Indeed, neuropharmacological studies across several species point to a role of opioids in mediating reward-related processing. Inhibition of mu-opioid signalling prevented newborn mice  and lambOverall, animal research indicates that opioid signalling impacts a large range of potentially rewarding behaviours such as feeding, bonding and play. Does the human opioid system exhibit similar modulation of reward processing? Due to methodological limitations, it is as yet unknown whether the human brain contains anything like the mu-opioid sensitive set of hedonic hotspots capable of enhancing food liking responses in rodents. PET imaging indicate rich expression of MORs in the human striatum, as well as in the thalamus, insulae, amygdala and anterior cingulate cortex Fig. b. OveralIn line with rodent work showing opioid modulation specifically of high reward options in the food and social domains, several human studies targeting mu-opioid receptors with systemic agonists and/or antagonists report drug modulation of high-value reward responses. As illustrated in Fig. Results from other labs are broadly consistent with the above pattern of results. Buchel et al.  used fMRfeeling of being connected to other people [Korb et al.  also repr people , 57. Ther people , at leasr people , 64, 70 Importantly, the human literature using antagonists unequivocally demonstrates that a substantial amount of reward liking can be observed see Fig.  even aftBeing able to adequately respond to threat cues in the environment is crucial for human social functioning. Hypersensitivity to threat and impaired threat-related learning processes are associated with affective disorders such as anxiety or PTSD , 98. RodThere is abundant evidence of MOR modulation of threat-related learning in rodents. Endogenous mu-opioid signalling inhibits fear conditioning  and faciThe MOR system also regulates stress responses. Early infant-attachment studies in different mammalian species showed that opioid blockade increased distress vocalisations in young animals after separation from their mothers . AdminiIn line with rodent evidence , 81, twoDoes human MOR signalling regulate sensitivity to negative affective cues outside of a fear learning context? There is some evidence in support of this notion from behavioural studies. Administration of a MOR agonist reduced sensitivity to fearful facial expressions in an emotion recognition task , whereasIn addition to mu-opioid fine-tuning of threat-related learning and perception of negative affective cues, endogenous opioid signalling also impacts the body’s stress response via its actions on the hypothalamic–pituitary–adrenal (HPA) axis , 72, 126The role of MOR for  social rejection was tested using PET: the bilateral amygdala, the ventral striatum, thalamus and the subgenual anterior cingulate cortex showed decreased binding after rejection . As has Overall, the evidence of acute opioid effects on stress and threat processing in healthy humans suggests a modest, protective function of endogenous opioid signalling—reducing the physiological stress response and fine-tuning sensitivity as well as learned responses to negative stimuli. Whether chronic dysregulation of endogenous opioid signalling, through e.g. chronic opioid drug use, could paradoxically reinforce stress responses and negative affect is of yet unknown. Current evidence indicates co-occurrence of opioid use disorder and anxiety-related symptoms , 79, 102Disrupted reward processing related to drug-induced disturbances of the mu-opioid (and dopamine) reward system is a key element in neurobiological theories of drug addiction  remain. With regard to threat processing human evidence points towards an inhibitory role of the MOR system in the acquisition of fear associations. However, whilst the direction of the effects is consistent with findings in rodents, the magnitude of effects is not comparable to those reported in rodent work. Results from perception studies are mixed but suggest a subtle reduction of sensitivity to negative affective stimuli via endogenous opioid signalling. Similarly, human studies investigating the regulatory role of the MOR system in stress responses indicate subtle effects on the subjective experience of stress, but large effects on the physiological level. We venture that the role of endogenous mu-opioids for human reward and threat is limited to fine-tuning the responses, and that the human brain also draws on other neurotransmitter systems such as the dopamine, serotonin and endocannabinoid systems in reward and threat-related processing.More research is warranted to gain a better understanding of opioid modulation of reward, threat and stress-related processing in the healthy human brain as well as in opioid users. Despite high co-occurrence of opioid misuse and anxiety, it is unclear whether anxiety predates or could result from chronic opioid exposure. Anhedonia is often, but not always observed in opioid using populations, but changes in reward sensitivity observed after chronic opioid use may be explained by other co-occurring vulnerability factors rather than opioid system dysregulation.


Schizophrenia disorder remains an unsolved puzzle. However, the integration of recent findings from genetics, molecular biology, neuroimaging, animal models and translational clinical research offers evidence that the synaptic overpruning hypothesis of schizophrenia needs to be reassessed. During a critical period of neurodevelopment and owing to an imbalance of excitatory glutamatergic pyramidal neurons and inhibitory GABAergic interneurons, a regionally-located glutamate storm might occur, triggering excessive dendritic pruning with the activation of local dendritic apoptosis machinery. The apoptotic loss of dendritic spines would be aggravated by microglia activation through a recently described signaling system from complement abnormalities and proteins of the MHC, thus implicating the immune system in schizophrenia. Overpruning of dendritic spines coupled with aberrant synaptic plasticity, an essential function for learning and memory, would lead to brain misconnections and synaptic inefficiency underlying the primary negative symptoms and cognitive deficits of schizophrenia. This driving hypothesis has relevant therapeutic implications, including the importance of pharmacological interventions during the prodromal phase or the transition to psychosis, targeting apoptosis, microglia cells or the glutamate storm. Future research on apoptosis and brain integrity should combine brain imaging, CSF biomarkers, animal models and cell biology. Part of the problem is that, in many ways, the neurobiology of schizophrenia remains an unsolved puzzle. To gain a more complete understanding of this disorder, it will be necessary to integrate findings from genetics and epigenetics, environmental factors, molecular biology, neuroimaging, animal models, and translational clinical research.Schizophrenia is a common and heterogeneous psychiatric disorder that severely affects the lives of roughly 1% of the population worldwide. It is characterized by the presence of positive psychotic symptoms , negative symptoms  as well as enduring neurocognitive deficits . While existing pharmacological treatments are generally able to ameliorate positive symptoms, there has been no way to target the negative and cognitive symptoms despite their clear association with long-term deleterious effects on real-world psychosocial function. For recent reviews see Marder and Cannon4, and Murray and Lewis5, or in the “2-hit model” proposed by Keshavan7, has been the most significant attempt to integrate different findings to explain the origin of this psychiatric disorder. According to this model, early neurodevelopmental insults, like impairment of neurogenesis, neuronal and interneuron migration, dendritic arborization or axonal outgrowth, may lead to abnormalities of specific neural circuits that account for clinical premorbid signs and symptoms, such as mild impairments in behavior, cognition, or social functioning, observed in individuals who later develop schizophrenia. During adolescence and young adulthood, postnatal brain maturation abnormalities like myelination defects, excessive pruning of dendritic spines and loss of synaptic plasticity could account for the emergence of symptoms11. Furthermore, the dopamine hypothesis formulated by Carlsson and Lindqvist in the sixties12, as an explanation for the genesis of positive psychotic symptoms, has given way to the glutamate hypothesis in recent decades. This newer view proposes that glutamatergic dysfunction may play a key role in schizophrenia pathophysiology, particularly regarding negative symptoms and cognitive dysfunction14.The neurodevelopmental hypothesis of schizophrenia, in its initial formulation by WeinbergerSchizophrenia Working Group of the Psychiatric Genomics Consortium15. These genetic findings have identified more than 100 loci associated with the risk of schizophrenia which converge into a coherent set of biological processes that align with the predominant etiological hypotheses of the disorder mentioned above16. The main genes that have been identified are those encoding synaptic proteins involved in synaptic plasticity, an essential function for learning and memory; genes encoding N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) glutamate receptors, the voltage-dependent calcium channel (VDCC), and dopamine receptor D2 (DRD2); and genetic loci variants in the major histocompatibility complex (MHC) of the immune system15. More recently, genetic variations in the complement component 4 (C4) gene have also been identified providing additional evidence of the implication of the immune system in schizophrenia18. At the same time, repeated neuroimaging studies have revealed gray matter loss as well as abnormal functional connectivity in schizophrenia patients21. In addition, postmortem studies of patients have reported reduced numbers of dendritic spines, especially on pyramidal neurons located in layer III in the prefrontal cortex (PFC), in the superior temporal gyrus (particularly in the primary auditory cortex), and in certain hippocampal (HPC) subfields26.Fortunately, in the last decade we have had access to the largest genomic study on schizophrenia published to date from the 26. Dendritic spines are protrusions from neuronal dendrites that act as hubs that facilitate excitatory synaptic communication between neurons. In the nineteenth century, the neuroanatomist Ramón y Cajal was the first to describe dendritic spines on neurons27. A dendritic spine contains an actin cytoskeleton, NMDA and AMPA glutamate receptors and other proteins regulating synaptic plasticity. From an evolutionary perspective, the appearance of a higher density of dendritic spines in humans led to an increase in our neural synaptic connections, providing a clear advantage that differentiated us from other primates28. In individuals with schizophrenia, the loss of dendritic spines may further impair synaptic plasticity  and long-term depression (LTD), both essential in learning and memory34. Long-term potentiation promotes the enlargement of spine heads, whereas LTD leads to spine shrinkage. Glutamate, via NMDAR and AMPAR, is the crucial regulator. However, as discussed below an excess of glutamate can be detrimental to the integrity of dendritic spines. In experimental studies, LTD induction by low-frequency glutamate stimuli resulted in a retraction of dendritic spines. By contrast, LTP induction by repetitive uncaging of glutamate produced a selective enlargement of the spines  associated with synaptic efficacy. Long-term potentiation is also characterized by the insertion of AMPA receptors into the spine’s surface. Small or thin spines are more dynamic and plastic than large ones and could function as “learning spines”, whereas large spines (mushroom spines) are more stable over time and could be the substrate for memory processes35. Both cognitive functions are seriously affected in schizophrenia.As noted above, excessive dendritic spine pruning during late adolescence and early adulthood may lead to the emergence of symptoms of schizophrenia during these critical periodsity Fig.  which is36. Dendritic pruning is a physiological process during neurodevelopment which leads the brain to strengthen some connections and eradicate others through experience-dependent plasticity. It typically overlaps with the period of onset for schizophrenia. However, the molecular mechanisms underlying dendritic pruning are not fully understood even in normal development38. Dendritic pruning shares several molecular features with apoptosis and it is hypothesized that components of the apoptotic machinery could be involved in this process41. Recently, it has been reported that microglia activation and molecules of the immune system also contribute to the observed reduction in dendritic spine density in schizophrenia42. In this article we discuss possible pathways toward dendritic apoptosis, which may well be one of the core pathophysiological mechanisms to help understand schizophrenia.Four decades ago, Feinberg was the first to suggest aberrant peri-adolescent pruning of synapses in schizophrenia14, NMDA glutamate receptor alterations, can produce a dysfunction of inhibitory GABAergic interneurons expressing parvalbumin (PV+) resulting in disinhibition of excitatory glutamatergic pyramidal cells . The fast-spiking PV + interneurons synchronize the firing of pyramidal neurons underlying the generation of gamma band oscillations, which is critical to optimal cognitive function. Electrophysiological studies in schizophrenia have shown disrupted synchronization of neural gamma oscillations associated with cognitive and negative symptoms43. GABAergic interneurons have diverse morphologies (chandelier or basket cells) but are typically aspiny and constitute 20% of all neurons in the cerebral cortex44. The PV + interneurons are highly sensitive to NMDA R antagonism as discussed below. They are also vulnerable to oxidative stress, needing a well-regulated antioxidant system to neutralize the overproduction of mitochondria-generated reactive oxygen species46. Moreover, in mice, it has been found that the deletion of the gene encoding the NR1 subunit of NMDA receptors from PV + interneurons leads to disinhibition of excitatory pyramidal cells and is enough to trigger schizophrenia-like symptoms47. Interestingly, in genetically predisposed individuals, cannabis abuse increases the risk of schizophrenia by enhancing GABAergic dysfunction in a subtype of interneurons containing high levels of cannabinoid receptor 149. Thus, we can see different paths that converge, leading to the same results.According to the glutamate hypothesis of schizophrenia52, there are translational models that can still shed light on the issue. The first is anti-NMDA receptor encephalitis, which can be considered a human translational model. This is a synaptic autoimmune disorder in which IgG autoantibodies target NMDARs in the brain . Patients frequently manifest with prominent psychiatric symptoms, particularly psychosis, early in the disease course55. It is worth noting that autoantibodies targeting AMPA receptors are prone to lead to limbic encephalitis . In a mouse model, the passive cerebroventricular infusion of NMDAR antibodies from patients over a two-week period caused changes in memory and behavior in parallel with an antibody-mediated reduction of synaptic NMDAR56. Thus, anti-NMDA receptor encephalitis represents a natural human model of psychosis and supports the glutamate hypothesis of schizophrenia.Although this hypothesis seems much more complex than its initial formulation57. Animal models have been used to investigate the post-natal administration of NMDA receptor antagonists such as phencyclidine60, or ketamine63, which inhibit GABAergic inhibitory interneurons. Ketamine exposure in postnatal mice has been shown to transiently disrupt NMDAR function, produce schizophrenia-like behavioral symptoms in adulthood and persistently induce changes in GABAergic interneurons, including a reduction in PV+ interneurons in the PFC and HPC, particularly in the dentate gyrus64. Furthermore, it is well known that the use of these glutamate NMDA receptor antagonists mimics the positive, negative and cognitive symptoms of schizophrenia in healthy volunteers and exacerbates psychotic symptoms and cognitive decline in individuals with the disease66. A recent meta-analysis of post-mortem studies found that PV+ interneurons were reduced in the PFC of patients with schizophrenia67. In addition, in an elegant mixed human-animal model, Schobel et al. administered the NMDA antagonist ketamine in mice, confirming the hypothesis that excess glutamate drives hippocampal hypermetabolism and atrophy in psychosis68.A second translational approach to help us better understand schizophrenia, involves NMDAR antagonists (glutamate storm) by excitatory glutamatergic pyramidal neurons in cortical areas and a subcortical hyperdopaminergic state (dopamine storm). Both of these phenomena might be due, in part, to lack of braking, although the long-range pyramidal cortical projection to the ventrotegmental area (VTA) could also contribute to the hyperdopaminergic state69. As mentioned above, increases in extracellular glutamate might act as a pathogenic driver in the brain68, and it is well known that glutamate-gated channels make neuronal cells susceptible to excitotoxicity39. On the other hand, the disinhibition of mesostriatal dopaminergic neurotransmission could cause aberrant salience and either premorbid (transient or attenuated) psychotic symptoms or, if sustained, the full-blown onset of a first psychotic episode70. In this sense, dopaminergic dysregulation appears to develop downstream of abnormalities in the glutamatergic system and it does not seem to be due to primary dysfunction73. Although this question goes beyond the scope of this article, it should be noted that increased DA transmission in the mesolimbic-striatal system probably runs in parallel with DA hypoactivity in the PFC.The dysfunction of GABAergic inhibitory interneurons expressing PV + could produce both a significant release of glutamate 74. Apoptosis is characterized by cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and cellular disintegration with phagocytosis. Another well-known hallmark of apoptosis is phosphatidylserine (PS) externalization in the cell membrane, which is a key signal for the removal of apoptotic cells by surrounding phagocytic cells, such as macrophages and neutrophils. Apoptosis occurs without inflammation and proceeds without a gliosis response. Interestingly, the absence of gliosis is a repeated finding in schizophrenia. Apoptosis is triggered by two principal pathways: the intrinsic  pathway and the extrinsic  pathway. Physiological apoptosis eliminates injured or diseased neurons during lifespan but also during crucial stages of normal brain development . Abnormal dendritic apoptosis has also been implicated in the early stages of Alzheimer’s disease. Specifically, the loss of dendritic spines and synapses in the HPC and cortex is an early event in Alzheimer preceding neuronal death39. Strangely enough, the increased apoptotic susceptibility could help explain the inverse epidemiological comorbidity that both neurodegenerative diseases and schizophrenia have with cancer. Patients with schizophrenia have a significantly lower risk of lung, prostate and colorectal cancers than do individuals without schizophrenia75.The glutamate storm and calcium overload, via glutamate receptors, would trigger neurotoxicity and accelerated dendritic pruning through local activation of the mitochondrial apoptosis pathway and caspase-3 cascade, leading to loss of density of dendritic spines. Apoptosis is a form of programmed cell death that is regulated by a complex cascade of pro- apoptotic (Bax) and anti-apoptotic (Bcl-2 and Bcl-XL) proteins in which caspases are the final executors and key drivers of the apoptotic program 79. Since then, in addition to glutamate excitotoxicity, other pathophysiological processes involved in schizophrenia, such as oxidative stress and lack of neurotrophic factors , have been potentially associated with apoptotic mechanisms81. Results from our own research group strongly support this hypothesis. We have observed increased apoptotic susceptibility in primary fibroblast cell cultures obtained from a skin biopsy of naïve patients with a first psychotic episode82. Using magnetic resonance imaging (MRI) and spectroscopy (1H-MRS), we also found a correlation between altered apoptotic markers and both the volume of certain brain regions and the concentration of glutamate plus glutamine neurometabolites83. In a recent microarray study, in which gene expression was analyzed in primary fibroblast cell cultures and in immortalized lymphocytes from the same patients and healthy controls, we also observed alterations in the expression of genes involved in the apoptotic pathways and other important biological functions such as cytoskeleton organization84.In recent years, it has been proposed that increased susceptibility to apoptosis might be the reason for synaptic or dendritic neuronal loss in individuals with schizophrenia. Two decades ago, the Columbia group was the first to propose this challenging hypothesis85. Generally speaking, cortical volume loss in schizophrenia occurs in the absence of neuronal cell loss and, at least in the PFC, HPC and non-mediodorsal thalamic nucleus, there are no changes in the number of pyramidal neurons86. But what is particularly interesting is that it is a local apoptosis that affects the branches but not the trunk of the tree. Animal models have shown local caspase activity in neurons in which caspases were confined to the dendritic compartment of pruning neurons and were not detected in the soma or axon41. Similarly, using an optogenetic approach, Ertürk and colleagues suggested that NMDA receptor activation can trigger this apoptotic pathway locally in dendrites87. The local activation of apoptosis through caspase-3 within distal dendrites is enough to prune dendritic spines and branches locally. Apoptosis inhibitory proteins and proteasomes act as brakes to limit the activation and prevent the spread of apoptotic events to the neuronal cell body, thereby preventing cell death.As mentioned above, an excess of glutamate could be the trigger of these phenomena of synaptic apoptosis which result in a loss of synapses and dendrites rather than neuronal death 89. In the animal experiments cited above, NMDA antagonists caused an extraordinary increase in extracellular glutamate in the PFC57. Interestingly, Schobel and colleagues in their aforementioned study, utilizing an in vivo glutamate biosensor method in multiple hippocampal subfields, found that ketamine causes a regionally preferential increase in extracellular glutamate in, for example, the CA1 and subiculum68. We could refer to this as a regionally-located glutamate storm. Looking at the timeline of dendritic spine loss, it is important to note that, in primates, excitatory neurons in layer 3 undergo the most dramatic elimination during the adolescent period26. In regard to the timing of apoptosis, it has also been suggested that NMDA receptor-mediated excitotoxicity is more common in young rodents, but not in adults, probably due to loss of caspase-3 expression in neurons with age39.The reason why dendrites and their spines are lost in some brain regions and not in others is not yet well understood, although it could be related to certain areas having a higher density of inhibitory interneurons and NMDA receptors, such as the dorsolateral PFC and HCP90. In terms of clinical translational findings, in different brain areas including the PFC, where synapses occupy 6% and their dendrites 30% of gray matter, respectively86, the loss of dendritic spines could be related to the accelerated gray matter loss observed in several longitudinal MRI studies, the primary negative symptoms and the cognitive deficits of the disease23.A recent meta-analysis of postmortem studies found lower levels of synaptic proteins, dendritic spines, and GABAergic and glutamatergic markers in patients with schizophrenia91. Using the same technique, in first episode psychosis, higher levels of glutamate in the anterior cingulate cortex were associated with a poor antipsychotic response92. Furthermore, the meta-analysis by Marsman and colleagues provides support that there is a progressive decrease of frontal glutamate and glutamine in patients with schizophrenia, possibly indicating a progressive loss of synaptic activity93. Taken together, these observations suggest that there is a decrease in glutamate levels after the glutamate storm occurring around the onset of schizophrenia, and that this runs in parallel with the glutamatergic synapse density decrease. We could refer to this as a transient glutamate storm associated with the early stages of schizophrenia.Recently, Bossong and colleagues, using 1H-MRS, found an association between increased baseline hippocampal glutamate levels and adverse outcomes in individuals at high risk for psychosis95. Once considered mere passive sentinels of immunity, microglia now seem to be crucial for pruning back dendrites during neurodevelopment, particularly in adolescence96. During dendritic apoptosis, caspases and other molecules, such as phosphatidylserine (PS), generate signals to attract microglia for rapid removal of synaptic debris. Thus, synaptic pruning involves microglial phagocytosis of synapses98.In recent years, microglia cells have attracted a great deal of attention. Microglia are the resident immune cells of the central nervous system, performing an essential function during neuroinflammation, while also playing a role in noninflammatory processes like axonal guidance, neurotrophic support, natural apoptosis of developing neurons and synaptic pruning99. It is not fully understood how microglia determine which synapses to prune and which to avoid, however it is known that microglial contact with apoptotic dendrites increases with higher electrical activity . Moreover, looking at analogies with the peripheral immune system, several elegant animal studies have shown that apoptotic dendrites release “find me” signals  and “eat me” signals 103. These findings are in accordance with the hypothesis presented here, combining the glutamate theory of schizophrenia and microglial synaptic pruning. At the same time, another class of immune molecules commonly called “don’t eat me” signals are essential to counterbalance the effects of “eat me” signals . This molecule protects synapses from excess microglia-mediated pruning during neurodevelopment104. Interestingly, recent genome-wide association studies have identified relevant risk loci for schizophrenia including the MHC and the complement C417. Thus, complement abnormalities and proteins of the MHC via microglia activation may contribute to the increase of dendritic apoptosis leading to an excess of synaptic pruning in schizophrenia. Recently, Sellgren and colleagues, using isolated synaptosomes based on patient-derived induced microglia-like cells (iMG), demonstrated increased synapse elimination that could be partly explained by schizophrenia risk variants at the complement C4 locus. They also showed that the antibiotic minocycline reduces microglia-mediated synapse uptake in vitro. Moreover, its clinical use was associated with a decrease, although modest, in the incidence of schizophrenia risk compared to other antibiotics in young adults identified using an electronic health record42. However, these preliminary results need confirmation in prospective randomized clinical trials. Besides cell models, positron emission tomography (PET) imaging could be another promising tool to evaluate in vivo microglial activation. It is assumed that when microglia cells are activated, this increases the expression of the 18-kDa translocator protein (TSPO) which can be measured with PET radiotracers. However, it is still not clear whether TSPO expression is specifically linked to microglia105. A recent meta-analysis of in vivo microglial PET imaging studies in individuals with schizophrenia gave inconclusive results, but further studies are needed with more specific PET radioligands107.During the developmental refinement of neural circuits in adolescence, there is an increase in the density of resident brain microglia cells for synaptic pruning/remodeling. The different timing of synaptic pruning in different cortical regions likely reflects differences in the onset of the critical periods of circuit refinement109.Taken together, the points discussed above seem to confirm that there is a noninflammatory role of the microglial activation. In recent years, elevated levels of some proinflammatory cytokines have been attributed to putative “inflammation” of the brain. However, this conclusion overlooks the fact that cytokines can also have noninflammatory roles related to plasticity, immunity, or brain homeostasis. Thus, although activated microglia and elevations of cytokines are often cited as a marker of “neuroinflammation”, we consider this term unfortunate when used to describe schizophrenia or other psychiatric disorders (for a further discussion of this issue see Estes and McAllister)110. To achieve this, several approaches could be further explored, especially at early stages of disease, targeting aberrant synaptic plasticity, the glutamate storm, dendritic apoptosis, calcium channel dysfunction and microglia activation , acting on microtubule polymerization in neurons, has shown preliminary effects improving memory and cognition in mice. Nevertheless, its efficacy in ongoing clinical trials awaits corroboration111. Raloxifene, a selective estrogen receptor modulator, can increase in vitro spine density in the PFC and improve cognitive symptoms when administered in healthy postmenopausal women and in schizophrenia patients, although results are inconsistent112. Some antipsychotic drugs, such as clozapine, the gold-standard treatment for treatment-resistant schizophrenia, promote the formation of dendritic spines, increasing some of the post-synaptic density proteins, such as spinophilin or shank1a puncta. Moreover, compared to haloperidol, clozapine increased the growth of the spines while changing their shape to be more like mushroom spines113. Finally, it should be mentioned that microRNAs (miRNAs), a class of nonprotein-coding RNAs, could serve as drugs for medical intervention. One example would be miRNAs that modulate gene-protein signaling pathways involved in the polymerization of F-actin filaments of the dendritic spine cytoskeleton. Inappropriate levels of mature miRNA result in the reduction of synaptic proteins, compromising the structure and functionality of the dendritic spines114;(1) Promoting dendritic spines and preventing their loss in individuals who are at high risk for the illness during the prodromal or transitional phase of psychosis has been proposed as a key target in the future treatment of schizophrenia115. Another potential therapeutic target to help correct dysfunctional GABAergic interneuron signaling is the alpha 5 subtype of the GABAA receptor, which is highly expressed in the human amygdala and HCP116. However, its potential efficacy remains to be seen. Evenamide (NW-3509), a selective inhibitor of voltage-gated sodium channels which attenuates glutamate release, is under trial as an adjunctive treatment for resistant schizophrenia117. Ionotropic AMPA glutamate receptors, which are involved in LTP and synaptic plasticity, could be another focus, although the ampakine CX-516 has been found not to be effective for improving cognitive symptoms in individuals with schizophrenia118. To prevent the glutamate storm, further research is needed with anti-glutamatergic drugs such as those that modulate type 2/3 metabotropic glutamate receptors (mGlu2/3), which are expressed on presynaptic nerve terminals and widely distributed in the brain, and which inhibit the presynaptic release of glutamate120. Some of these, such as LY35474063, LY2140023121, or JNJ40411813, the positive allosteric modulator (PAM) of the mGlu2 receptor123, may have failed to treat established cases of schizophrenia, but it could be helpful to investigate their possible benefits during critical early periods of the disease such as the prodromal stage or during the transition to psychosis. Along these lines, we have recently found that treatment of adult mice with JNJ-46356479, a new PAM of the mGlu2 receptor124, partially improves neuropathological deficits and schizophrenia-like behaviors in a postnatal ketamine mice model64, and we are currently investigating the administration of this PAM in adolescent mice. Impeding the glutamate storm may be particularly effective during early stages of schizophrenia to prevent or slow the progression of the illness according to the disease modification hypothesis110;(2) Modulation of the upstream glutamate and GABA systems that regulate dopamine firing could be another approach. In an animal model, a pharmacological modulation of the hippocampal GABAergic system has been reported87. We must bear in mind that modulation of apoptosis demands great caution, especially if such treatments are applied in the early phases of schizophrenia. It is well known that in some cell types and tissues, excessive apoptosis could induce atrophy, while a reduced rate of apoptosis may lead to uncontrolled cell proliferation such as cancer. In preclinical studies, inhibitors of caspase-3 such as Q-VD-OPh and Z-VAD-fmk have been explored in some classic neurodegenerative disorders39. Another focus could be blockers of cytochrome c release in the mitochondrial apoptotic pathway. Such blockers have been found to prevent dopaminergic neuronal death by apoptosis in a rat model of Parkinson’s disease125. It is also worth mentioning here that certain antipsychotics could have a neuroprotective role in vitro through an antiapoptotic action, as was shown in a previous study from our group using a neuroblastoma cell model126. A more recent study also found that clozapine may have a protective/anti-apoptotic effect on adult neural stem cells from ketamine-induced cell death in correlation with decreased apoptosis and autophagy127;(3) Owing to the plausible relevance of dendritic apoptosis in the pathophysiology of schizophrenia, a challenging approach that remains to be investigated involves trying to prevent dendrite removal with the inhibitors of caspase-3 or other components of the mitochondrial apoptosis pathway128;(4) VDCC, found in dendrites and dendritic spines of cortical neurons, could be another potential target, although no evidence has yet been found of the efficacy of any typical calcium channel antagonist such as gabapentin or pregabalin129. Minocycline, as reported earlier, has been found to reduce microglia-mediated synapse uptake in a schizophrenia patient-derived model of synaptic pruning42. Moreover, twelve months of add-on minocycline in recent-onset schizophrenia seems to protect from gray matter loss in the fronto-temporal cortical regions129, although its potential clinical efficacy remains to be established in clinical trials. Another approach could be blockers of the microglial phagocytic receptors  that recognize “eat me” signals  or “don’t eat me” signals 104. Again, caution is required when manipulating the microglial function because it is fairly well-established that microglia have both inflammatory and non-inflammatory physiological roles130.(5) Specific pharmacological interventions targeting microglial elimination of synapses should also be examined, particularly those preventing aberrant synaptic pruning which occurs in the early stages of schizophrenia. These could include the tetracycline antibiotic minocycline or blockers of the microglial phagocytic receptors131.(6) Finally, as depicted in Table 132. This is why a concerted effort should be made to examine central cerebrospinal fluid (CSF) biomarkers of dendritic pruning in both first-episode psychosis as well as in longitudinal studies133. Given the heterogeneity of the illness, stratification of individuals according to future clinical outcomes is crucial. Neurogranin, which is expressed exclusively in the brain, particularly in dendritic spines, seems to be a promising novel CSF biomarker for synaptic loss135. Another potential biomarker may be levels of kynurenic acid, which is an endogenous antagonist of the NMDA glycine modulatory site137. Other approaches will come from new genetic animal models and optogenetic studies in rodents69. Of particular interest are knockout mutant mice that have genes knocked out of microglia cells but no other cells. In animal models, refined imaging techniques such as intravital Multiphoton Microscopy now provide the opportunity for high resolution detection of microglia cells in the living brain138. Finally, another approach that could be investigated is human cellular models, by applying cellular reprogramming methods to create patient-specific in vitro models of dendritic pruning42.Peripheral biomarkers do not fully capture the disease process which primarily occurs in the brain141. The findings, in turn, will allow us to clarify a long-standing hypothesis, not yet proven, suggesting that active psychosis could be neurotoxic142. To accomplish this aim in vivo, we need to explore central CSF biomarkers, find more specific PET radioligands with reliable binding at the NMDA receptor, or identify PET ligands for in vivo evaluation of apoptosis phenomena or microglial activation106. It was thought that labeled annexin-V as PET radiotracer, could detect in vivo externalized phosphatidylserine (PS), one hallmark of apoptosis, but this approach did not live up to its promise. Imaging cell death by apoptosis remains an unsolved problem in clinical molecular imaging, and efforts are needed to overcome this limitation143. One promising approach that is currently under investigation involves using caspase-based specific PET radiotracers to detect caspase-3 activation144. Regarding 1H-MRS, further studies are needed using more sophisticated functional MRS protocols to evaluate brain glutamate levels and discern whether observed increases are due to increased neurotransmission rather than metabolism, since it is difficult to disentangle the peaks from glutamate and glutamine145. A combination of brain imaging, especially PET, 1H-MRS, CSF biomarkers, and cell biology may help improve our understanding of these relevant clinical issues.Last but not least, since psychotic relapses, especially those occurring in the early stages, have been associated with gray matter loss, treatment resistance and poor outcome, it would be interesting to evaluate what happens during a psychotic relapse in terms of brain integrity133. At the same time, considering that schizophrenia is a multifactorial and highly polygenic disorder that shares many risk genes with other psychiatric illnesses including bipolar disorder, depression, intellectual disability and autism spectrum disorders, it is to be expected that these diseases may also share some of these same mechanisms which, in each case, may disrupt different key brain circuits. This driving hypothesis has relevant therapeutic implications. Specifically, it suggests that pharmacological interventions during critical periods of the disease, such as the prodromal stage or during the transition to psychosis, should target dendritic apoptosis, microglia or preventing the glutamate storm. Future research on dendritic apoptosis, brain integrity and clinical outcomes should combine brain imaging techniques, central CSF biomarkers, animal models and cell biology.In conclusion, in this article we have highlighted some of the pathways toward dendritic apoptosis that could stem from a transient glutamate storm and might be aggravated by microglial activation. This links the glutamate hypothesis with the intervention of the immune system in the etiology of schizophrenia. These phenomena could explain the exaggerated synaptic pruning that occurs during late adolescence or early adulthood which might be a core feature underlying schizophrenia. Of course, many other crucial questions remain, such as the timeline of events, the exact nature and location of the cells and neural circuits affected, as well as details about possible cause-effect relationships . Additionally, it should be remembered that the mechanisms mentioned here may be neither exclusive to schizophrenia nor representative of all types of the disease, given its great heterogeneity