Maternal smoking of traditional or electronic cigarettes during pregnancy, which constitutes developmental nicotine exposure (DNE), heightens the risk of neurodevelopmental disorders including ADHD, autism, and schizophrenia in children. Modeling the intergenerationally transmissible impacts of smoking during pregnancy, we previously demonstrated that both the first- and second-generation adolescent offspring of nicotine-exposed female mice exhibit enhanced nicotine preference, hyperactivity and risk-taking behaviors, aberrant rhythmicity of home cage activity, nicotinic acetylcholine receptor and dopamine transporter dysfunction, impaired furin-mediated proBDNF proteolysis, hypocorticosteronemia-related glucocorticoid receptor hypoactivity, and global DNA hypomethylation in the frontal cortices and striata. This ensemble of multigenerational DNE-induced behavioral, neuropharmacological, neurotrophic, neuroendocrine, and DNA methylomic anomalies recapitulates the pathosymptomatology of neurodevelopmental disorders such as ADHD, autism, and schizophrenia. Further probing the epigenetic bases of DNE-induced multigenerational phenotypic aberrations, the present study examined the expression and phosphorylation of key epigenetic factors via an array of immunoblot experiments.394) but not MeCP2 (Ser421) phosphorylation in the frontal cortices, striata, and hippocampi.Data indicate that DNE confers intergenerational deficits in corticostriatal DNA methyltransferase 3A (DNMT3A) expression accompanied by downregulation of methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) in the frontal cortices and hippocampi, while the expression of ten-eleven translocase methylcytosine dioxygenase 2 (TET2) is unaltered. Moreover, DNE evokes multigenerational abnormalities in HDAC2 (SerIn light of the extensive gene regulatory roles of DNMT3A, MeCP2, and HDAC2, the findings of this study that DNE elicits downregulation and aberrant posttranslational modification of these factors in both first- and second-generation DNE mice suggest that epigenetic perturbations may constitute a mechanistic hub for the intergenerational transmission of DNE-induced neurodevelopmental disorder-like phenotypes. One in ten women in the United States disclose smoking conventional cigarettes during pregnancy, and 14% report consuming electronic cigarette during pregnancy . ConcernDNA methylation is a fundamental epigenetic regulator of gene expression which modulates behavior, influences cholinergic, dopaminergic, neurotrophic, and glucocorticoid signaling, and is disrupted in neurodevelopmental disorders, DNE children, and animal models thereof –66. For Contemporary research indicates that DNE alters the transcription and expression of the epigenetic factors DNA methyltransferase 3A (DNMT3A) and ten-eleven translocase methylcytosine dioxygenase 2 (TET2) in rodent offspring , 71, 72.Beyond the presumptive roles of DNA-methylating and DNA-demethylating enzymes, the literature suggests that additional epigenetic factors may be involved in the intergenerational transmission of phenotypic aberrations elicited by developmental exposure to nicotine and other drugs of abuse , 100. Th421) phosphorylation facilitates neurodevelopment and neuronal function, experience-dependent chromatin remodeling, dendritic outgrowth and spine maturation, BDNF expression, and the neurocircuitry of behavior [394) phosphorylation disinhibits oxidative stress-induced neuroapoptosis and may thereby contribute to the aberrant neuroinflammatory and neurodegenerative processes implicated in the pathophysiology of ADHD, autism, and schizophrenia [In addition to the putative roles of perturbed MeCP2 and HDAC2 expression in neurodevelopmental disorders, aberrant posttranslational modifications of MeCP2 and HDAC2 may alter the function of these epigenetic factors and thereby further contribute to neurodevelopmental disorders as well as the multigenerational impacts of DNE. Therein, MeCP2 , region , and a significant group × region interaction  were detected. Relative to F1 Veh mice, F1 NIC and F2 NIC mice exhibit DNMT3A downregulation in the  and , but no DNE-induced alterations in DNMT3A abundance were identified in the hippocampi , region , and a significant group × region × measure interaction  were detected. Compared to F1 Veh mice, F1 NIC and F2 NIC mice have reduced MeCP2 content in the  and , but not in the striata  phosphorylation regulates neurodevelopment and neuronal function, experience-dependent chromatin remodeling, dendritic outgrowth and spine maturation, BDNF expression, and the neural circuitry of behavior [421) phosphorylation. Examining this possibility, we quantified fractional MeCP2 (Ser421) phosphorylation in the frontal cortices, striata, and hippocampi of DNE offspring and grandoffspring compared to F1 Veh control mice. Discordant with our hypothesis, no DNE-induced alterations in MeCP2 (Ser421) phosphorylation were detected in the frontal cortices , region , measure , a significant group × region interaction , a significant group × measure interaction , a significant region × measure interaction , and a significant group × region × measure interaction  were detected. Compared to F1 Veh mice, F1 NIC and F2 NIC mice display decreased HDAC2 abundance in the  and , while no DNE-induced alterations in HDAC2 content were apparent in the striata  derepresses oxidative neuroinflammation and neuroapoptosis and may thereby contribute to the pathophysiology of ADHD, autism, and schizophrenia [394) phosphorylation. To this end, immunoblots were conducted to assess fractional HDAC2 (Ser394) phosphorylation in the frontal cortices, striata, and hippocampi of DNE offspring and grandoffspring compared to F1 Veh controls. In contrast to F1 Veh mice, F1 NIC and F2 NIC mice have increased fractional HDAC2 (Ser394) phosphorylation in the  and  as well as reduced fractional HDAC2 (Ser394) phosphorylation in the .Of additional pertinence to neurodevelopmental disorders and the multigenerational impacts of DNE, phospho-HDAC2  and HDAC2 (Ser394) phosphorylation in the adolescent frontal cortices, striata, and hippocampi. Importantly, a noteworthy limitation in the experimental design of this study is the omission of a second-generation developmental vehicle-exposed control group for direct comparison to second-generation DNE mice. However, prior independent research detected no intergenerational impacts of developmental saccharin (vehicle) exposure on various neurodevelopmental disorder-related phenotypes such as locomotor activity, risk-taking behaviors, and dopamine signaling, the latter of which findings are corroborated by preliminary data from our lab [Toward an improved understanding of the epigenetic mechanisms underlying the association of DNE with neurodevelopmental disorders in children and grandchildren, the present study addresses a void in the literature concerning the hitherto unknown multigenerational impacts of DNE on DNMT3A, TET2, MeCP2, and HDAC2 expression as well as MeCP2  phosphorylation hinders neurodevelopment, experience-dependent chromatin remodeling, dendritic outgrowth and spine maturation, and BDNF expression, resulting in anomalous behavioral phenotypes which are concordant with ADHD, autism, schizophrenia, and smoking during pregnancy [421) content, MeCP2 (Ser421) phosphorylation was unaltered in all brain regions of DNE mice assayed. These data indicate that aberrant phosphorylation of MeCP2 (Ser421) is not a mechanism whereby DNE elicits neurodevelopmental disorder-like brain and behavioral alterations.Complementing the relevance of altered MeCP2 expression to neurodevelopmental disorders and the multigenerational consequences of DNE, atypical MeCP2  content is elevated in the frontal cortices and hippocampi but decreased in the striata of both first- and second-generation adolescent DNE progeny. These results recapitulate the pathophysiology of ADHD, autism, and schizophrenia. Considering that HDAC2 (Ser394) hyperphosphorylation disinhibits oxidative stress-induced neuronal inflammation and apoptosis [394) phosphorylation may disinhibit neuroinflammation and oxidative neurodegeneration and thereby impede neurodevelopment in the frontal cortices and hippocampi, but not the striata [Lastly, the current study is the first to demonstrate that phospho-HDAC2  phosphorylation, while TET2 expression and MeCP2 (Ser421) phosphorylation were unaltered. Cumulatively, these findings imply that DNE differentially impacts discrete epigenetic factors in a brain region-selective fashion. Moreover, the findings of this study suggest that DNE-induced, brain region-selective epigenetic alterations may comprise nexuses for the behavioral, neuropharmacological, neurotrophic, and neuroendocrine anomalies that we previously reported in DNE offspring and grandoffspring [The results of the present study reveal that DNE precipitates intergenerational transmission of corticostriatal DNMT3A deficiency, downregulation of MeCP2 and HDAC2 in the frontal cortices and hippocampi, and aberrant corticostriatal and hippocampal HDAC2 Ser4 phosphoffspring . From a All experimental and housing conditions were reviewed and pre-authorized by the Institutional Animal Care and Utilization Committee at the University of Colorado Boulder and conform to the guidelines for animal care and use established by the NIH and the Guide for the Care and Use of Laboratory Animals (8th Ed.). All mice were maintained in the same animal facility on a standard 12 h light/dark cycle (lights on at 07:00) and were provided food  and water ad libitum. As diagrammed in Fig. Importantly, the 200 µg/mL oral nicotine dosage utilized herein yields a nicotine pharmacokinetic profile in C57BL/6J mice which is comparable to that of regular smokers, confers behavioral and neurobiological anomalies, hinders neurodevelopment, and is widely implemented across the DNE literature, thereby augmenting the generalizability of the DNE paradigm employed herein , 127–130Tissue collection was performed as previously described , 40. BriImmediately following collection, tissue dissectants were homogenized and nuclear-enriched proteins fractionated using a NE-PER kit  according to manufacturer protocol.Protein concentrations for all nuclear-enriched lysates were determined using a Pierce bicinchoninic acid (BCA) Assay Kit  according to manufacturer protocol. Upon determination of protein concentration, samples were diluted to 2 µg/µL, separated into 15 µL (30 µg) aliquots, and stored at − 80 °C until subsequent immunoblot assays.421 MeCP2 (PhosphoSolutions Cat# p1205-421), anti-HDAC2 (ProteinTech Cat# 12922-1-AP), and anti-phospho-Ser394 HDAC2 (PhosphoSolutions Cat# p11421-394). After primary antibody incubation, membranes were washed 5 × 5 min in 0.15% TBST (with agitation). Following washing, membranes were incubated for one hour at RT (with agitation) in secondary antibody solution containing horseradish peroxidase-conjugated donkey anti-rabbit IgG  diluted to a concentration of 1:3000 in blocking solution. Following secondary antibody incubation, membranes were washed 5 × 5 min in 0.15% TBST with agitation, and were then incubated (without agitation) for 5 min in chemiluminescent substrate . The chemiluminescent signals emitted from all membranes were captured using a FluorChem Imager .Immunoblot experiments were conducted via a standard protocol as previously described , 132. Br421) and HDAC2 (Ser394), corresponding relative optical density values for the phospho-specific bands were divided by those for the pan-specific  bands. Notably, and consistent with previous reports, the anti-TET2 antibody used for this study detected three discrete TET2 isoforms [421) and phospho-HDAC2 (Ser394), were transformed to, respectively, percentages relative optical density and percentages fractional phosphorylation versus the mean control (F1 Veh) value for each sample [Unprocessed 8-bit immunoblot images were uploaded to ImageJ 1.52a for densitometry . Backgroisoforms . Howeverh sample , 134, inFor DNMT3A and TET2 immunoblots, percentages relative optical density versus the mean F1 Veh control value were analyzed by mixed ANOVA with the between-subjects factor group  and the within-subjects factor region . For total and phosphorylated MeCP2 and HDAC2 datasets, percentages relative optical density and percentages fractional phosphorylation versus the mean control (F1 Veh) value were analyzed by mixed ANOVA with the between-subjects factor group  and the within-subjects factors region  and measure .Q = 1%), and verified outliers were omitted from analyses as indicated. A maximum of one outlier was excluded per group for each dataset. All data were first analyzed by multivariate ANOVA to assess potential effects of sex, breeder, or litter. No main effects of or interactions with these variables were detected for any outcome measure, and data were therefore collapsed accordingly.Statistical analyses were performed using SPSS , and data were visualized via GraphPad Prism 7.04 . Prior to statistical analyses, data were screened for outliers using the ROUT test (


Mycobacterium tuberculosis (Mtb), the causative infectious agent of tuberculosis (TB), kills more individuals per year than any other infectious agent. Granulomas, the hallmark of Mtb infection, are complex structures that form in lungs, composed of immune cells surrounding bacteria, infected cells, and a caseous necrotic core. While granulomas serve to physically contain and immunologically restrain bacteria growth, some granulomas are unable to control Mtb growth, leading to bacteria and infected cells leaving the granuloma and disseminating, either resulting in additional granuloma formation  or spread to airways or lymph nodes. Dissemination is associated with development of active TB. It is challenging to experimentally address specific mechanisms driving dissemination from TB lung granulomas. Herein, we develop a novel hybrid multi-scale computational model, MultiGran, that tracks Mtb infection within multiple granulomas in an entire lung. MultiGran follows cells, cytokines, and bacterial populations within each lung granuloma throughout the course of infection and is calibrated to multiple non-human primate (NHP) cellular, granuloma, and whole-lung datasets. We show that MultiGran can recapitulate patterns of in vivo local and non-local dissemination, predict likelihood of dissemination, and predict a crucial role for multifunctional CD8+ T cells and macrophage dynamics for preventing dissemination. Mycobacterium tuberculosis (Mtb) and kills 3 people per minute worldwide. Granulomas, spherical structures composed of immune cells surrounding bacteria, are the hallmark of Mtb infection and sometimes fail to contain the bacteria and disseminate, leading to further granuloma growth within the lung environment. To date, the mechanisms that determine granuloma dissemination events have not been characterized. We present a computational multi-scale model of granuloma formation and dissemination within primate lungs. Our computational model is calibrated to multiple experimental datasets across the cellular, granuloma, and whole-lung scales of non-human primates. We match to both individual granuloma and granuloma-population datasets, predict likelihood of dissemination events, and predict a critical role for multifunctional CD8+ T cells and macrophage-bacteria interactions to prevent infection dissemination.Tuberculosis (TB) is caused by infection with  Mycobacterium tuberculosis (Mtb) develop active symptomatic disease [Tuberculosis (TB) kills more individuals per year than any other infectious disease and treatment remains a global challenge . Only a  disease , while t disease . Of key  disease . If Mtb GranSim, our computational model that allows simulation of the formation and function of a single granuloma using a hybrid agent-based model framework, has offered strategies for drug treatment and vaccine development [GranSim, which considers thousands of cells and bacteria as “agents” in the simulation and tracks diffusion of multiple immune mediators , is computationally intensive, limiting our ability to simultaneously simulate multiple granulomas present in an entire lung during infection. In contrast, Prats et al. [It is difficult to experimentally address specific mechanisms operating within lungs that drive different granuloma outcomes in primates, although it is known through interventional studies that certain factors, such as TNF, CD4+ T cells, and CD8+ T cells are important in controlling early and established Mtb infection –8. As a elopment ,14,22–24s et al.  utilizedIn order to study the formation of new granulomas after initial establishment of infection, referred to as dissemination, the evolution of individual granulomas must be captured over time. Recently, research on Mtb-infected NHPs provided data on disseminating granulomas . Of all MultiGran, to track Mtb infection at the scale of the entire lung, including capturing multiple granulomas and their individual outcomes as well as the formation of new granulomas. MultiGran is an agent-based model that follows cells, cytokines, and bacterial populations across multiple lung granulomas throughout the course of infection. Each granuloma is now formulated as a single agent, and each agent contains within it a system of non-linear ordinary differential equations (ODEs) that capture individual granuloma dynamics. MultiGran follows the steps observed through the course of Mtb infection: (1) initial granuloma establishment with Mtb that have been virtually barcoded and placed within the lung environment, (2) granuloma development across time, (3) the possibility of granuloma dissemination with barcoded bacteria moving to a new location, and (4) granuloma merging by granulomas that have formed close together and whose individual boundaries are indistinguishable, or those that grow in size and thus merge into a granuloma cluster (that may have multiple barcoded bacteria IDs). We use MultiGran to address three outstanding questions about dissemination: what mechanisms are consistent with granuloma dissemination and merging patterns seen in vivo? What is the likelihood of a granuloma to disseminate? Can we predict factors that lead to dissemination?Herein, we develop a novel multi-scale hybrid model, th Edition) as mandated by the U.S. Public Health Service Policy.All experimental manipulations, protocols, and care of the animals were approved by the University of Pittsburgh School of Medicine Institutional Animal Care and Use Committee (IACUC). The protocol assurance number for our IACUC is A3187-01. Our specific protocol approval numbers for this project are 1280653, 12126588, 11110045, 19024273, 15066174, 16017309 and 18124275. The IACUC adheres to national guidelines established in the Animal Welfare Act (7 U.S.C. Sections 2131–2159) and the Guide for the Care and Use of Laboratory Animals . These are rotated on a regular basis. Puzzle feeders foraging boards, and cardboard tubes containing small food items also are placed in the cage to stimulate foraging behaviors. Adjustable mirrors accessible to the animals stimulate interaction between animals. Second, routine interaction between humans and macaques are encouraged. These interactions occur daily and consist mainly of small food objects offered as enrichment and adhere to established safety protocols. Animal caretakers are encouraged to interact with the animals  while performing tasks in the housing area. Routine procedures  are done on a strict schedule to allow the animals to acclimate to a routine daily schedule. Third, all macaques are provided with a variety of visual and auditory stimulation. Housing areas contain either radios or TV/video equipment that play cartoons or other formats designed for children for at least 3 hours each day. The videos and radios are rotated between animal rooms so that the same enrichment is not played repetitively for the same group of animals.All macaques used in this study were housed at the University of Pittsburgh in rooms with autonomously controlled temperature, humidity, and lighting. Animals were singly housed in caging at least 2 square meters apart that allowed visual and tactile contact with neighboring conspecifics. The macaques were fed twice daily with biscuits formulated for NHPs, supplemented at least 4 days/week with large pieces of fresh fruits or vegetables. Animals had access to water All animals are checked at least twice daily to assess appetite, attitude, activity level, hydration status, etc. Following Mtb infection, the animals are monitored closely for evidence of disease . Physical exams, including weights, are performed on a regular basis. Animals are sedated prior to all veterinary procedures  using ketamine or other approved drugs. Regular PET/CT imaging is conducted on most of our macaques following infection and has proved very useful for monitoring disease progression. Our veterinary technicians monitor animals especially closely for any signs of pain or distress. If any are noted, appropriate supportive care  and clinical treatments  are given. Any animal considered to have advanced disease or intractable pain or distress from any cause is sedated with ketamine and then humanely euthanatized using sodium pentobarbital.Macaca fascicularis), infected with low dose Mtb  as previously described [18F-FDG PET-CT scan was performed on every animal 1–3 days prior to necropsy to measure disease progression and identify individual granulomas and other pathologies as described [Experimental data specifically for this study were obtained from seven cynomolgus macaques , 5 weeks (n = 2), 7 weeks (n = 2) and 9 weeks (n = 1). In addition, an animal without Mtb infection was also included in this study as a control. To obtain accurate cell number measurements, enzymatic digestion  was performed on excised granulomas using gentleMACS octo dissociator. The single cell suspension obtained by enzymatic digestion was processed for bacterial burden and cell numbers enumeration . Single http://malthus.micro.med.umich.edu/lab/movies/MultiGran/ Table: Granuloma_CFU_counts) and ranged from 4–17 weeks post Mtb infection.In addition, bacterial burden data of 623 granulomas from 38 NHP that were controls in other studies .To allow us to test whether the distribution of granulomas in our virtual lungs matched that observed in NHP lungs, we refer to the distribution of granulomas arising from barcoded bacteria derived from our previously published data in Martin et al. . In thatThree of the four maps are shown in MultiGran is a novel multi-scale, hybrid agent-based model that describes the formation, function, and dissemination of lung granulomas containing Mtb  is placed stochastically within the boundary of the lung environment based on a set of rules. Within each agent, a system of ODEs is linked internally and solved simultaneously to update concentrations of cells, cytokines, and bacterial burdens within each granuloma at every time step. Additionally, within every time step, each granuloma is given the opportunity to disseminate locally and non-locally. Local dissemination involves a new granuloma being initialized nearby, while non-local dissemination allows initialization anywhere within the lung environment. At the lung scale, the model tracks the development, location, and quantity of granulomas, and determines whether each granuloma is either alone or a member of a larger granuloma cluster. At the granuloma scale, dissemination-event decisions, rules for granuloma formation, and concentrations of all granuloma components are tracked and defined. As is occasionally done when a flexible agent size is needed [ning Mtb . It usess needed , our agein silico experiment using MultiGran is designed to replicate an in vivo experiment. To replicate the studies by Martin et al. [Each n et al. , our simn et al. . Each grhttp://malthus.micro.med.umich.edu/lab/movies/MultiGran/ has pseudocode and implementation descriptions, as well as simulation videos.Code is written in MATLAB, with Bash script for submission to run on computer clusters. ODEs are solved using MATLAB’s ode15s with the NonNegative option for all terms, and we define the start and end time interval to be the size of the agent time step. To avoid complications with the random number generator seed being reset with the initialization of each MATLAB instance, the Bash script executes code that generates a randomized seed list for the simulation to use. The website A granuloma is initialized when Mtb is deposited into the lung environment. Based on our previous publications ,25, we aIndivGranID(i), which is assigned in chronological order of initialization i = 1,2,…N , as well as the individual granuloma ID of its parent, so the lineage of each of the founder Mtb can be tracked throughout the course of infection. Each granuloma is also given a position on a continuous grid.Granulomas are agents, so at initialization we assign parameter values to each granuloma and its infecting Mtb, as well as counts and concentrations of all cell types and cytokines. Every granuloma is assigned unique identification markers. These include being given a unique individual granuloma ID The development of each individual granuloma “agent” is captured by a set of ODEs with 16 equations for 16 state variables capturing bacterial, T cell, macrophage and cytokine dynamics  to reproduce multiple scenarios and datasets –48, redu GranSim ,37–40,49 GranSim  to samplMultiGran that matched the NHP whole lung outcome datasets , a global sensitivity analysis index , to idenMultiGran model  with the tissue scale ABM (multiple granulomas) to form the multi-scale an model . LinkingMultiGran, that captures the behavior of Mtb infection leading to the development of multiple granulomas via initial infection and then dissemination of bacteria from existing granulomas. We calibrate and validate MultiGran with unique datasets derived from NHPs, the animal model that most closely mimics the features of human infection. We then use the model to identify dissemination rates and to predict mechanisms leading to dissemination within the calibrated parameter space.We present a whole lung model, We calibrated our single-granuloma model, comprised of a system of non-linear ODEs, to data derived from NHP studies. We compared bacterial load (CFU), T cell counts, and macrophage counts over time per granuloma. Our CFU dataset consists of 623 granulomas from 38 NHPs  of each granuloma, and allows for merging of nearby granulomas to form a cluster. Each granuloma has a unique parameter set chosen from the ranges in in vivo, we use our dataset consisting of four NHPs in that were inoculated with uniquely identifiable Mtb , the median number of granulomas at necropsy (20.5), the median percentage of Mtb barcodes that were found in multiple granulomas (14.3%), and the median percentage of granulomas that contained multiple Mtb barcodes (17.5%) across the four NHP from Martin et al. [From our repository of 200 n et al. .http://malthus.micro.med.umich.edu/lab/movies/MultiGran/.These five simulations represent the best matches to the NHP used in Martin et al. . We compMultiGran simulated lungs on four whole-lung outcomes of interest: the number of dissemination events, the total number of granuloma clusters at the end of the simulation, the percentage of granuloma clusters that contain multiple barcodes, and the percentage of granulomas that occupy the initially-inoculated lung at the end of the simulation. We quantify the contributions of each model parameters to the outcomes of interest by calculating partial rank correlation coefficients (PRCC) at the end of the simulation (250 days). This analysis provides information about the contribution of each model parameter within our parameter space that was calibrated to multiple NHP datasets across cellular, granuloma, and whole-lung datasets. Our analysis reveals one parameter as the main driver of these four whole lung outcomes  and there will be fewer dissemination events and fewer granulomas overall. CD8+ T cells are a key host cell in a functional immune response to Mtb infection, and if the subpopulation that can perform multiple roles within the complex microenvironment of a granuloma increased, it would certainly benefit the host.To predict the mechanisms that lead to dissemination events within lungs, we perform global sensitivity analysis using our repository of 200 outcomes . ParametCD8MultiFun from the analysis, we reveal secondary contributions of other parameters to the whole lung outcomes . We addressed this by allowing T cells to leave the parent granuloma to travel to a daughter granuloma in a local dissemination event, expecting this to sterilize new granulomas. Surprisingly, this was largely ineffective. Instead, it is more likely that the lung parenchyma in infected individuals has increased numbers of Mtb-specific T cells and possibly activated macrophages, so that new granulomas form in a completely different immune environment, compared to the initial granulomas that form in an immunologically naïve environment. This notion is supported by our data in NHP models demonstrating that primary ongoing infection protects against reinfection . MultiGr GranSim , could bMultiGran, the first multi-scale model of granuloma dissemination and formation, that was calibrated and validated to NHP data and we make predictions about the rate of dissemination and the role of specific immune cells in granuloma dissemination. In particular, we discovered roles for multifunctional CD8+ T cells and macrophage dynamics in preventing local and non-local dissemination within the lungs. Altogether, we argue that MultiGran, together with NHP experimental approaches, offers great potential to understand and predict dissemination events within Mtb infected lungs.In summary, we utilized a systems biology approach that combined computational modeling and NHP datasets to better understand mechanisms of granuloma dissemination. We present S1 Equations(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig. The black box designates inoculation region (row 1), wherein the specific within-lung region destined for inoculation is highlighted in green (row 2). The third row demonstrates successful inoculation of a single bacterium–the black box was sampled randomly until the sampled coordinates lie within the green region. Cases 2 and 3 define granuloma placement following dissemination. Case2 –non-local dissemination. When non-local dissemination occurs, a bacterium escapes a single granuloma (row 1) and can be placed in any region (shown in black in row 2) that encompasses the entire lung. The green highlighted region is the area in which the bacterial placement will be accepted. Row 3 shows three trial placements: two realizations of accepted bacterial placement (black arrows) and one unaccepted placement (red arrow) at . Case 3– local dissemination. Local dissemination is the only form of granuloma placement which does not utilize random placement within a region of lung space. Rather, an infected macrophage from the parent granuloma is placed in a random direction away from the parent granuloma. Row 2 shows several options for granuloma infected macrophage placement. Note that the arrows are of different length to represent our assumption that local dissemination likely follows a normal distribution with respect to parent granuloma location. Here, the green and black arrows show valid directions for the new placement for the infected macrophage, while red arrows show invalid directions. A new granuloma will begin to develop in the chosen (green) valid location (Row 3). Note that in both (A) and (B) bacteria, granulomas, and infected macrophages are not to scale. Lung image from Servier Medical Art.Inoculation deposits bacteria in a specific lung region at position (TIF)Click here for additional data file.


Tuberculosis (TB) remains as one of the world's deadliest infectious diseases despite the use of standardized antibiotic therapies. Recommended therapy for drug-susceptible TB is up to 6 months of antibiotics. Factors that contribute to lengthy regimens include antibiotic underexposure in lesions due to poor pharmacokinetics (PK) and complex granuloma compositions, but it is difficult to quantify how individual antibiotics are affected by these factors and to what extent these impact treatments. We use our next-generation multi-scale computational model to simulate granuloma formation and function together with antibiotic pharmacokinetics and pharmacodynamics, allowing us to predict conditions leading to granuloma sterilization. In this work, we focus on how PK variability, determined from human PK data, and granuloma heterogeneity each quantitatively impact granuloma sterilization. We focus on treatment with the standard regimen for TB of four first-line antibiotics: isoniazid, rifampin, ethambutol, and pyrazinamide. We find that low levels of antibiotic concentration due to naturally occurring PK variability and complex granulomas leads to longer granuloma sterilization times. Additionally, the ability of antibiotics to distribute in granulomas and kill different subpopulations of bacteria contributes to their specialization in the more efficacious combination therapy. These results can inform strategies to improve antibiotic therapy for TB. Mycobacterium tuberculosis (Mtb), TB most commonly presents as a pulmonary disease in adults when individuals inhale aerosolized Mtb transmitted by other infected individuals. The immune response in the lungs leads to the formation of multiple lesions called granulomas, collections of immune cells that act to contain the infection both immunologically and physically but also present a barrier to antibiotic diffusion and delivery (Tuberculosis (TB) continues to be one of the world's deadliest infectious diseases, leading to the death of 1.3 million people in 2017, about 2–3 people per minute . Caused delivery . UnderstThe current recommended regimen to treat active, drug-susceptible TB disease requires up to six months of multiple antibiotics . For theThere are two key factors outside of drug resistance that have been identified as contributing to drug failure in TB: pharmacokinetic (PK) variability  and granuloma heterogeneity . How these factors interact to affect both the rate and extent of sterilization during treatment is not well-understood. PK variability is defined as differences in plasma antibiotic exposure, typically measured as variability in plasma area under the curve (AUC) measurements. Population PK models can help determine appropriate dosing of TB antibiotics and represent this variability based on distributions of PK model parameters . These dHost-scale heterogeneity encompasses host-level variations in granuloma number, size, and composition. Granuloma size and composition can lead to slower diffusion of antibiotics, spatial gradients of concentration, and underexposure at the host tissue scale . GranuloCapturing both PK variability at the population scale and granuloma heterogeneity at the host scale in a computational model can help predict granuloma sterilization and design antibiotic regimens. Our group previously developed a computational model that incorporates the host formation of granulomas and antibiotic PK to predict the sterilization of granulomas using different regimens with INH and RIF . Using tHere we use our hybrid, multi-scale agent-based model to capture PK variability and granuloma heterogeneity and to simulate antibiotic treatment of primary, lung granulomas. For the first time with this model, we simulate treatment based on human PK and with the combination of the four first-line antibiotics used to treat TB: INH, RIF, EMB, and PZA. We also present a sequential calibration scheme that captures spatial distributions of antibiotics within granulomas and known PK variability that exists across the population scale and at the host scale within granulomas. Using this highly detailed model, we discuss the role of first-line antibiotics (HRZE) in sterilizing granulomas and how PK variability and granuloma heterogeneity impact distributions of sterilization times.GranSim is a well-established hybrid, multi-scale computational model that produces the emergent behavior of granuloma formation in Mtb infection . GranSim simulates lung granulomas that form due to primary, pulmonary infection in adults and captures a wide diversity of granulomas through variations in host immune system parameters and stochastic events in the agent-based model. The boundary of the granuloma is defined by regions of high cell density, and outlines of the granuloma are drawn to enclose these regions. Parameters that were varied to generate our library of heterogeneous granulomas are listed in nfection . Brieflynfection . Non-repnfection . A more We generate two distinct libraries of granulomas that are heterogeneous in bacterial load and cellular composition: one categorized as low-CFU  granulomas and the other as high-CFU granulomas. The low-CFU granulomas are smaller in size and have CFU/granuloma that are more stable over time, whereas the high-CFU granulomas are larger in size, have increasing CFU over time, and have higher levels of caseum. To generate the low-CFU granulomas, we sampled 500 parameter sets based on ranges for host immune system parameters listed in The plasma PK model is comprised of a two-compartmental model with one or two transit compartments that simulate oral absorption. INH and RIF follow a two-absorption compartment model based on previously developed PK models, whereas EMB and PZA are simulated with one absorption compartment based on best fits and other PK models . The twoThe plasma PK model is linked to the agent-based environment through blood vessels placed on the simulation grid . AntibioGranSim  is dependent on the variable concentration (C), the maximum killing rate constant (maxE), the concentration at half maximal killing (50C), and the Hill curve constant (h). The concentration used to determine the antibiotic killing rate constant is based only on free drug concentration. Parameters maxE, 50C, and h need to be determined for each antibiotic and for each bacterial subpopulation . To fit these parameters, we use in vitro dose-response assays from individual experiments from the literature of Mtb growth/death under varying antibiotic concentrations (see The killing rate constant ions see . This asin silico granulomas that have formed 300 days post infection (in the absence of antibiotics). The doses for the standard regimen are based on CDC recommended adult doses for each of the four antibiotics: INH, 5 mg/kg; RIF, 10 mg/kg; EMB, 17 mg/kg; PZA 21 mg/kg (10 (CFU) per day. For example, the EBA for 0–2 days is calculated as (log10(CFU day 0)-log10(CFU day 2))/2. We simulate four groups of granulomas to incorporate PK variability and granuloma heterogeneity  can lead to differences in antibiotic exposure at the lesion level. To capture both sources of heterogeneity, which occur at different length scales, we devised a sequential calibration scheme to calibrate the PK model from data  (The benefit of combination therapy is more dramatic for high-CFU granulomas (Group 2 of via the immune response.During combination therapy (HRZE), a majority of bacterial death is due to antibiotics; antibiotics are responsible for roughly an order of magnitude more bacterial death than the immune response, and two orders more than bacterial death in caseum representing a lack of oxygen and nutrients , medium (sterilize between 90 and 150 days), high (sterilize after 150 days), and unsterilized. For each of these groups, we compared characteristics of the granulomas before treatment to see what types of granulomas have different levels of risk. Unsterilized and high-risk granulomas tend to be higher in CFU, size, and amount of caseation /day for each treatment. Many of our simulated estimates are near the clinically measured EBA values . For simulated EBA estimates that do not match clinical results, our simulations tend to predict lower EBA values than those observed clinically. Our EBA estimates account for the entire granuloma's CFU count, and it is possible that we predict lower EBAs as our simulations detect more remaining bacteria than those that would be detected clinically in sputum due to limitations of detection in assays used.To place the findings of our work into better context with clinical evidence that has been gathered on first-line TB antibiotics, we compared our simulations for single-drug treatments and combination treatments to early bactericidal activities (EBA) measured in multiple studies  contributed to the conceptualization of this work, analysis of results, and writing and editing.This research was supported by the following grants from the National Institutes of Health: U01HL131072 , R01AI123093 (to DK), R01AI106398, and 1S10OD018072-01A1 (to VD). Simulations used resources of the National Energy Research Scientific Computing Center, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. ACI-1053575 and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation grant MCB140228.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.001, AUC 0.96). Next, a murine DCIS model was combined with ultrasound molecular imaging of B7-H3 targeted microbubbles to differentiate normal glands from those harboring DCIS 634Mul, AUC 0.89). Finally, photoacoustic and fluorescence molecular imaging with an anti-B7-H3 antibody-indocyanine green conjugate were utilized for DCIS detection (n = 53). Molecular imaging of B7-H3 expression may allow for active surveillance of DCIS.Ductal carcinoma in situ (DCIS) will account for 62,930 cases of breast cancer in 2019. DCIS is a pre-invasive lesion which may not progress to invasive carcinoma, yet surgery remains the mainstay treatment. Molecular imaging of a specific marker for DCIS grade for detection and active surveillance are critically needed to reduce potential overtreatment. First, breast cancer marker B7-H3 (CD276) expression was evaluated by immunohistochemical staining in 123 human specimens including benign epithelium (H-score 10.0 ± 8.2) and low (20.8 ± 17.7), intermediate (87.1 ± 69.5), and high (159.1 ± 87.6) grade DCIS, showing a positive association with DCIS nuclear grade ( Widely implemented advanced screening methods, such as mammography, ultrasound, and magnetic resonance imaging, have significantly reduced breast cancer mortality due to earlier detection of carcinomas and, increasingly, pre-invasive lesions such as ductal carcinoma in situ (DCIS) and earlier precursors4. With an estimated 62,930 new cases in 2019, DCIS now accounts for approximately 20% of all breast cancers detected during screening exams1. Once diagnosed, the vast majority of patients with DCIS undergo treatment including breast-conserving surgery with radiation or mastectomy. However, it is estimated that only 16% of low grade DCIS and 60% of high-grade DCIS cases will progress to invasive carcinomas2. Given this wide range of biologic behavior, it is debated whether there is a role for “watchful waiting” for low-risk DCIS, and large clinical trials to determine the best treatment options are currently underway5. Currently, only a select minority of patients adopt a watchful waiting approach with regular imaging surveillance and possibly hormonal therapy. For the cases that will never progress to invasive disease, overtreatment of DCIS comes with its own morbidity risks6 and represents a large, and potentially unnecessary, burden on the US medical system5. Currently, over $800 million is spent annually on DCIS surgical and medical treatment7. Therefore, if a cost-effective and noninvasive method was available to accurately risk-stratify and monitor DCIS for signs of progression7, clinicians and patients might make more informed decisions on treatment, reducing patient morbidity and healthcare costs.Breast cancer remains the second leading cause of cancer-related death in women with ~268,600 new cases and 41,760 deaths predicted in 201910. Microbubbles are small (1–2 µm), non-toxic, lipid shell encapsulated gas bubbles which have targeting moieties attached to the surface. These MBs can circulate within the vasculature12 and attach to molecular targets associated with neoangiogenesis, one of the hallmarks of cancer. MB binding can then be detected noninvasively with contrast-enhanced ultrasound imaging (CEUS), representing a cost-effective, real-time, and noninvasive screening method. Previously, the use of US molecular imaging has been shown to be able to detect invasive breast carcinomas both clinically13 and preclinically17. US molecular imaging represents an ideal method to monitor DCIS for progression, if an ideal molecular target can be determined.Ultrasound (US) molecular imaging, which utilizes molecularly targeted microbubbles (MBs), has recently been explored as a potential method to increase the specificity of ultrasound breast screening, particularly in women with dense breast tissue19, was found to be specifically overexpressed in the four molecular subtypes of breast cancer , and insignificantly expressed in benign epithelium and various fibrocystic changes15. B7-H3 expression is correlated with worse disease outcomes and may play multiple roles in cancers beyond immune regulation, participating in progression, proliferation, and metastasis23. B7-H3 shows both epithelial and endothelial expression, making it an ideal target for various contrast agents that bind either in the vasculature or on the cancer cells24. The expression of B7-H3 in DCIS may not only allow for stratification between low and high-grade lesions, which have different progression rates, but monitoring expression levels over time may allow clinicians to intervene with progression as appropriate. Active surveillance may reduce unnecessary prophylactic procedures, patient morbidity, and burden to the healthcare system.Previously, B7-H3 (CD276), a member of the B7 family of immunoregulators25. Currently, almost one-quarter of patients will undergo additional surgical interventions due to positive or insufficient surgical margins25. Therefore, a real-time, high resolution, and highly specific method for intraoperative tissue assessment is critically needed.Treatment of early disease typically includes less aggressive treatment, such as breast-conserving surgery. For optimal surgical outcomes, it is imperative that the surgeon is able to visualize the borders of the primary tumor, including involvement of DCIS, to minimize recurrence with negative margins26. Photoacoustic imaging, utilizing the photoacoustic effect of generating sound waves subsequent to absorption of nanosecond pulses of light28, allows for high-resolution imaging of optical absorption at depth (up to 5 cm), while fluorescence imaging, lacking background signal, provides high sensitivity to the contrast agent and mimics a surgeon’s field of view. In addition, as optical absorption is wavelength dependent, PA imaging allows for spectral analysis of relative concentrations of tissue chromophores in the tissue, suppressing background signal, and allowing for highly specific imaging of exogenous contrast agents30. Combined with a “smart” contrast agent, such as B7-H3-ICG, which undergoes endocytosis-mediated shifts in optical absorption spectra detectable by PA imaging24, these modalities may represent the optimal method to guide surgeons in intraoperative assessment of tumor margins, reduce the occurrence of positive margins and the need for additional surgical procedures, and improve patient outcomes.It was shown previously that combined photoacoustic (PA) and fluorescent molecular imaging using an anti-B7-H3 antibody-near infrared dye (indocyanine green) conjugate (B7-H3-ICG) could differentiate clinically actionable breast carcinomas from normal mammary tissues in a transgenic murine model of breast cancerThe purpose of this study is multifold as depicted in Fig. n = 57) and three nuclear grades of DCIS: low (n = 18), intermediate (n = 23), and high (n = 16). B7-H3 expression was predominantly membranous. Overall, B7-H3 expression was significantly (P < 0.001) higher in high-grade DCIS (H-Score = 159.1 ± 87.6) compared with normal tissue (H-Score = 10 ± 8.2), low grade DCIS (H-Score = 20.8 ± 17.7), and intermediate grade DCIS (H-Score = 87.1 ± 69.5). Intermediate and low grades were found to have statistically significantly higher expression of B7-H3 than normal mammary tissues (P < 0.001). ROC analysis indicated that B7-H3 immunostaining could distinguish high grade from low grade DCIS with an AUC of 0.96 , and high grade from intermediate grade DCIS with an AUC of 0.73  for all tissues sampled. Representative immunohistochemical staining images and summary graphs are depicted in Fig. B7-H3 expression was assessed via immunohistochemical staining on human tissues including normal epithelium . Similarly, ROC analysis showed an AUC of 1.00  for the ability to distinguish disease state by QIF staining. Representative micrographs and summary graphs are presented in Fig. B7-H3 expression in endothelial cells associated with normal murine mammary tissue and those demonstrating DCIS was assessed by quantitative immunofluorescence staining. Normal mammary glands showed minimal endothelial expression of B7-H3 as measured by histology composite score (CS = 0.01 ± 0.02 a.u.) while DCIS containing tissues had statistically significantly higher endothelial expression  showed a statistically significant increase in CEUS signal (P < 0.001) compared with murine mammary glands containing normal tissues (5.0 ± 3.5 a.u.). ROC analysis of the ability of ultrasound molecular imaging to differentiate between normal tissues and DCIS provided an AUC of 0.89 . Sample B-mode and contrast-enhanced US molecular imaging images and summary graphs are provided in Fig. Next, the ability of CEUS molecular imaging to detect and differentiate B7-H3 expression in DCIS as compared with normal murine mammary tissues with the use of a B7-H3 targeted microbubble was assessed. After injection of MBP < 0.05) compared with normal murine mammary glands which showed an average of 3.2 ± 2.0 a.u molecular imaging signal. ROC analysis of the ability of PA molecular imaging to differentiate between normal tissues and those harboring DCIS provided an AUC of 0.93 . Representative images and summary graphs are presented in Fig. Photoacoustic molecular imaging combined with an antibody-dye contrast agent was evaluated for its ability to image B7-H3 expression in DCIS and differentiate expression levels from normal mammary tissues in a transgenic model of breast cancer development. After injection of B7-H3-ICG, glands with histologically confirmed DCIS showed a statistically significantly higher average sPA molecular B7-H3 signal /(µW/cm2)) compared with normal mammary glands (7.0 × 107 ± 1.0 × 107 (p/s)/(µW/cm2)) but these differences were not statistically significant (P = 0.09). ROC analysis of the ability of fluorescence molecular imaging to differentiate between normal tissues and DCIS provided an AUC of 0.66 . Example fluorescence images and summary graphs are presented in Fig. Next, fluorescence molecular imaging combined with the same antibody-dye contrast agent was evaluated for its ability to image B7-H3 expression in DCIS compared with normal mammary tissues. After injection of B7-H3-ICG, glands with histologically confirmed DCIS showed an increase in average of fluorescence signal  combined with a B7-H3 targeted microbubble was utilized in a screening capacity to image and differentiate DCIS lesions from normal mammary glands in a transgenic mouse model of breast cancer development. Finally, photoacoustic and fluorescence molecular imaging combined with a B7-H3-ICG antibody-dye contrast agent were utilized in an intraoperative scenario to detect small foci of DCIS in mammary glands of the murine breast cancer model.32, and is generally considered to be correlated with poorer outcomes35. B7-H3 is a T-cell modulator, which in part functions to prevent immune invasion within the tumor stroma36. However, B7-H3 expression in early in situ breast carcinomas had not been characterized. Moreover, any association with grade, and therefore associated risk of progression to invasive disease, was unknown. It was found that low-grade lesions had statistically significantly lower B7-H3 protein expression by H-score (taking into consideration both intensity and percent tumor staining), than high-grade DCIS. In addition, low-grade lesions had moderately higher expression than normal epithelium. B7-H3 expression was found to be an excellent method to differentiate between low- and high-grade lesions with an AUC of 0.96. Membranous B7-H3 expression in tumor cells makes it an ideal target for various contrast agents that bind both intravascularly and on the cell surface. Therefore, B7-H3 could be an optimal marker to detect intermediate and high nuclear grade DCIS and serially monitor all grades of DCIS lesions for increasing B7-H3 expression using multiple noninvasive molecular imaging techniques.Previously, B7-H3 has been shown by us and others to be expressed in numerous cancer types including breast10. When ultrasound imaging is combined with molecularly targeted microbubbles there is a dramatic increase in the specificity of the modality. Here, USMI combined with an anti-B7-H3 microbubble was able to differentiate murine DCIS from normal mammary glands with an AUC of 0.89. Initial clinical trials using anti-VEGFR2 targeted microbubbles combined with USMI have been shown safe and hold great promise in cancer detection13. However, the VEGFR2 receptor has only shown a moderate ability to distinguish benign and malignant lesions (AUC of 0.71) in human tissues37 and only preliminary studies into the expression of VEGFR2 on high-grade DCIS have occurred38. Therefore, the USMI of the B7-H3 receptor and its high specificity and ability to differentiate between normal, DCIS, and invasive lesions represents an optimal modality for longitudinal monitoring of DCIS to help plan surgical treatment.A highly specific and sensitive, noninvasive imaging technique is critical to allow for monitoring of patients diagnosed with DCIS over time. Such a technique should be non-radiative, cost-effective, and rapid, all qualities of ultrasound imaging. Currently, ultrasound imaging is being studied for its ability to detect cancers in situ, and while B-mode ultrasound is highly sensitive, it lacks specificity for malignant lesions41. Here, photoacoustic molecular imaging combined with the B7-H3-ICG contrast agent was shown to be able to detect small foci of DCIS in a murine model of breast cancer development. Direct correlation between imaging signal and histological stage  was determined, and photoacoustic molecular imaging was able to differentiate B7-H3-ICG accumulation within small (<1 mm) foci of DCIS from normal murine mammary glands with an AUC of 0.93. The high specificity of photoacoustic imaging of the B7-H3 agent arises from the dynamic optical absorption spectrum of the B7-H3-ICG when binding to its molecular target. Photoacoustic imaging has the sensitivity to detect the changes in the optical absorption spectrum and suppress background signal from blood and unbound agent and this ability was verified through a multi-control study26. While the required imaging depth was limited in this study due to the superficial and small nature of murine mammary glands, photoacoustic imaging was able to provide high-resolution images of optical contrast at depth within the glands. Currently, clinical photoacoustic systems are emerging on the market that are optimized for human application and imaging depths up to 5 cm46, making clinical photoacoustic imaging, especially during intraoperative scenarios, a feasible molecular imaging technique to detect and monitor DCIS in humans.Aside from detecting and monitoring DCIS for screening purposes, it is also critical to be able to do so in the intraoperative setting to ensure negative margins. Significant research effort is currently dedicated to implementing fluorescence and photoacoustic imaging for intraoperative guidance26. Fluorescence imaging has larger filter bandwidth (~40 nm) for excitation and emission, and therefore taking advantage of the shifting ICG spectrum is difficult for the modality compared with photoacoustic imaging, and consequently separating blood pool signal from B7-H3-specific signal proved difficult. Currently, fluorescence guided surgery is a large area of ongoing research with multiple applications and available agents being developed41. The familiar “bird’s eye view” of fluorescence imaging makes it highly compatible with surgical applications and user friendly for surgeons. The high sensitivity of fluorescence imaging can detect small amounts of tracer due to limited background signal, and fluorescence can easily detect areas of agent accumulation away from the primary site of interest, critical in surgical settings, a limitation of ultrasound and photoacoustic techniques. Fundamentally, when used together, photoacoustics and fluorescence may offer a high-resolution and high-sensitivity molecular imaging at depth and with a wide field of view all with one molecularly targeted contrast agent for multiple purposes including intraoperative guidance.While photoacoustic imaging provides high-resolution images at depth within a surgical field, it remains a focal imaging scan that transects the imaging plane visible to the surgeon. Fluorescence imaging provides a wide field of view that corresponds directly to the surgeon’s, making the two modalities highly complementary for intraoperative molecular imaging. However, in this study fluorescence imaging of the B7-H3-ICG agent was not able to reliably detect significant differences between DCIS and normal tissues, indicating an early stage disease sensitivity limit for the modality in this application. Previously, the modality has been able to differentiate invasive carcinoma in a similar situationThere are several limitations of this study. First, as most patients undergo treatment after diagnosis with DCIS, determining progression of disease or changes in B7-H3 expression with progression was not possible within the patient population studied here. Second, the murine model of breast cancer progression used in these studies is transgenic with a virus promoter of Polyoma Virus middle T antigen (PyVT). Therefore, mammary glands will always progress to invasive carcinomas with time and it is not possible to grade murine DCIS as in human disease. There is currently no natural model of murine DCIS to use to correlate B7-H3 expression levels with progression probabilities. Furthermore, disease development in this model is multifocal, which, even in early disease such as DCIS, could indicate higher disease burden within the mammary glands. Increased areas of DCIS could overestimate the ability of the imaging modalities to detect early disease. However, for the imaging studies here, each gland was analyzed as a single source of imaging signal, including the surrounding normal tissues, potentially minimizing molecular imaging signal from DCIS. Finally, there is no need to accommodate depth during in vivo mouse imaging studies due to their small size. Typically, optical modalities suffer from depth limitations, and while photoacoustics can image up to 5 cm deep, intraoperative use, as suggested here, highlights an optimal application due to reduced depth requirement.In conclusion, the results of this study suggest that B7-H3 protein expression levels in human DCIS strongly correlate with nuclear grade and, therefore, likelihood of progression to invasive carcinoma. In addition, ultrasound, photoacoustic, and fluorescence molecular imaging techniques combined with appropriate B7-H3 targeted contrast agents were able to detect DCIS in a murine model of breast cancer development. In the future, these imaging strategies may allow for active surveillance of DCIS or detection of DCIS in an intraoperative scenario minimizing unnecessary surgical interventions.Human breast tissue samples were obtained retrospectively and were selected under an HIPAA compliant, Institutional Review Board-approved protocol at the University of California San Francisco which superseded the need for informed consent. A total of 57 samples of DCIS of low, intermediate, or high nuclear grade and an additional 57 cases of normal breast epithelium were evaluated utilizing tissue microarray (TMA). In brief, TMA cases were constructed from patient excisions after characterization by a dedicated breast pathologist. Three 2-mm punch biopsy tissue cores were obtained from each case for analysis. Positive and negative on-slide controls consisted of normal breast and invasive ductal carcinoma of no special type.47, incorporating intensity and percent positive membranous staining (1 * % with low intensity + 2 * % with moderate intensity + 3 * % with strong intensity), resulting in ranges of values between 0 and 300. Replicate samples (1–3 per patient) were averaged. The H-scores from within each category  are presented as means ± standard deviation.Immunohistochemical staining and scoring of collected human samples was performed using anti-human B7-H3 antibody  on 5 µm sections of paraffin-embedded breast tissues using heat induced antigen retrieval program at pH 9.0. A dedicated breast pathologist scored all immunohistochemically stained sections by H-score48. The MMTV-PyMT model closely recapitulates human disease by spontaneously progressing through several stages of breast cancer disease including hyperplasia, DCIS, and finally invasive carcinoma between 5 and 12 weeks of age, independently in each mammary gland. By 5–7 weeks most of the 10 mammary glands in a given animal will contain multifocal DCIS48. Glands that were found to contain either hyperplasia or invasive carcinoma, as assessed by ultrasound imaging and hematoxylin and eosin (H&E) staining, were excluded from the study. Normal mammary glands from littermates that were negative for the transgene were used as controls. Fur covering the mammary glands was removed with the use of depilatory cream. During US molecular imaging studies, mice were anesthetized with 2% isoflurane in oxygen flowing at 2 L/min. Heart rate, ECG, and respiration rate were monitored using an Advanced Physiological Monitoring Unit, and the body temperature was maintained at 37 °C with the use of a heated stages and pre-warmed ultrasound gel. For photoacoustic and fluorescence molecular imaging, animals were humanely euthanized immediately before imaging.All experiments involving animals were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. A transgenic mouse model of breast cancer (FVB/N-Tg(MMTVPyMT)634Mul) was used49 and solid, cribriform and/or comedo architecture with cells demonstrating intermediate to high nuclear grade. Comedonecrosis was focally identified. Glands containing invasive carcinoma or hyperplasia  were excluded from the study. Pathological assessment was used to classify mammary glands for molecular imaging analysis.Immediately after imaging, mammary tissues from transgene positive animals, and select mammary glands from transgene negative animals were excised, formalin-fixed and paraffin-embedded. Tissue sections (10 μm) were stained with H&E via standard protocol. Histological diagnosis of DCIS versus normal was rendered blinded to the imaging findings and age of the animal. Normal mammary tissue was comprised of primarily adipose inclusive of a few, well-organized ducts. DCIS was defined as an increase of acinar cell clusters with completely expanded intraductal epithelial proliferation24. Briefly, mammary glands known to harbor DCIS (n = 7) and normal tissues (n = 5) on H&E-stained sections were split before fixation, frozen in optimal cutting temperature (OCT) compound, sectioned (10 μm), rinsed with PBS for 5 min, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.05% Triton in PBS for 15 min. Sections were blocked with 5% BSA, 5% goat serum in PBS for 1 h at room temperature. Primary antibodies were applied (rabbit anti-mouse B7-H3 and rat anti-mouse CD31 antibodies (Abcam Inc.)) overnight at 4 °C at dilutions of 1:50 and 1:100, respectively, and were visualized with labeled secondary antibodies (AlexaFluor-488 conjugated goat anti-rabbit or AlexaFluor-546 anti-rat secondary antibodies (1:300) respectively) . Fluorescent images were acquired with a LSM 510 Meta confocal microscope  at ×200 magnification.Murine B7-H3 expression of the transgenic animal model was confirmed with ex vivo immunofluorescence staining as described previously24, only endothelial expression was analyzed to provide comparable ROIs between normal  and DCIS . In random fields of view, vessels were visualized with CD31 staining and quantified using ImageJ (version 1.46r) software. ROIs were determined by converting to 8 bit images then applying a threshold to create binary images. These binary images were used as masks on the B7-H3 channel images, and fluorescence intensity was measured using mean fluorescence intensity (a.u.)15. Data are presented as means ± standard deviation and differences were assessed by a two-sided t-test, with P values of <0.05 considered indicative of statistically significant differences between the means. The ability of B7-H3 expression to differentiate normal glands and those glands demonstrating DCIS was examined with receiver operating characteristic (ROC) analysis presented as area under the curve (AUC) and 95% confidence intervals.Though B7-H3 is expressed on both endothelial and epithelial cells in diseased murine mammary tissuen = 4 mice, 36 glands shown to be normal on histology) or those suspected of containing DCIS due to age  underwent molecular imaging immediately following injection of B7-H3 targeted microbubbles.For ultrasound molecular imaging of B7-H3 expression in murine DCIS versus normal, animals with normal mammary glands (B7-H3) as described previously15. Briefly, each vial of lyophilized streptavidin-coated microbubbles was suspended in 1 mL of sterile saline (0.9% sodium chloride). To add the targeting antibody to the surface, 6 μg of biotinylated rat anti-mouse B7-H3 antibodies  were incubated with 5 × 107 MBs for 10 min at room temperature. Bubbles were used immediately.Commercially available preclinical streptavidin-coated, perfluorocarbon containing lipid-shelled microbubbles  were used to generate B7-H3-targeted microbubbles  was delivered to clear all MBs within the field of view. Finally, the MB replenishment curve (indicative of freely circulating MBs) was monitored by collecting 200 additional frames of CEUS. Multiple glands can be imaged with a single injection of MBs 52.Targeted CEUS was performed in contrast mode using a dedicated small-animal high-resolution ultrasound imaging system  equipped with an 18 MHz transducer . Mammary glands were imaged in the transverse cross section at the location of maximal gland diameter. To differentiate between the acoustic signal from adherent microbubbles and the signal from freely circulating microbubbles, destruction and replenishment curves were obtained as previously described26. Briefly, per batch, 100 µg B7-H3-targeted antibody  (ab134161) in 200 µl PBS was incubated with a 20x molar equivalent of ICG-NHS  in 50 μl dimethyl sulfoxide (DMSO) to form bonds via NHS ester labeling of amino groups of the lysine peptides on the antibodies. Labeled antibodies were separated from free ICG dye using a Sephadex™ PD-10 gel filtration column as a visible fraction. Protein concentrations were determined through spectrophotometric analysis at 280 and 560 nm with correction factor53. Ab-dye conjugates were concentrated to 0.33 mg/ml in PBS using 30,000 Da molecular weight cutoff filters centrifuged at 2500 × g for 5 min. For photoacoustic and fluorescence molecular imaging techniques, each animal was injected via tail vein with 100 µl (33 µg) of the B7-H3-ICG contrast agent 24 h before imaging to allow for sufficient accumulation at the target sites26.B7-H3-targeted antibody-dye conjugation and characterization was carried out as previously describedn = 2 mice, 17 glands) or those harboring DCIS  underwent molecular imaging before and 24 h after injection of the B7-H3-ICG contrast agent and immediately after humane euthanasia and removal of the skin over the mammary glands to allow for simulation of intraoperative imaging. The VisualSonics VevoLAZR  was used to obtain transverse, single-plane ultrasound images to identify the maximal gland diameter and maintain imaging consistency between time points. Co-registered, multiwavelength  photoacoustic images were then obtained. The transducer surface was maintained at 9 mm from the surface of the skin coupled with clear, bubble free, colorless gel to maintain even light illumination throughout the gland and imaging consistency. Images were saved in the .RAW format for subsequent processing.For photoacoustic molecular imaging of B7-H3 expression, animals with normal mammary glands  or those harboring DCIS  underwent molecular imaging 24 h after injection of the B7-H3-ICG contrast agent and immediately after humane euthanasia and removal of the skin covering the mammary glands to simulate intraoperative conditions. Fluorescence images were collected using the IVIS Spectrum Preclinical Imaging System (PerkinsElmer) in epifluorescence mode equipped with 710/30 nm and 820/20 nm filters for excitation and emission, respectively. Data were saved for subsequent image analysis.For fluorescence molecular imaging of B7-H3 expression, animals with normal mammary glands . ROIs were drawn over imaged mammary glands and the value of the differential targeted enhancement (T.E. (a.u.)) was calculated, defined as the average pre-destruction imaging signal (a.u.) divided by the average post-destruction signal (a.u.) as described previously54. Data are presented as means ± standard deviation.Ultrasound molecular imaging signal, corresponding to the volume of MB56. Briefly, the co-registered B-mode ultrasound images were used to guide selection of a region of interest (ROI) over the whole mammary gland. Then, the relative concentrations of each photoabsorber  were calculated by comparing wavelength dependent PA signal changes to known optical absorption properties of the chromophores with linear regression techniques. Photoacoustic molecular imaging signal is displayed as the ratio of ICG signal 24 h after injection compared with before injection imaging within the ROI. Data are presented as means ± standard deviation.Photoacoustic molecular imaging signal corresponding to relative B7-H3 expression levels was determined using a custom MATLAB-based linear least squares error classification algorithm that has been described in detail elsewhere2/sr/µW/cm2) was measured. The background blood pool signal was measured with an ROI over the sternum. Fluorescence signal ratios were calculated as the radiant efficiency in the mammary gland over the background signal. Data are presented as means ± standard deviation.Fluorescence molecular imaging signal, quantitatively measured and corresponding to B7-H3-ICG accumulation in the mammary gland, was evaluated using Living Image Software 4.0 (PerkinElmer). Fixed sized ROIs were placed over each mammary gland guided by co-registered photographs and average radiant efficiency  software. Associated manuscript data in Prism GraphPad file format are available from the metadata entry57.Receiver operating curves  were used to elucidate the ability of the various molecular imaging modalities and agents to differentiate normal tissues from DCIS and are presented as the AUC followed by the 95% confidence interval. Statistical significance was determine through two-sided Reporting-Summary


Electrospinning is one of the most widely used techniques for the fabrication of nano/microparticles and nano/microfibers, induced by a high voltage applied to the drug-loaded solution. The modification of environmental conditions, solution properties or operation parameters results in different fiber properties, thus enabling the fine-tuning of functionality-related characteristics of the final product. The latter includes the alteration of the rate and extent of the solubility of drugs, hence the rapid or prolonged onset of absorption.This Special Issue serves to highlight and capture the contemporary progress of electrospinning techniques, with particular attention to their further pharmaceutical application as conventional and novel drug delivery systems or for tissue regeneration purposes. It comprises a series of 12 research articles and one review, illustrating the versatile researches and teams from 13 different countries, making profound contributions to the field.Palo et al. investigated a combined technique for the fabrication of bi-layered carriers from a blend of polyvinyl alcohol (PVA) and sodium alginate (SA). The bi-layered carriers were prepared by solvent casting in combination with two surface modification approaches, electrospinning or three-dimensional (3D) printing. An initial inkjet printing trial for the incorporation of bioactive substances for drug delivery purposes was performed. The solvent cast (SC) film served as a robust base layer. The bi-layered carriers with electrospun nanofibers (NFs) as the surface layer showed improved physical durability and decreased adhesiveness compared to the SC film and bi-layered carriers with a patterned three-dimensional (3D) printed layer. The bi-layered carriers presented favorable properties for dermal use with minimal tissue damage. In addition, electrospun NFs on SC films (bi-layered SC/NF carrier) provided the best physical structure for cell adhesion, and proliferation as the highest cell viability was measured compared to the SC film and the carrier with a patterned 3D printed layer (bi-layered SC/3D carrier) .Cherwin et al. developed a novel oxygen therapeutic made from a pectin-based hydrogel microcapsule carrier mimicking red blood cells. The study focused on three main criteria for developing the oxygen therapeutic to mimic red blood cells: Size (5–10 μm), morphology (biconcave shape), and functionality ). The hydrogel carriers were generated via the electrospraying of the pectin-based solution into an oligochitosan crosslinking solution using an electrospinning setup. The pectin-based solution was investigated first to develop the simplest possible formulation for electrospray. The production process of the hydrogel microcapsules was also optimized. The microcapsule with the desired morphology and size was successfully prepared under the optimized condition. The encapsulation of Hb into the microcapsule did not adversely affect the microcapsule preparation process, and the encapsulation efficiency remained high (99.99%). The produced hydrogel microcapsule system offers a promising alternative for creating a novel oxygen therapeutic .Staphylococcus aureus and Escherichia coli. Consequently, PCL-GEL/CLV nanofiber mats can be potential candidates for antibiotic-free wound healing applications [Unalan et al. prepared antibacterial poly(ε-caprolactone) (PCL)-gelatin (GEL) electrospun nanofiber mats containing clove essential oil (CLV) using glacial acetic acid (GAA) solvent. The addition of CLV increased the fiber diameter from 241 ± 96 nm to 305 ± 82 nm. Along with the increase of the CLV content of nanofibers, the wettability of PCL-GEL nanofiber mats was also increased. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the presence of CLV, and the actual content of CLV was determined by gas chromatography–mass spectrometry (GC-MS). It was confirmed that the CLV-loaded PCL-GEL nanofiber mats did not have cytotoxic effects on normal human dermal fibroblast (NHDF) cells, while the fibers exhibited antibacterial activity against Viet Nguyen et al. developed novel amphiphilic electrospun nanofibers (NFs) loaded with haemanthamine (HAE), phosphatidylcholine (PC), and polyvinylpyrrolidone (PVP), intended for a stabilizing platform of self-assembled liposomes of the active agent. The NFs were fabricated with a solvent-based electrospinning method. The HAE-loaded fibers showed a nanoscale size ranging from 197 nm to 534 nm. The liposomes with a diameter between 63 nm and 401 nm were spontaneously formed as the NFs were exposed to water. HAE dispersed inside liposomes showed a tri-modal dissolution behavior. Amphiphilic NFs loaded with HAE are an alternative approach for the formulation of a liposomal drug delivery system and stabilization of the liposomes of the chemically instable and poorly water-soluble alkaloid [v/v) and formic acid solution (90% v/v) . The fiber diameter and morphology of the nanofibrous DDSs were modulated by varying ultrasonic parameters in the USES process . The authors found that the USES technology produced nanofibers with a higher fiber diameter (402 ± 127 nm) than TES (77 ± 21 nm). An increase in burst count in USES increased the fiber diameter (555 ± 265 nm) and the variation in fiber size. The slight-to-moderate changes in a solid state  were detected in comparison to the nanofibers generated by TES and USES. In conclusion, USES provides a promising alternative for aqueous-based fabrication of nanofibrous DDSs for various pharmaceutical and biomedical applications [Hakkarainen et al. investigated nozzleless ultrasound-enhanced electrospinning (USES) as a means to generate nanofibrous drug delivery systems (DDSs) for pharmaceutical and biomedical applications. Traditional electrospinning (TES) equipped with a conventional spinneret was used as a reference method. High-molecular polyethylene oxide (PEO) and chitosan were used as carrier polymers and theophylline anhydrate as a water-soluble model drug. The nanofibers were electrospun with the diluted mixture (7:3) of aqueous acetic acid -loaded polymeric nanofibers in situ and determined the solid-state of the drug during dissolution. Hydroxypropyl methylcellulose (HPMC) and polydextrose (PD) were used as carrier polymers for electrospinning (ES). The initial-stage dissolution of the nanofibers was monitored in situ with three-dimensional white light microscopic interferometry (SWLI) and high-resolution optical microscopy. They confirmed that PRX recrystallizes in a microcrystalline form immediately after wetting of nanofibers, which could lead to enhanced dissolution of the drug. Initiation of crystal formation was detected by SWLI, indicating that PRX was partially released from the nanofibers, and the solid-state form of PRX changed from amorphous to crystalline. The amount, shape, and size of the PRX crystals depended on the carrier polymer used in the nanofibers and the pH of the dissolution media. The PRX-loaded nanofibers exhibited a quasi-dynamic dissolution via recrystallization. SWLI enabled a rapid, non-contacting, and non-destructive method for in situ monitoring of the early-stage dissolution of nanofibers and regional mapping of crystalline changes during wetting .Preem et al. tested and compared different drug release model systems for electrospun chloramphenicol (CAM)-loaded nanofiber (polycaprolactone (PCL)) and microfiber (PCL in combination with polyethylene oxide) mats with different drug release profiles. The CAM release and its antibacterial effects in disc diffusion assay were assessed by bacterial bioreporters. The release into buffer solution showed larger differences in the drug release rate between differently designed mats compared to the hydrogel release tests. The UV imaging method provided an insight into the interactions with an agarose hydrogel mimicking wound tissue, thus providing information about early drug release from the mat. Bacterial bioreporters showed clear correlations between the drug release into gel and antibacterial activity of the electrospun CAM-loaded mats .Lactobacillus and one of Lactococcus were characterized. The electrospinning of polymer solutions containing ~10 log colony forming units (CFU)/mL of lactic acid bacteria enabled the successful incorporation of all bacterial species tested, from the smallest  to the largest , into poly(ethylene oxide) nanofibers with an average diameter of ~100 nm. All of these lactobacilli were viable after incorporation into nanofibers, with 0 to 3 log CFU/mg loss in viability, depending on the species. Viability correlated with the hydrophobicity and extreme length of lactic acid bacteria, whereas a horizontal or vertical electrospinning set-up did not have any role. Therefore, electrospinning represents a promising method for the incorporation of lactic acid bacteria into solid delivery systems, while drying the bacterial dispersion at the same time [Zupančič et al. investigated the effect of electrospinning on the viability of bacteria incorporated into nanofibers. The morphology, zeta potential, hydrophobicity, average cell mass, and growth characteristics of nine different species of w/w triethanolamine, 15% w/w PVPK90) were found to be suitable for obtaining orally dissolving webs of fast dissolution and potential oral absorption [Sipos et al. formulated aceclofenac-loaded poly(vinyl-pyrrolidone)-based nanofibers by electrospinning to obtain drug-loaded orally disintegrating webs to enhance the solubility and dissolution rate of the poorly soluble anti-inflammatory active that belongs to the Biopharmaceutical Classification System (BCS) Class-II. Triethanolamine-containing ternary composite of aceclofenac-poly(vinylpyrrolidone) nanofibers was formulated to exert the synergistic effect on the drug-dissolution improvement. The nanofibrous formulations had diameters in the range of a few hundred nanometers. FT-IR spectra and DSC thermograms indicated the amorphization of aceclofenac, which resulted in a rapid release of the active substance. The characteristics of the selected ternary fiber composition . The presence of crystalline mannitol and the low water content enabled appropriate grinding of the fibrous sample without secondary drying. The ground powder was mixed with commonly used tabletting excipients and was successfully directly compressed. β-galactosidase remained stable in the course of the whole processing and after one year of storage at room temperature in the tablets. The results demonstrate that high-speed electrospinning is a viable alternative to traditional biopharmaceutical drying methods, especially for heat-sensitive molecules, and further processing of electrospun fibers to tablets can be successfully achieved [Vass et al. developed a processable, electrospun formulation of a model biopharmaceutical drug, β-galactosidase. They demonstrated that higher production rates of drug-loaded fibers could be achieved by using high-speed electrospinning compared to traditional electrospinning techniques. An aqueous solution of 7.6 achieved .d,l-lactide-co-glycolide) (PLGA)/gelatin polymer solutions for the simultaneous release of recombinant human epidermal growth factor (rhEGF) and gentamicin sulfate at the wound site to hasten the process of diabetic wound healing and inactivation of bacterial growth. The bacterial inhibition percentage and detailed in vivo biocompatibility for wound healing efficiency was performed on diabetic C57BL6 mice with dorsal wounds. The scaffolds exhibited excellent wound healing and continuous proliferation of cells for 12 days, thus providing a promising means for the rapid healing of diabetic wounds and ulcers [Dwivedi et al. designed a nanocomposite carrier using Poly(d ulcers .co-polycaprolactone (PLA-PCL) electrospun nanofibers; quantification and in vitro drug release profiles in static and dynamic conditions were investigated. The kinetics of the GS release from nanofibers was studied using mathematical models. A preliminary microbiological test was carried out towards Staphylococcus aureus and Escherichia coli bacteria. The prolonged effect of the antibiotic at the site of action can reduce administration frequency and improve patient compliance [Pisani et al. studied electrospun nanofibers as antibiotic release devices for preventing bacteria biofilm formation after surgical operation. In their work gentamicin sulfate (GS) was loaded into polylactide-mpliance . Pant et al. summarized that electrospinning has emerged as a potential technique for producing nanofibers. The use of electrospun nanofibers in drug delivery has increased rapidly over recent years due to their valuable properties, which include a large surface area, high porosity, small pore size, superior mechanical properties, and ease of surface modification. A drug-loaded nanofiber membrane can be prepared via electrospinning using a model drug and polymer solution; however, the release of the drug from the nanofiber membrane in a safe and controlled way is challenging as a result of the initial burst release. Employing a core-sheath design provides a promising solution for controlling the initial burst release. This paper summarizes the physical phenomena, the effects of various parameters in coaxial electrospinning, and the usefulness of core-sheath nanofibers in drug delivery. It also highlights the future challenges involved in utilizing core-sheath nanofibers for drug delivery applications . All the articles presented in this Special Issue represent a small fraction of the great research interest in the field of nanofibrous system applications as drug delivery bases or for tissue engineering purposes. Their diverse and tunable features enable a wide variety of use, which opens a new dimension in the case of their feasible scaling-up.


Clinical assessments were undertaken at baseline and follow-up (median 15 months). Striatal dopamine synthesis capacity predicted the worsening of psychotic symptoms at follow-up , but not transition to a psychotic disorder (p = 0.22), and was not significantly related to hippocampal glutamate concentration (p = 0.13). There were no differences in either glutamate (p = 0.5) or dopamine (p = 0.5) measures in the total patient group relative to controls. Striatal dopamine synthesis capacity at presentation predicts the subsequent worsening of sub-clinical total and psychotic symptoms, consistent with a role for dopamine in the development of psychotic symptoms, but is not strongly linked to hippocampal glutamate concentrations.Preclinical models of psychosis propose that hippocampal glutamatergic neuron hyperactivity drives increased striatal dopaminergic activity, which underlies the development of psychotic symptoms. The aim of this study was to examine the relationship between hippocampal glutamate and subcortical dopaminergic function in people at clinical high risk for psychosis, and to assess the association with the development of psychotic symptoms.  Psychotic disorders are typically preceded by a prodromal period of 1–5 years, characterised by worsening sub-clinical psychotic symptoms and a decline in overall functioning . People n = 14), the first aim of this study was to test the hypothesis that striatal dopamine synthesis capacity and hippocampal glutamate measures are related in an independent and larger sample of clinical high-risk subjects.The dopamine and glutamate hypotheses are two leading theories for the pathophysiology of psychosis . AlthougIn vivo imaging studies of dopaminergic function show that dopamine synthesis and release capacity are elevated in patients with psychosis , 23, andThe study comprised a case–control comparison of patients with healthy controls with a longitudinal, naturalistic clinical follow-up of patients to determine the relationship between baseline imaging measures and clinical outcome. All volunteers received clinical and imaging measures. In addition, the patients received follow-up to determine clinical outcome as described below. Ethical permission was given by the local research ethics committee. All participants provided written informed consent to participate.Patients were recruited from services for people at clinical high risk of psychosis in South England. Inclusion criteria were (1) met operationalized criteria for being at clinical high risk of psychosis based on a standardised, semi-structured clinical assessment as described by Yung et al. ; (2) no Healthy controls were recruited from the same geographical area via adverts in the local media and met the following inclusion criteria: (1) no history of current or past mental disorder assessed using the structured clinical interview for diagnosis ; (2) no Exclusion criteria for all subjects were any of (1) a history of significant head trauma, (2) dependence on illicit substances, (3) medical co-morbidity  and (4) contraindications to scanning (such as pregnancy).At baseline, all subjects were assessed using the Global Assessment of Function (GAF ) to measn = 3), PANSS-positive severity ratings (after subtracting seven to correct for the non-zero floor [Patients received a follow-up assessment at a median of 15.0 (interquartile range (IQR) = 11–23) months post baseline measures. Transition to a psychotic disorder was determined using the structured clinical interview for diagnosis . The perro floor ) were us18F-DOPA was administered by bolus intravenous injection 30 s after the start of the dynamic PET scan. PET data were acquired in 32 frames of increasing duration, over the 95-min scan . Our primary measure was the whole striatal influx constant , and refrain from alcohol for 12 h prior to scan and not to smoke for 2 h prior to the scan . Imagingications ).18F-DOPA template [icer was calculated using the Patlak–Gjedde graphical approach adapted for a reference tissue input function [Image analysis was conducted blind to group status. A mutual information algorithm was used to correct for head movement . SPM8 watemplate , togethetemplate . The whotemplate , we condfunction . We havefunction .http://adni.loni.usc.edu/methods/mri-analysis/mri-acquisition/). Structural T1 images were segmented into grey matter, white matter and cerebrospinal fluid (CSF) using Statistical Parametric Mapping software  to allow correction of the 1H-MRS data for partial volume CSF contamination. 1H-MRS spectra  were acquired in the left hippocampus ; see Supplementary Fig. Scanning was conducted on a General Electric  Signa HDxt 3Tesla MRI scanner. Structural images were acquired using a whole-brain three-dimensional sagittal T1-weighted scan, with parameters based on the Alzheimer’s Disease Neuroimaging Initiative (ADNI) . An in-house script was used to identify the relative distribution of white matter, grey matter and cerebrospinal fluid in the voxel. Metabolite values were corrected for the CSF content of the voxel using the formula Mcorr = M × (WM + 1.28 GM + 1.55 CSF)/(WM + GM), where M is the uncorrected metabolite value, and WM, GM and CSF are the white matter, grey matter and cerebrospinal fluid fractions of the voxel, respectively [1H-MRS data are a sub-set of a larger sample recently reported [1H-MRS data have not been previously reported. The voxel tissue content and imaging quality control variables are summarised in Supplementary Table LC-model 6.3-I0  was usedectively . The 1H-reported , but thep < 0.05 (two tailed). Baseline clinico-demographic variables were compared using independent t tests or ANOVA for the continuous data, and the chi-square test for categorical variables.All statistical analyses were performed using R version 3.3.2 . SignifiTo test our first hypothesis that there was an inverse relationship between glutamate and dopamine in the high-risk sample as a whole, a linear regression analysis was performed with whole striatal dopamine synthesis capacity as the dependent variable, and glutamate concentrations as the predictor. We investigated whether this relationship was altered following the addition of potentially confounding variables  to the model, given possible effects of age , sex 5151, 52 anTo test our second hypothesis that dopamine synthesis capacity at baseline predicted worsening of symptoms, we performed linear regressions, analysing both the relationship between dopamine synthesis capacity and percentage change in symptoms. Given possible effects of age , sex 5151, 52 ant tests and ANCOVA.In addition, we conducted exploratory analyses to determine if dopamine synthesis capacity predicted persistent functional impairment. We also investigated whether baseline dopamine levels were different from controls using independent p = 0.82). There were no significant differences between patient and control groups in terms of age, gender, ethnicity or estimated premorbid IQ (see Table Fifty-one patients and 19 controls participated in the study. Forty-seven patients received both PET and MRI scans (one received only an MRI scan and three received only a PET scan), and all controls received both PET and MRI scans. Time between scans was similar for patients  and controls   or hippocampal glutamate concentrations . There were also no significant differences in dopamine synthesis capacity between groups for any of the striatal subdivisions , including in the adjusted model ), or in controls in either the bivariate  or adjusted  models. No significant relationships were seen for any of the striatal subdivisions either, in either patients or controls  . The relationship was significant for the associative subdivision, but not sensorimotor or limbic subdivisions  and adjusted models .Dopamine synthesis capacity at baseline was associated with subsequent worsening of psychotic (positive) symptoms .Ten individuals  developed a psychotic disorder during the follow-up period after the PET and MRS scans. There was no significant difference in baseline dopamine synthesis capacity between the transition (mean (SD): 0.0122 (0.001)) and non-transition (mean (SD): 0.0127 (0.001)) sub-groups  was much larger than in the previous study (n = 14), and had >80% power to detect the anticipated moderate or larger (r > 0.4) relationship between dopamine and glutamate indices. Thus, our study was well powered to detect the anticipated effect size, although it is possible that there is a smaller effect. It should be recognised that the MRS glutamate signal at 3 T is a composite of intra and extrasynaptic glutamate, and glutamine [Our finding that there was no significant relationship between hippocampal glutamate levels and striatal dopamine synthesis capacity contrasts with our previous finding in subjects at clinical high risk of psychosis, which found a negative relationship with r = 0.54 . It is plutamine . Thus, wlutamine , 18–20, lutamine , and thilutamine . UnfortuHowever, an alternative hypothesis is that it is disinhibition of hippocampal glutamatergic output neurons, as opposed to glutamatergic drive in the hippocampus, that leads to striatal dopaminergic dysregulation ,We did not find a difference in striatal dopaminergic function between clinical high-risk subjects overall and controls, in contrast to previous findings , 29, 66.A number of subjects were lost to follow-up, which could introduce bias into the outcomes. However, the clinical and demographic characteristics of these subjects were not significantly different from those in the other groups, indicating this unlikely to be a major bias. It should be recognised that some non-transition subjects might subsequently develop a psychotic disorder. However, as the peak period for transition to psychosis is within the first year of follow-up , it is lOur finding that dopamine synthesis capacity predicted the worsening of psychotic symptoms but was not linked to transition suggests that other factors are involved in the diagnosis of psychotic disorder. One interpretation could be that dopamine dysfunction underlies the development of psychotic symptoms, but whether these have a functional impact depends on additional factors, such as the coping skills, and psychological response of the individual, and their social support, consistent with psychosocio-biological models of psychosis –71. AnotOur finding that hippocampal glutamate is not linked to striatal dopamine dysfunction does not support the hypothesis that elevated hippocampal glutamatergic drive is driving striatal dopamine dysfunction, but is consistent with models that disinhibition of glutamatergic projections could drive striatal dopamine dysregulation. This predicts increased glutamate levels in targets of glutamatergic projections from the hippocampus, including the striatum. Elevated glutamate levels have been reported in the striatum in people at risk of psychosis, and linked to the transition to psychosis , 73. UnfStriatal dopamine synthesis capacity predicts worsening of psychotic-like symptoms, but is not strongly related to transition to psychosis or hippocampal glutamate levels, indicating a role for dopamine in the development of symptoms but that other factors contribute to the transition to a psychotic disorder.This study was funded by Medical Research Council-UK, and Wellcome Trust grants and the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. Dr Howes has received investigator-initiated research funding from and/or participated in advisory/speaker meetings organised by Angellini, Astra-Zeneca, Autifony, Biogen, BMS, Eli Lilly, Heptares, Jansenn, Lundbeck, Lyden-Delta, Otsuka, Servier, Sunovion, Rand and Roche. Neither Dr Howes nor his family have been employed by or have holdings/a financial stake in any pharmaceutical company. The other authors have no relevant conflicts of interest.Supplementary material