Technology-Based Smoking Cessation for Youth Exiting Foster Care: A Pilot Randomized Trial.

Despite high rates of cigarette use, little attention has been paid to screening and cessation services for youth in foster care. Study aims were to test the feasibility, acceptability, and preliminary efficacy of a technology-based smoking cessation intervention. Study enrollment, satisfaction, and engagement were high in the intervention arm, where readiness to change also significantly increased over time. Intervention and control participants significantly reduced cigarette use at 6-month follow up, though groups did not differ. Technology-based interventions appear to be attractive and offer a potentially scalable link to health care that this vulnerable population may not otherwise seek.

Survey of Anti-Pathogen Antibody Levels in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Infectious pathogens are implicated in the etiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) because of the occurrence of outbreaks of the disease. While a number of different infectious agents have been associated with the onset of ME/CFS, the identity of a specific organism has been difficult to determine in individual cases. The aim of our study is to survey ME/CFS subjects for evidence of an infectious trigger and/or evidence of immune dysregulation via serological testing of plasma samples for antibodies to 122 different pathogen antigens. Immune profiles were compared to age-, sex-, and BMI-matched controls to provide a basis for comparison. Antibody levels to individual antigens surveyed in this study do not implicate any one of the pathogens in ME/CFS, nor do they rule out common pathogens that frequently infect the US population. However, our results revealed sex-based differences in steady-state humoral immunity, both within the ME/CFS cohort and when compared to trends seen in the healthy control cohort.

Inability of ovarian cancers to upregulate their MHC-class I surface expression marks their aggressiveness and increased susceptibility to NK cell-mediated cytotoxicity.

We extended our previous observations with other tumor models to study seven ovarian tumor cell lines-OVCAR3, OVCAR4, OVCAR8, SKOV3, Kuramochi, OAW28, and CaOV3. We found that NK cells targeted and killed poorly differentiated OVCAR8 and CAOV3; these two tumor lines express lower MHC-class I and higher CD44 surface receptors. OVCAR3 and OVCAR4 were more resistant to NK cell-mediated cytotoxicity, and SKOV3, Kuramochi and OAW28 had intermediate sensitivity to NK cell-mediated cytotoxicity, likely representing well-differentiated and moderately differentiated ovarian tumor cell lines, respectively. Similar trends were observed for secretion of IFN-γ by the NK cells when co-cultured with different ovarian tumor cell lines. Treatment with both IFN-γ and TNF-α upregulated MHC-class I in all ovarian tumor cell lines and resulted in tumor resistance to NK cell-mediated cytotoxicity and decreased secretion of IFN-γ in co-cultures of NK cells with tumors cells with the exception of OVCAR8 and CAOV3 which did not upregulate MHC-class I and remained sensitive to NK cell-mediated cytotoxicity and increased secretion of IFN-γ when co-cultured with NK cells. Similarly, treatment with NK cell supernatants induced resistance to NK cell-mediated cytotoxicity in OVCAR4 but not in OVCAR8, and the resistance to killing was correlated with the increased surface expression of MHC-class I in OVCAR4 but not in OVCAR8. In addition, OVCAR4 was found to be carboplatin sensitive before and after treatment with IFN-γ and NK cell supernatants, whereas OVCAR8 remained carboplatin resistant with and without treatment with IFN-γ and NK cell supernatants. Overall, sensitivity to NK cell-mediated killing correlated with the levels of tumor differentiation and aggressiveness, and more importantly, poorly differentiated ovarian tumors were unable to upregulate MHC-class I under the activating conditions for MHC-class I, a feature that was not seen in other tumor models and may likely be specific to ovarian tumors. Such tumors may also pose a significant challenge in elimination by the T cells; however, NK cells are capable of targeting such tumors and can be exploited to eliminate these tumors in immunotherapeutic strategies.

Efficacy of birinapant in combination with carboplatin in targeting platinum­resistant epithelial ovarian cancers.

Patients diagnosed with epithelial ovarian cancers (EOCs) often suffer from disease relapse associated with the emergence of resistance to standard platinum­based chemotherapy. Treatment of patients with chemo­resistant disease remains a clinical challenge. One mechanism of chemoresistance includes overexpression of pro­survival proteins called inhibitors of apoptosis (IAP) which enable cancer cells to evade apoptosis. Due to their anti­apoptotic activity, association with poor prognosis, and correlation with therapy resistance in multiple malignancies, IAP proteins have become an attractive target for development of anticancer therapeutics. Second mitochondrial activator of caspase (SMAC) mimetics are the most widely used IAP antagonists currently being tested in clinical trials as a monotherapy and in combination with different chemotherapeutic drugs to target different types of cancer. In the present study, the antitumor efficacy of combination therapy with birinapant, a bivalent SMAC mimetic compound, and carboplatin to target platinum­resistant EOC cells was investigated. A 3D organoid bioassay was utilized to test the efficacy of the combination therapy in a panel of 7 EOC cell lines and 10 platinum­resistant primary patient tumor samples. Findings from the in vitro studies demonstrated that the birinapant and carboplatin combination was effective in targeting a subset of ovarian cancer cell lines and platinum­resistant primary patient tumor samples. This combination therapy was also effective in vitro and in vivo in targeting a platinum­resistant patient­derived xenograft (PDX) model established from one of the patient tumors tested. Overall, our study demonstrated that birinapant and carboplatin combination could target a subset of platinum­resistant ovarian cancers and also highlights the potential of the 3D organoid bioassay as a preclinical tool to assess the response to chemotherapy or targeted therapies in ovarian cancer.

cfTrack: A Method of Exome-Wide Mutation Analysis of Cell-free DNA to Simultaneously Monitor the Full Spectrum of Cancer Treatment Outcomes Including MRD, Recurrence, and Evolution.

PURPOSE: Cell-free DNA (cfDNA) offers a noninvasive approach to monitor cancer. Here we develop a method using whole-exome sequencing (WES) of cfDNA for simultaneously monitoring the full spectrum of cancer treatment outcomes, including minimal residual disease (MRD), recurrence, evolution, and second primary cancers. EXPERIMENTAL DESIGN: Three simulation datasets were generated from 26 patients with cancer to benchmark the detection performance of MRD/recurrence and second primary cancers. For further validation, cfDNA samples (n = 76) from patients with cancer (n = 35) with six different cancer types were used for performance validation during various treatments. RESULTS: We present a cfDNA-based cancer monitoring method, named cfTrack. Taking advantage of the broad genome coverage of WES data, cfTrack can sensitively detect MRD and cancer recurrence by integrating signals across known clonal tumor mutations of a patient. In addition, cfTrack detects tumor evolution and second primary cancers by de novo identifying emerging tumor mutations. A series of machine learning and statistical denoising techniques are applied to enhance the detection power. On the simulation data, cfTrack achieved an average AUC of 99% on the validation dataset and 100% on the independent dataset in detecting recurrence in samples with tumor fractions ≥0.05%. In addition, cfTrack yielded an average AUC of 88% in detecting second primary cancers in samples with tumor fractions ≥0.2%. On real data, cfTrack accurately monitors tumor evolution during treatment, which cannot be accomplished by previous methods. CONCLUSIONS: Our results demonstrated that cfTrack can sensitively and specifically monitor the full spectrum of cancer treatment outcomes using exome-wide mutation analysis of cfDNA.

Combining ReACp53 with Carboplatin to Target High-Grade Serous Ovarian Cancers.

Ovarian malignancies are a leading cause of cancer-related death for US women. High-grade serous ovarian carcinomas (HGSOCs), the most common ovarian cancer subtype, are aggressive tumors with poor outcomes. Mutations in TP53 are common in HGSOCs, with a subset resulting in p53 aggregation and misregulation. ReACp53 is a peptide designed to inhibit mutant p53 aggregation and has been shown efficacious in targeting cancer cells in vitro and in vivo. As p53 regulates apoptosis, combining ReACp53 with carboplatin represents a logical therapeutic strategy. The efficacy of this combinatorial approach was tested in eight ovarian cancer cell lines and 10 patient HGSOC samples using an in vitro organoid drug assay, with the SynergyFinder tool utilized for calculating drug interactions. Results demonstrate that the addition of ReACp53 to carboplatin enhanced tumor cell targeting in the majority of samples tested, with synergistic effects measured in 2 samples, additivity measured in 14 samples, and antagonism measured in 1 sample. This combination was found to be synergistic in OVCAR3 ovarian cancer cells in vitro through enhanced apoptosis, and survival of mice bearing OVCAR3 intraperitoneal xenografts was extended when treated with the addition of ReACp53 to carboplatin versus carboplatin alone. Results suggest that carboplatin and ReACp53 may be a potential strategy in targeting a subset of HGSOCs.

The Enterovirus Theory of Disease Etiology in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Critical Review.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex, multi-system disease whose etiological basis has not been established. Enteroviruses (EVs) as a cause of ME/CFS have sometimes been proposed, as they are known agents of acute respiratory and gastrointestinal infections that may persist in secondary infection sites, including the central nervous system, muscle, and heart. To date, the body of research that has investigated enterovirus infections in relation to ME/CFS supports an increased prevalence of chronic or persistent enteroviral infections in ME/CFS patient cohorts than in healthy individuals. Nevertheless, inconsistent results have fueled a decline in related studies over the past two decades. This review covers the aspects of ME/CFS pathophysiology that are consistent with a chronic enterovirus infection and critically reviews methodologies and approaches used in past EV-related ME/CFS studies. We describe the prior sample types that were interrogated, the methods used and the limitations to the approaches that were chosen. We conclude that there is considerable evidence that prior outbreaks of ME/CFS were caused by one or more enterovirus groups. Furthermore, we find that the methods used in prior studies were inadequate to rule out the presence of chronic enteroviral infections in individuals with ME/CFS. Given the possibility that such infections could be contributing to morbidity and preventing recovery, further studies of appropriate biological samples with the latest molecular methods are urgently needed.

Targeting monoamine oxidase A-regulated tumor-associated macrophage polarization for cancer immunotherapy.

Targeting tumor-associated macrophages (TAMs) is a promising strategy to modify the immunosuppressive tumor microenvironment and improve cancer immunotherapy. Monoamine oxidase A (MAO-A) is an enzyme best known for its function in the brain; small molecule MAO inhibitors (MAOIs) are clinically used for treating neurological disorders. Here we observe MAO-A induction in mouse and human TAMs. MAO-A-deficient mice exhibit decreased TAM immunosuppressive functions corresponding with enhanced antitumor immunity. MAOI treatment induces TAM reprogramming and suppresses tumor growth in preclinical mouse syngeneic and human xenograft tumor models. Combining MAOI and anti-PD-1 treatments results in synergistic tumor suppression. Clinical data correlation studies associate high intratumoral MAOA expression with poor patient survival in a broad range of cancers. We further demonstrate that MAO-A promotes TAM immunosuppressive polarization via upregulating oxidative stress. Together, these data identify MAO-A as a critical regulator of TAMs and support repurposing MAOIs for TAM reprogramming to improve cancer immunotherapy.

Matched sequential tumor molecular profiling in solid malignancies may impact clinical practice.

OBJECTIVES: To determine if performing repeat tumor molecular profiling in solid malignancies over time can identify new findings that impact clinical care. METHODS: All patients with a solid malignancy and more than 1 tumor molecular analysis were identified at a single institution. Each test report was examined to identify the genomic alterations. Chart review was performed to determine subsequent therapies following each test result and the impact of tumor profiling on clinical practice. RESULTS: At a single institution, 110 patients were identified with having more than 1 tumor molecular analysis, with 98 subjects having test results available for review. Eighty-seven patients had differences in reported results at the time of subsequent analysis. These differences may reflect changes in tumor biology, be attributed to intra-patient or intra-tumor heterogeneity or be due to technical updates of the next generation sequencing platforms. Among the 98 subjects with solid tumors, the median time between tests was 10 months (range 0.5-66 months), with the majority of tests performed at the time of disease progression or recurrence. In this population, a total of 30 patients received targeted therapies that were associated with actionable findings on any tumor molecular analysis. Of these, 6 patients had new genomic findings identified on sequential testing that affected treatment. CONCLUSIONS: The future of cancer care must include precision medicine approaches. Evolution of next generation sequencing has contributed to this effort. Results of this single institution study summarize the reported findings on tumor molecular testing and suggest that subsequent testing may impact clinical care in a subset of patients. While only 6% of patients in this study saw a change in treatment based on new findings on sequential testing reports, this approach may be more clinically relevant in the future with the development of novel targeted therapies. This may be especially significant in a patient population that has progressed on standard therapies and where treatment options are limited.

Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls. METHODS: We included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed. RESULTS: ME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association. CONCLUSIONS: Elevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CFS.

Expression of Stromal Progesterone Receptor and Differential Methylation Patterns in the Endometrium May Correlate with Response to Progesterone Therapy in Endometrial Complex Atypical Hyperplasia.

Progesterone therapy is a viable treatment for complex atypical hyperplasia (CAH) and endometrial adenocarcinoma, though reliable molecular determinants of response are not available. To explore if analysis of pre-therapy endometrial biopsies could yield biomarkers of response to progesterone, patients with CAH or adenocarcinoma undergoing treatment with progestins were included in this cross-sectional study. Immunohistochemistry for progesterone receptor (PR) was performed. Manual PR expression scores (PRES) were first calculated for biopsies by counting PR-positive nuclei in 12 sensitive vs 9 resistant samples. Significant differences in manual PRES were detected in the stroma (p < 0.01) and total endometrium (p < 0.01) for sensitive vs resistant patients. Manual PRES in the stroma had the highest accuracy in segregating sensitive vs resistant patients (96%). Differences in epithelial PRES were not significant. To validate these findings, a correlation between manual PRES and visual PRES was performed in the 21 patients. An additional 11 patients were analyzed to test if visual PRES would be predictive of response to progesterone. Visual PRES in epithelia and stroma in the 32 specimens was calculated. Significant differences in visual PRES were detected in the stroma for sensitive vs resistant samples (p < 0.01), while differences in epithelial and total endometrium were not significant. Whole genome bisulfite sequencing was performed on DNA isolated using pre-therapy biopsies from 6 sensitive and 6 resistant patients in this cohort. Differentially methylated regions were identified in the stroma and epithelium when evaluating sensitive vs resistant samples. Pathways involved in cell adhesion demonstrated the greatest difference in methylation in these samples.

Exploring the Potential of Drug Response Assays for Precision Medicine in Ovarian Cancer.

One of the major challenges in the treatment of cancer are differential responses of patients to existing standard of care anti-cancer drugs. These differential responses may, in part, be due to a diverse range of genomic, epigenomic, proteomic, and metabolic alterations among individuals suffering from the same type of cancer. Precision medicine is an emerging approach in cancer therapeutics that takes into account specific molecular alterations, environmental factors as well as lifestyle of individual patients. This approach allows clinicians and researchers to select or predict treatments that would most likely benefit the patient based on their individual tumor characteristics. One class of precision medicine tools are predictive, in vitro drug-response assays designed to test the sensitivity of patient tumor cells to existing or novel therapies. These assays have the potential to rapidly identify the most effective treatments for cancer patients and thus hold great promise in the field of precision medicine. In this review, we have highlighted several drug-response assays developed in ovarian cancer and discussed the current challenges and future prospects of these assays in the clinical management of this disease.

Combustible Cigarette Smoking and Alternative Tobacco Use in a Sample of Youth Transitioning from Foster Care.

Among the struggles faced by youth currently in or recently exiting foster care, tobacco use remains a low priority for practitioners and researchers, alike. Indeed, despite the dramatically altered landscape of tobacco products on the market, there have been no studies evaluating the use of alternative tobacco products among this vulnerable population. The current study aimed to determine the prevalence of lifetime and current combustible and non-combustible tobacco use among youth exiting foster care, and report on the prevalence of nicotine dependence, motivation to quit, and preferred methods of tobacco cessation. Youth aged 18-24 (M = 20.13, SD = 1.16) who were transitioning from foster care (N = 154) completed a survey of tobacco product use adapted from the Population Assessment of Tobacco and Health Baseline Survey. Most participants (76%) reported lifetime use of combustible cigarettes, while almost half (42%) were current combustible cigarette smokers. Current use of electronic cigarettes was comparable to general population rates. Many participants (76%) reported interest in quitting and willingness to try through patches/gum (56%) and technology-based (61%) approaches. Youth exiting foster care are at high risk for smoking and other tobacco product use, as well as dependence, yet are rarely screened for use or advised to quit. As tobacco use remains among the most preventable causes of mortality and morbidity, future work should involve implementation of screening within child welfare and tailoring interventions to the unique needs of this population. The current results underscore a missed opportunity to promote public health in a vulnerable population.

Mitochondrial GTP Links Nutrient Sensing to ß Cell Health, Mitochondrial Morphology, and Insulin Secretion Independent of OxPhos.

Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.

Developing a tailored substance use intervention for youth exiting foster care.

Youth who are aging out of the foster care system face significant barriers to accessing substance use treatment. Mobile interventions have shown efficacy for several mental and physical health issues and may be helpful in overcoming barriers facing foster youth with substance use problems. A program (iHeLP) for substance use reduction was developed that used a computerized screening and brief intervention (SBI) followed by six months of dynamically-tailored text messages. The program was shown to focus groups of youth (N = 24) ages 18-19 who recently left foster care and had moderate to severe substance use risk. Focus group feedback was used to modify iHeLP prior to delivery in an open trial (N = 16). Both study phases included assessments of feasibility and acceptability; the open trial also included assessments of substance use outcomes at 3 and 6 months. Focus groups indicated a high level of acceptability for the proposed intervention components. Of those screened for the open trial, 43% were eligible and 74% of those eligible enrolled, indicating good feasibility. Retention through the final follow-up was 59%, and drop out was associated with involvement in the criminal justice system. Participant ratings for liking, ease of working with, interest in and respectfulness of the SBI were high. Satisfaction ratings for the texting component were also high. A computerized brief screening intervention for substance use risk reduction together with tailored text messaging is both feasible and highly acceptable among youth who have recently aged-out of foster care.

Feeling Heard and Not Judged: Perspectives on Substance Use Services Among Youth Formerly in Foster Care.

Youth in foster care have limited access to substance use services for a variety of reasons. Attempts to unpack this health disparity have focused on foster care systems, administrators, providers, and foster parents. This study seeks to understand the perspectives of youth themselves, with the hope of understanding their experiences with and preferences for such services. Analyses of focus groups with youth who had recently left foster care suggested concrete and perceptual facilitators/barriers to treatment. Concrete facilitators/barriers included the need for expanding social support, access to multiple service options, and tailored intervention approaches. Perceptual concerns revolved around understanding each individual's readiness to change, feeling judged by authority figures, and desiring help from people with lived experience. Participants also described novel intervention ideas, including a focus on technology-based approaches. By relying on youth voices, we can improve upon the current state of substance use interventions within foster care.

Incomplete Ionization of a 110 meV Unintentional Donor in ß-Ga2O3 and its Effect on Power Devices.

Understanding the origin of unintentional doping in Ga2O3 is key to increasing breakdown voltages of Ga2O3 based power devices. Therefore, transport and capacitance spectroscopy studies have been performed to better understand the origin of unintentional doping in Ga2O3. Previously unobserved unintentional donors in commercially available [Formula: see text] Ga2O3 substrates have been electrically characterized via temperature dependent Hall effect measurements up to 1000 K and found to have a donor energy of 110 meV. The existence of the unintentional donor is confirmed by temperature dependent admittance spectroscopy, with an activation energy of 131 meV determined via that technique, in agreement with Hall effect measurements. With the concentration of this donor determined to be in the mid to high 1016 cm-3 range, elimination of this donor from the drift layer of Ga2O3 power electronics devices will be key to pushing the limits of device performance. Indeed, analytical assessment of the specific on-resistance (Ronsp) and breakdown voltage of Schottky diodes containing the 110 meV donor indicates that incomplete ionization increases Ronsp and decreases breakdown voltage as compared to Ga2O3 Schottky diodes containing only the shallow donor. The reduced performance due to incomplete ionization occurs in addition to the usual tradeoff between Ronsp and breakdown voltage.

A Comparison of Long- vs. Short-Term Recall of Substance Use and HIV Risk Behaviors.

OBJECTIVE: The Timeline Follow-back (TLFB) questionnaire has become a pre-eminent tool in substance use and human immunodeficiency virus (HIV) risk research, allowing researchers to assess fine-grained changes in risk behavior over long periods. However, data on accuracy of recall over long (12-month) periods are sparse, especially combined data on HIV risk and substance use from post-treatment samples. Studies on the development of substance use and HIV risk stand to benefit from data on the accurate recall of such behavior over longer retroactive spans of time. METHOD: The present study offers data on the test-retest reliability of current TLFB assessment versus 6- and 12-month delayed TLFB assessment, using a post-treatment sample (n = 50). RESULTS: Long-term reliability of TLFB data on HIV risk was predominantly good to excellent, with 13 of 20 assessed variables in that range. TLFB data on substance use was similar, with 22 of 26 variables resulting in good/excellent reliability. CONCLUSIONS: Our findings support the notion that, notable exceptions aside, the TLFB may be effectively used to assess retroactive HIV risk and substance use in periods of 12 months.

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations.

Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.

Real-time imaging of intracellular hydrogen peroxide in pancreatic islets.

A real-time method to measure intracellular hydrogen peroxide (H2O2) would be very impactful in characterizing rapid changes that occur in physiologic and pathophysiologic states. Current methods do not provide the sensitivity, specificity and spatiotemporal resolution needed for such experiments on intact cells. We developed the use of HyPer, a genetic indicator for H2O2 that can be expressed in the cytosol (cyto-HyPer) or the mitochondria (mito-HyPer) of live cells. INS-1 cells or islets were permeabilized and the cytosolic HyPer signal was a linear function of extracellular H2O2, allowing fluorescent cyto-HyPer signals to be converted into H2O2 concentrations. Glucose increased cytosolic H2O2, an effect that was suppressed by overexpression of catalase. Large perturbations in pH can influence the HyPer signal, but inclusion of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] in the perfusate prevented pH changes, but did not affect glucose-induced cyto-HyPer signals, suggesting that this effect is largely pH-independent. Using the assay, two fundamental questions were addressed. Knockdown of superoxide dismutase 2 (SOD2), the mitochondrial form of SOD, completely suppressed glucose-induced H2O2 Furthermore, glucose also induced mitochondrial superoxide and H2O2 production, which preceded the appearance of cytosolic H2O2 Therefore, glucose-induced H2O2 largely originated from mitochondria. Finally, the glucose-induced HyPer signal was less than 1/20th of that induced by toxic levels of H2O2 Overall, the use of HyPer for real-time imaging allowed resolution of acute changes in intracellular levels of H2O2 and will have great utility for islet studies involving mechanisms of H2O2-mediated signaling and oxidative stress.