Antagonism of the brain P2X7 ion channel attenuates repeated social defeat induced microglia reactivity, monocyte recruitment and anxiety-like behavior in male mice.

Chronic stress is linked to increased anxiety. Repeated social defeat (RSD) in mice causes anxiety that is dependent on activated neurons, reactive microglia, and accumulation of monocytes in the brain. This response requires interactions between the immune system and central nervous system (CNS). Neuronal activation within threat appraisal regions is a key response to RSD, however, it is unclear how microglia become activated. One potential explanation is that microglia express a purinergic non-selective ligand gated adenosine-triphosphate (ATP) receptor 7 (P2X7). Activation of P2X7 promotes the release of chemokines and cytokines, and recruitment of monocytes to the brain. Thus, the purpose of this study was to determine if a novel P2X7 antagonist blocked neuronal and microglia interactions and the corresponding anxiety following RSD. Male mice were administered (i.p.) a P2X7 antagonist, JNJ-54471300, prior to each cycle of RSD. Fourteen hours after RSD, behavioral deficits including social avoidance and anxiety-like were determined. Moreover, several immune parameters were assessed. RSD caused neuronal activation in stress-responsive regions, monocyte production and release, splenomegaly, and social avoidance. These parameters were unaffected by P2X7 antagonism. RSD-associated proportional area of Iba-1+ microglia, monocyte accumulation in the brain, IL-1ß mRNA expression in enriched myeloid cells, plasma IL-6, and anxiety-like behavior were ameliorated by P2X7 antagonism. Gene expression analysis in the hippocampus and amygdala showed regional specific responses to RSD and some were reversed with P2X7 antagonism. Overall, blocking P2X7 activation attenuated RSD-induced microglia reactivity with corresponding reduction in neuroinflammation, monocyte accumulation, and anxiety-like behavior in male mice.

IL-1 Receptor-1 on Vglut2 + neurons in the hippocampus is critical for neuronal and behavioral sensitization after repeated social stress.

Myriad findings connect stress and inflammation to mood disorders. Social defeat in mice promotes the convergence of neuronal, central inflammatory (microglia), and peripheral immune (monocytes) pathways causing anxiety, social avoidance, and "stress-sensitization." Stress-sensitization results in augmented inflammation and the recurrence of anxiety after re-exposure to social stress. Different cell compartments, including neurons, may be uniquely sensitized by social defeat-induced interleukin-1 (IL-1) signaling. Therefore, the aim of this study was to determine if glutamatergic neuronal IL-1 receptor signaling was essential in promoting stress-sensitization after social defeat. Here, wild-type (IL-1R1+/+) mice and mice with IL-1 receptor-1 deleted selectively in glutamatergic neurons (Vglut2-IL-1R1-/-) were stress-sensitized by social defeat (6-cycles) and then exposed to acute defeat (1-cycle) at day 30. Acute defeat-induced neuronal activation (ΔFosB and phospo-CREB) in the hippocampus of stress-sensitized mice was dependent on neuronal IL-1R1. Moreover, acute defeat-induced social withdrawal and working memory impairment in stress-sensitized mice were also dependent on neuronal IL-1R1. To address region and time dependency, an AAV2-IL-1 receptor antagonist construct was administered into the hippocampus after sensitization, but prior to acute defeat at day 30. Although stress-sensitized mice had increased hippocampal pCREB and decreased working memory after stress re-exposure, these events were not influenced by AAV2-IL-1 receptor antagonist. Hippocampal ΔFosB induction and corresponding social withdrawal in stress-sensitized mice after stress re-exposure were prevented by the AAV2-IL-1 receptor antagonist. Collectively, IL-1 signaling in glutamatergic neurons of the hippocampus was essential in neuronal-sensitization after social defeat and the recall of social withdrawal.

Unique brain endothelial profiles activated by social stress promote cell adhesion, prostaglandin E2 signaling, hypothalamic-pituitary-adrenal axis modulation, and anxiety.

Chronic stress may precipitate psychiatric disorders including anxiety. We reported that Repeated Social Defeat (RSD) in mice increased accumulation of inflammatory monocytes within the brain vasculature, which corresponded with increased interleukin (IL)-1 Receptor 1-mediated activation of endothelia, and augmented anxiety-like behavior. One unknown, however, is the role of immune-activated endothelia in regulating the physiological and behavioral responses to social stress. Thus, we sought to determine the RNA profile of activated endothelia and delineate the pathways by which these endothelia communicate within the brain to influence key responses to social stress. First, endothelial-specific RiboTag mice were exposed to RSD and brain endothelial mRNA profiles from the whole brain and prefrontal cortex were determined using RNAseq. RSD increased expression of cell adhesion molecules (Icam1), inflammatory genes (Lrg1, Lcn2, Ackr1, Il1r1), and cyclooxygenase-2 (Ptgs2/COX-2). In studies with IL-1R1KO mice, there was clear dependence on IL-1R1 on endothelia-associated transcripts including Lrg1, Icam1, Lcn2. Moreover, prostaglandin (PG)E2 was increased in the brain after RSD and Ptgs2 was localized to endothelia, especially within the hypothalamus. Next, a selective COX-2 inhibitor, Celecoxib (CCB), was used with social stress. RSD increased PGE2 in the brain and this was abrogated by CCB. Moreover, CCB reduced RSD-induced Hypothalamic-Pituitary-Adrenal (HPA) axis activation with attenuation of hypothalamic paraventricular neuron activation, hypothalamic Crh expression, and corticosterone in circulation. Production, release, and accumulation of inflammatory monocytes after RSD was COX-2 independent. Nonetheless, CCB blocked anxiety-like behavior in response to RSD. Collectively, social stress stimulated specific endothelia RNA profiles associated with increased cell adhesion, IL-1 and prostaglandin signaling, HPA axis activation, and anxiety.

The neutral red assay can be used to evaluate cell viability during autophagy or in an acidic microenvironment in vitro.

Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pHe), inactivate some chemotherapies, which results in limited or no cytotoxicity. Standard MTT, ATPlite and protease assays that are used to determine the potency of newly developed drugs often give erroneous results when applied under hypoxic or acidic conditions. Therefore, development of a cytotoxicity assay that does not yield false positive or false negative results under circumstances of both hypoxia and acidic pHe is needed. We evaluated currently used cell viability assays as well as neutral red staining to assess viability of ovarian and pancreatic cancer cells grown in an acidic pHe microenvironment after treatment with carboplatin, gemcitabine or chloroquine. We validated cell viability using western blotting of pro-caspase-9 and cleaved-caspase-9, and LC3-I and - II. Standard cell viability assays indicated cell viability accurately at pHe 7.4, but was not correlated with induction of apoptosis or autophagy at acidic pHe. By contrast, our modified neutral red assay detected cell viability accurately over a range of pHe as demonstrated by its correlation with induction of apoptosis and autophagy. Neutral red staining is effective for evaluating the effect of chemotherapeutic agents on cell viability under acidic pHe or hypoxic conditions.

Actively Targeted Nanodelivery of Echinomycin Induces Autophagy-Mediated Death in Chemoresistant Pancreatic Cancer In Vivo.

Pancreatic cancer remains a recalcitrant neoplasm associated with chemoresistance and high fatality. Because it is frequently resistant to apoptosis, exploiting autophagic cell death could offer a new treatment approach. We repurpose echinomycin, an antibiotic encapsulated within a syndecan-1 actively targeted nanoparticle, for treatment of pancreatic cancer. Tumor-specific uptake, biodistribution, efficacy of nanodelivered echinomycin, and mechanism of cell death were assessed in aggressive, metastatic models of pancreatic cancer. In these autophagic-dependent pancreatic cancer models, echinomycin treatment resulted in autophagic cell death noted by high levels of LC3 among other autophagy markers, but without hallmarks of apoptosis, e.g., caspase activation and chromatin fragmentation, or necrosis, e.g., plasma membrane degradation and chromatin condensation/degrading. In vivo, biodistribution of syndecan-1-targeted nanoparticles indicated preferential S2VP10 or S2CP9 tumor uptake compared to the liver and kidney (S2VP10 p = 0.0016, p = 0.00004 and S2CP9 p = 0.0009, p = 0.0001). Actively targeted nanodelivered echinomycin resulted in significant survival increases compared to Gemzar (S2VP10 p = 0.0003, S2CP9 p = 0.0017) or echinomycin only (S2VP10 p = 0.0096, S2CP9 p = 0.0073). We demonstrate that actively targeted nanodelivery of echinomycin results in autophagic cell death in pancreatic and potentially other high-autophagy, apoptosis-resistant tumors. Collectively, these findings support syndecan-1-targeted delivery of echinomycin and dysregulation of autophagy to induce cell death in pancreatic cancer.

Repeated social defeat in female mice induces anxiety-like behavior associated with enhanced myelopoiesis and increased monocyte accumulation in the brain.

Anxiety and mood disorders affect both men and women. The majority of experimental models of stress, however, are completed using only male animals. For repeated social defeat (RSD), a rodent model, this is due to the inherent difficulty in eliciting male aggression toward female mice. To address this limitation, a recent study showed that a DREADD-based activation of the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) was effective in inducing aggressive behavior in male mice towards females in a social defeat paradigm. Therefore, the goal of this study was to determine if this modified version of RSD in females elicited behavioral, physiological, and immune responses similar to those reported in males. Here, we show that female mice subjected to RSD with the male DREADD aggressor developed anxiety-like behavior and social avoidance. These behavioral alterations coincided with enhanced neuronal and microglial activation in threat-appraisal regions of the brain. Moreover, stressed female mice had an enhanced peripheral immune response characterized by increased myelopoiesis, release of myeloid cells into circulation, and monocyte accumulation in the spleen and brain. These results are consistent with previously reported findings that male mice exposed to RSD exhibited increased fear and threat appraisal responses, enhanced myelopoiesis, myeloid cell release and trafficking, and anxiety-like behavior. These findings validate that RSD is a relevant model to study stress responses in female mice.

The Influence of Microglial Elimination and Repopulation on Stress Sensitization Induced by Repeated Social Defeat.

BACKGROUND: Stress is associated with an increased prevalence of anxiety and depression. Repeated social defeat (RSD) stress in mice increases the release of monocytes from the bone marrow that are recruited to the brain by microglia. These monocytes enhance inflammatory signaling and augment anxiety. Moreover, RSD promotes stress sensitization, in which exposure to acute stress 24 days after cessation of RSD causes anxiety recurrence. The purpose of this study was to determine whether microglia were critical to stress sensitization and exhibited increased reactivity to subsequent acute stress or immune challenge. METHODS: Mice were exposed to RSD, microglia were eliminated by colony-stimulating factor 1 receptor antagonism (PLX5622) and allowed to repopulate, and responses to acute stress or immune challenge (lipopolysaccharide) were determined 24 days after RSD sensitization. RESULTS: Microglia maintained a unique messenger RNA signature 24 days after RSD. Moreover, elimination of RSD-sensitized microglia prevented monocyte accumulation in the brain and blocked anxiety recurrence following acute stress (24 days). When microglia were eliminated prior to RSD and repopulated and mice were subjected to acute stress, there was monocyte accumulation in the brain and anxiety in RSD-sensitized mice. These responses were unaffected by microglial elimination/repopulation. This may be related to neuronal sensitization that persisted 24 days after RSD. Following immune challenge, there was robust microglial reactivity in RSD-sensitized mice associated with prolonged sickness behavior. Here, microglial elimination/repopulation prevented the amplified immune reactivity ex vivo and in vivo in RSD-sensitized mice. CONCLUSIONS: Microglia and neurons remain sensitized weeks after RSD, and only the immune reactivity component of RSD-sensitized microglia was prevented by elimination/repopulation.

Social Stress Mobilizes Hematopoietic Stem Cells to Establish Persistent Splenic Myelopoiesis.

Psychosocial stress accelerates myelopoietic production of monocytes and neutrophils that contributes to a variety of health complications ranging from atherosclerosis to anxiety. Here, we show that social stress in mice mobilizes hematopoietic stem progenitor cells (HSPCs) from the bone marrow that enter circulation, engraft into the spleen, and establish a persistent extramedullary hematopoietic depot. These splenic progenitors actively proliferate and differentiate into multiple cell types, including monocytes, neutrophils, and erythrocytes. Splenic erythropoiesis partially abrogates stress-induced anemia. Repeated injection with isoprenaline induces progenitor mobilization to the spleen, identifying a key role for ß-adrenergic signaling. Moreover, protracted splenic production of CD11b+ cells persists for at least 24 days after the cessation of social stress. Thus, chronic stress establishes a persistent extramedullary hematopoietic depot that can modify a wide range of chronic disease processes and alter homeostasis of the bi-directional regulatory axis between the nervous and immune systems.

Partner notification in cooperation with community-based organizations among HIV-positive men who have sex with men in two Chinese cities.

The epidemic of HIV/AIDS among Chinese men who have sex with men (MSM) is rapidly escalating. We implemented partner notification among HIV-infected MSM, cooperating with MSM-serving community-based organizations (CBOs) in two Chinese cities from June 2014 to May 2015. CBOs participated in identifying new HIV-positive MSM utilizing rapid HIV tests and partner notification among index cases. 253 index cases were recruited and 275 sexual partners were notified and tested with 10.5% screened positive. Compared with previously identified index cases, the proportion of contactable sexual partners of newly identified index cases was higher, but the testing rate was lower (p < 0.001). Overall, 83.7% of sexual partners were casual with a contactable rate of 24.9% and a HIV testing rate of 71.1%. Having no contact information for sexual partners and fear of disclosure of HIV status are the main reasons for declining partner notification. It is feasible and effective to perform partner notification in cooperation with CBOs serving Chinese MSM.

Property Values as a Measure of Neighborhoods: An Application of Hedonic Price Theory.

BACKGROUND: Researchers measuring relationships between neighborhoods and health have begun using property appraisal data as a source of information about neighborhoods. Economists have developed a rich tool kit to understand how neighborhood characteristics are quantified in appraisal values. This tool kit principally relies on hedonic (implicit) price models and has much to offer regarding the interpretation and operationalization of property appraisal data-derived neighborhood measures, which goes beyond the use of appraisal data as a measure of neighborhood socioeconomic status. METHODS: We develop a theoretically informed hedonic-based neighborhood measure using residuals of a hedonic price regression applied to appraisal data in a single metropolitan area. We describe its characteristics, reliability in different types of neighborhoods, and correlation with other neighborhood measures (i.e., raw neighborhood appraisal values, census block group poverty, and observed property characteristics). We examine the association between all neighborhood measures and body mass index. RESULTS: The hedonic-based neighborhood measure was correlated in the expected direction with block group poverty rate and observed property characteristics. The neighborhood measure and average raw neighborhood appraisal value, but not census block group poverty, were associated with individual body mass index. CONCLUSION: We draw theoretically consistent methodology from the economics literature on hedonic price models to demonstrate how to leverage the implicit valuation of neighborhoods contained in publicly available appraisal data. Consistent measurement and application of the hedonic-based neighborhood measures in epidemiology will improve understanding of the relationships between neighborhoods and health. Researchers should proceed with a careful use of appraisal values utilizing theoretically informed methods such as this one.

Tumor specific liposomes improve detection of pancreatic adenocarcinoma in vivo using optoacoustic tomography.

BACKGROUND: Pancreatic cancer often goes undiagnosed until late stage disease due in part to suboptimal early detection. Our goal was to develop a Syndecan-1 tagged liposome containing fluorescent dye as an improved contrast agent for detection of pancreatic adenocarcinoma in vivo using multispectral optoacoustic tomography. RESULTS: The diagnostic capabilities and specificity to pancreatic adenocarcinoma of Syndecan-1 targeted liposomes were evaluated both in vitro and in vivo. Immunocytochemistry showed that liposomes preferentially bound to and released their contents into cells expressing high levels of insulin-like growth factor 1 receptor. We determined that the contents of the liposome were released into the cell as noted by the change in propidium iodide fluorescence from green to red based upon nucleic acid binding. In an orthotopic mouse model, the liposomes preferentially targeted the pancreatic tumor with little off-target binding in the liver and spleen. Peak accumulation of the liposomes in the tumor occurred at 8 h post-injection. Multispectral optoacoustic tomographic imaging was able to provide high-resolution 3D images of the tumor and liposome location. Ex vivo analysis showed that non-targeted liposomes accumulated in the liver, suggesting that specificity of the liposomes for pancreatic adenocarcinoma was due to the presence of the Syndecan-1 ligand. CONCLUSIONS: This study demonstrated that Syndecan-1 liposomes were able to release cargo into IGF1-R expressing tumor cells. The Syndecan-1 liposomes demonstrated tumor specificity in orthotopic pancreatic cancer as observed using multispectral optoacoustic tomography with reduced kidney and liver uptake. By targeting the liposome with Syndecan-1, this nanovehicle has potential as a targeted theranostic nanoparticle for both drug and contrast agent delivery to pancreatic tumors.

[Allocation analysis of central government AIDS special funding in priority sites of HIV/AIDS prevention and control].

OBJECTIVE: To analyze the allocation and trend of central government AIDS special funding in 4 priority sites of HIV/AIDS prevention and control across calendar years. METHODS: Information about the allocation of central government special AIDS funding and cumulative HIV/AIDS survivor numbers of Z city, D prefecture, L prefecture and D prefecture were collected until 2013. Data were collected from 2004-2013 for Z city and D prefecture, and data from 2009-2013 were collected for L and Y prefecture. Funding allocation among all working areas and their trend over time were analyzed. RESULTS: From 2004-2013, the total amount of special funding in Z prefecture was 110.15 million RMB. The largest three areas of allocation were key population response (29%, 3 190/11 015), surveillance and testing (23%, 2 535/11 015) and human resource (13%, 1 498/11 015). The least area of allocation was follow-up and prevention of discordant couple transmission (2%, 251/11 015). The total amount of special funding in D prefecture from 2004-2013 was 109.77 million RMB. The largest three areas of allocation were treatment and care (25%, 2 691/10 977), follow-up and prevention of discordant couple transmission (17%, 1 843/10 977) and surveillance and testing (15%, 1 656/10 977). The least area was blood safety (1%, 135/10 977). From 2009 to 2013, the total amount of special funding in L prefecture was 55 million RMB. The largest three areas of allocation were surveillance and testing (60%, 3 298/5 500), high risk population intervention (14%, 768/5 500) and follow up and prevention of discordant couple transmission (12%, 675/5 500). The least area was blood safety (0.1%, 8/5 500). From 2009-2013, the total amount of special funding in Y prefecture was 55 million RMB and the largest three areas of allocation were project management and others (28%, 1 527/5 500), key population response (19%, 1 046/5 500) and high risk population intervention (17%, 922/5 500). The least area of special funding was blood safety (2%, 106/5 500). Among three HIV/AIDS epidemic related key areas (surveillance and testing, follow-up and prevention of discordant couple transmission, treatment and care), 2004-2013, allocated funds were between 2.96-3.36, 0-0.37, 0.37-1.97 million RMB in Z city; 0.64-2.35, 0.00-3.00, 2.00-4.70 million RMB in D prefecture; 2009-2013, allocated funds were between 2.67-8.85, 0.41-2.39, 0.35-1.84 million RMB in L prefecture, 1.18-2.84, 0.70-1.05, 0.46-0.89 million RMB in Y prefecture. CONCLUSION: The allocation patterns of central government AIDS special funding among key working areas were different across 4 different sites; in each individual site, the trend of special funding allocation was stable among HIV epidemic related key areas over calendar years.

Who uses methadone services in China? Monitoring the world's largest methadone programme.

AIMS: To describe the data collected by the Chinese methadone maintenance treatment (MMT) system and the characteristics of clients entering the programme. DESIGN: Descriptive study using routinely collected data from the MMT data management system for the period March 2004 and March 2010. SETTING: All MMT clinics in China. PARTICIPANTS: Clients who enrolled for services between March 2004 and March 2010. MEASUREMENTS: Routinely collected data included: demographic information; drug use, sexual and criminal behaviours; status of infection with human immunodeficiency virus (HIV), hepatitis C virus (HCV) and syphilis; random urine-opiate test results; and the daily methadone dose received. Differences among clients by year were examined. FINDINGS: During the period examined, there were 251,974 clients attending 684 clinics in 27 provinces. Overall, the mean age was 34.4 years, 83.8% were male, 70.2% were unemployed, 75% had ever injected drugs, 17% had shared needles and 7.4% were HIV-positive. The profile of clients changed over time, with fewer HIV-positive individuals, fewer injecting drug users, fewer needle-sharers, fewer females and fewer unemployed. Half the clients dropped out within 6 months. The average final dose received was 49.4 mg. The estimated probability of interrupting treatment before 6 months was 52.5%. CONCLUSIONS: The profile of clients enrolling in methadone maintenance treatment in China is continually changing and appears to be associated with reduced risk of HIV. High dropout in the programme may limit its effectiveness. The availability of a centralized, real-time data system was extremely useful for monitoring the progress of the Chinese methadone maintenance treatment programme.

Factors associated with depression and anxiety among patients attending community-based methadone maintenance treatment in China.

AIM: To estimate the prevalence of, and identify factors associated with, depression and anxiety among community-based methadone maintenance treatment (MMT) clients in China. DESIGN: A cross-sectional survey. SETTING: Nine MMT clinics, three each from three Chinese provinces (Yunnan, Anhui and Jiangsu) between October 2008 and February 2009. PARTICIPANTS: A total of 1301 MMT clients. MEASUREMENTS: A questionnaire, including the Zung Self-Rating Depression Scale (SDS) and Zung Self-Rating Anxiety Scale (SAS), and on-site urine drug testing. FINDINGS: The prevalence of depression (SDS score≥53) and anxiety (SAS score≥50) in our sample was 38.3% [95% confidence interval (CI)=35.7, 40.9] and 18.4% (95% CI=16.3, 20.5), respectively, with 14.2% (95% CI=12.3, 16.1) displaying symptoms of both. Sample prevalence rates for depression [mean=49.69, standard deviation (SD)=10.34] and anxiety (mean=40.98, SD=10.66) were higher than the national average for each (t(0.05/2, 1300)=19.2, P<0.001 and t(0.05/2, 1300)=8.0, P<0.001, respectively). Employing multi-level modelling techniques, gender (P=0.03) and employment status (P<0.001) were found to be associated significantly with depression in a single-level model; however, in a multi-level mixed model, only employment status (P<0.001) was associated with depression. Gender (P=0.03), education level (P=0.02), marital status (P=0.04), employment status (P<0.001), positive urine drug test results (P=0.02) and daily methadone dose (P<0.001) were found to be associated significantly with anxiety in a single-level model, while only employment status (P<0.01) and positive results for the urine drug test (P=0.04) were associated with anxiety in a multi-level mixed model. CONCLUSIONS: A considerable proportion of methadone maintenance treatment clients in China have experienced depression and anxiety during treatment. There is a need to provide tailored mental health interventions for this high-risk population.

Retention and its predictors among methadone maintenance treatment clients in China: a six-year cohort study.

BACKGROUND: To investigate factors associated with retention among the first cohort of drug users attending methadone maintenance treatment (MMT) clinics in China. METHODS: A six-year cohort of 1511 drug users was enrolled in the first 8 MMT clinics in China in 2004 and followed between March, 2004 and December, 2010. Six-year retention rates were calculated and compared by methadone dosage, clinic location, and length of follow-up. Factors associated with retention were evaluated using Cox proportional hazard regression models. RESULTS: The overall retention rate over 6-year was 35.7%. Highest drop-out occurred within the first 12 months of treatment. The retention rates for the 6-year by low (≤ 30 mg/day), medium (31-60 mg/day), and high (>60 mg/day) methadone dosage groups were 20.8%, 34.8% and 53.2%, respectively (p < 0.001). The highest 6-year retention rate among eight clinics was 43.8%, while the lowest one was 17.7% (p < 0.001). The results from Cox proportional hazard models indicate that clients having >30 mg daily methadone dosage (p < 0.001), having relatives receiving MMT (p = 0.027), and having >10% urine morphine positive result (p < 0.001) were more likely to be retained in MMT over the six-year period. It has also found that drug injection (p = 0.005) and needle sharing (p < 0.0001) were significantly associated with better retention. CONCLUSIONS: Health workers should adjust individuals' methadone dosages as a method to prevent early treatment termination. Further studies are needed to explore the impact of severity of opiate addiction on long-term retention of MMT clients. Careful assessment of drug users upon MMT enrollment may be helpful for providing additional care to clients.

Targeted noninvasive imaging of EGFR-expressing orthotopic pancreatic cancer using multispectral optoacoustic tomography.

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe before MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a nonspecific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at 6 hours postinjection, demonstrating specific intratumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of the EGF receptor.

Chloroquine-mediated cell death in metastatic pancreatic adenocarcinoma through inhibition of autophagy.

CONTEXT: Cells in the interior of pancreatic tumors are believed to undergo continual autophagy to maintain homeostasis during unregulated growth in hypoxia caused by impaired vascularity. We hypothesize that treating metastatic cells with chloroquine, an inhibitor of autophagy, in hypoxia will decrease cell viability and induce cell death. DESIGN: MiaPaCa2 (non-metastatic) and S2VP10 (metastatic) cell lines were treated with 25 and 50 µM chloroquine for 24 and 48 hours in normoxia and hypoxia (5-10% O2). Viability was measured using ATPlite™. After treatment, the cell stress was analyzed, and protein was lysed and quantified using the Bradford assay. Autophagy-associated protein levels were determined by Western blot. RESULTS: S2VP10 cells treated for 48 hours with 50 µM chloroquine in hypoxia had 24% viability compared to normoxia control, with loss of 10% viability caused by low O2 alone. MiaPaCa2 cells under these conditions had 60% viability compared to normoxia control, with loss of 25% viability caused by low O2 alone. Analysis of cell stress pathways and dosimetry of Western blot data suggest that chloroquine inhibits the autophagy pathway in the metastatic S2VP10 cells. CONCLUSION: Autophagy blockage with chloroquine or similar-acting drugs may serve as a viable therapy for highly metastatic pancreatic cancers.

Mortality among methadone maintenance clients in China: a six-year cohort study.

OBJECTIVE: To assess the overall mortality of methadone maintenance treatment (MMT) clients in China and its associated factors. METHODS: A total of 1,511 MMT clients, all of whom enrolled in China's first eight MMT clinics between March and December 2004, were included in this cohort study and followed for approximately six years, until June 2010. Mortality and its predictors were examined using Cox proportional hazards regression models. RESULTS: A total of 154 deaths were observed within 5,391 person-years (PY) of follow-up for an all-cause mortality rate of 28.6 per 1,000 PY. The leading causes of death were drug overdose (33.8%), HIV/AIDS-unrelated disease (21.4%), and HIV/AIDS (16.9%). The all-cause mortality rate of clients engaged in MMT for one year or less was roughly three times that of clients who stayed in MMT for four years or more (14.0 vs. 4.6, p<0.0001), HIV-positive subjects was nearly four times mortality rate than that of HIV-negative individuals (28.1 vs.6.8, p<0.0001). ART-naive HIV-positive subjects had approximately two times higher mortality rate than those receiving ART (31.2 vs. 17.3, <0.0001). After adjusting for confounding variables, we found that being male (HR = 1.63, CI: 1.03-2.57, p = 0.0355) and being HIV-positive (HR = 5.16, CI: 3.70-7.10, p<0.0001) were both associated with higher risk of death whereas increased durations of methadone treatment were associated with a lower risk of death (HR = 0.26, CI: 0.18-0.38, p<0.0001 for two to three years, HR = 0.08, CI: 0.05-0.14, p<0.0001 for four or more years). CONCLUSION: Overall mortality was high among MMT clients in China. Specific interventions aimed at decreasing mortality among MMT clients are needed. Our study supports the need for keeping client at MMT longer and for expanding ART coverage and suggests the potential benefits of integrated MMT and ART services for drug users in China.