A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

Towards enhanced understanding of idiopathic ketotic hypoglycemia: a literature review and introduction of the patient organization, Ketotic Hypoglycemia International.

BACKGROUND: Idiopathic Ketotic hypoglycemia (IKH) is a diagnosis of exclusion. Although considered as the most frequent cause of hypoglycemia in childhood, little progress has been made to advance the understanding of IKH since the medical term was coined in 1964. We aimed to review the literature on ketotic hypoglycemia (KH) and introduce a novel patient organization, Ketotic Hypoglycemia International (KHI). RESULTS: IKH may be diagnosed after the exclusion of various metabolic and hormonal diseases with KH. Although often mild and self-limiting, more severe and long-lasting IKH occurs. We therefore divide IKH in physiological KH and pathological KH, the latter defined as recurrent symptomatic, or occasionally symptomatic, episodes with beta-hydroxybutyrate ≥ 1.0 mmol/L and blood glucose < 70 mg/dL (3.9 mol/L), in the absence of prolonged fasting, acute infections and chronic diseases known to cause KH. Pathological KH may represent undiscovered diseases, e.g. glycogen storage disease IXa, Silver-Russel syndrome, and ketone transporter defects, or suggested novel disease entities identified by exome sequencing. The management of KH aims to prevent hypoglycemia, fatty acid oxidation and protein deficiency by supplying adequate amounts of carbohydrates and protein, including nutritional therapy, uncooked cornstarch, and sometimes continuous tube feeding by night. Still, intravenous dextrose may be needed in acute KH episodes. Failure to acknowledge that IKH can be more than normal variation may lead to under-treatment. KHI is a non-profit, patient-centric, global organization established in 2020. The organization was created by adult IKH patients, patient family members, and volunteers. The mission of KHI is to enhance the understanding of IKH while advocating for patients, their families and the continued research into KH. CONCLUSION: IKH is a heterogeneous disorder including physiological KH and pathological KH. IKH may represent missed diagnoses or novel disease entities, but shares common management principles to prevent fatty acid oxygenation. KHI, a novel patient organization, aims to enhance the understanding of IKH by supporting IKH families and research into IKH.

Biomechanical Contributions to Macrophage Activation in the Tumor Microenvironment.

Alterations in extracellular matrix composition and organization are known to promote tumor growth and metastatic progression in breast cancer through interactions with tumor cells as well as stromal cell populations. Macrophages display a spectrum of behaviors from tumor-suppressive to tumor-promoting, and their function is spatially and temporally dependent upon integrated signals from the tumor microenvironment including, but not limited to, cytokines, metabolites, and hypoxia. Through years of investigation, the specific biochemical cues that recruit and activate tumor-promoting macrophage functions within the tumor microenvironment are becoming clear. In contrast, the impact of biomechanical stimuli on macrophage activation has been largely underappreciated, however there is a growing body of evidence that physical cues from the extracellular matrix can influence macrophage migration and behavior. While the complex, heterogeneous nature of the extracellular matrix and the transient nature of macrophage activation make studying macrophages in their native tumor microenvironment challenging, this review highlights the importance of investigating how the extracellular matrix directly and indirectly impacts tumor-associated macrophage activation. Additionally, recent advances in investigating macrophages in the tumor microenvironment and future directions regarding mechano-immunomodulation in cancer will also be discussed.

Ambient urban dust particulate matter reduces pathologic T cells in the CNS and severity of EAE.

BACKGROUND: Autoimmune diseases have increased in incidence and prevalence worldwide. While genetic predispositions play a role, environmental factors are a major contributor. Atmospheric particulate matter (PM) is a complex mixture composed of metals, nitrates, sulfates and diverse adsorbed organic compounds like polycyclic aromatic hydrocarbons (PAHs) and dioxins. Exposure to atmospheric PM aggravates autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, among others. PAHs and dioxins are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and directs the balance between effector and regulatory T cells in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify pathways that contribute to autoimmune disease and their potential use as therapeutic targets to alleviate symptoms and the need for global immunosuppression. This study tests the hypothesis that atmospheric PM enhances effector T cell differentiation and aggravates autoimmune disease. RESULTS: An atmospheric ambient urban dust PM sample, standard reference material (SRM)1649b, was tested for its effects on autoimmunity. SRM1649b PM enhanced Th17 differentiation in an AHR-dependent manner in vitro, however intranasal treatment of SRM1649b PM delayed onset of EAE and reduced cumulative and peak clinical scores. Chronic and acute intranasal exposure of SRM1649b PM delayed onset of EAE. Chronic intranasal exposure did not reduce severity of EAE while acute intranasal exposure significantly reduced severity of disease. Acute intranasal treatment of low dose SRM1649b PM had no effect on clinical score or day of onset in EAE. Delayed onset of EAE by intranasal SRM1649b PM was AHR-dependent in vivo. Oral gavage of SRM1649b PM, in the absence of AHR ligands in the diet, had no effect on day of disease onset or severity of EAE. Day 10 analysis of T cells in the CNS after intranasal treatment of SRM1649b PM showed a reduction of pathologic T cell subsets in vivo. Moreover, MOG-specific splenocytes require AHR to generate or maintain IL-10 producing cells and reduce IFNγ producing cells in vitro. CONCLUSIONS: These results identify the AHR pathway as a potential target for driving targeted immunosuppression in the CNS in the context of atmospheric PM-mediated autoimmune disease. The effects of SRM1649b PM on EAE are dependent on route of exposure, with intranasal treatment reducing severity of EAE and delaying disease onset while oral gavage has no effect. Intranasal SRM1649b PM reduces pathologic T cells in the CNS, specifically Th1 cells and Th1Th17 double positive cells, leading to reduced severity of EAE and AHR-dependent delayed disease onset. Additionally, SRM1649b PM treatment of antigen-specific T cells leads to AHR-dependent increase in percent IL-10 positive cells in vitro. These findings may shed light on the known increase of infection after exposure to atmospheric PM and serve as the first step in identifying components of the AHR pathway responsible for Th1-mediated immunosuppression in response to atmospheric PM exposure.

Polycyclic aromatic hydrocarbons (PAHs) present in ambient urban dust drive proinflammatory T cell and dendritic cell responses via the aryl hydrocarbon receptor (AHR) in vitro.

Atmospheric particulate matter (PM) is a complex component of air pollution that is a composed of inorganic and organic constituents. The chemically-extracted organic fraction (OF) of PM excludes inorganics but retains most organic constituents like polycyclic aromatic hydrocarbons (PAHs). PAHs are ubiquitous environmental toxicants and known aryl hydrocarbon receptor (AHR) ligands. The AHR is a ligand activated transcription factor that responds to endogenous ligands and exogenous ligands including PAHs. Activation of the AHR leads to upregulation of cytochrome P450 (CYP) metabolizing enzymes which are important for the biotransformation of toxicants to less toxic, or in the case of PAHs, more toxic intermediates. Additionally, the AHR plays an important role in balancing regulatory and effector T cell responses. This study aimed to determine whether PAHs present in PM aggravate inflammation by driving inflammatory T cell and dendritic cell (DC) responses and their mechanism of action. This study tests the hypothesis that PAHs present in PM activate the AHR and alter the immune balance shifting from regulation to inflammation. To test this, the effects of SRM1649b OF on T cell differentiation and DC function were measured in vitro. SRM1649b OF enhanced Th17 differentiation in an AHR and CYP-dependent manner and increased the percent of IFNγ positive DCs in an AHR-dependent manner. SRM1649b PAH mixtures enhanced Th17 differentiation in an AHR-dependent but CYP-independent manner and increased the percent of IFNγ positive DCs. Cumulatively, these results suggest that PAHs present in PM are active components that contribute to immune responses in both T cells and BMDCs through the AHR and CYP metabolism. Understanding the role of AHR and CYP metabolism of PAHs in immune cells after PM exposure will shed light on new targets that will shift the immune balance from inflammation to regulation.

Differential effects of diesel exhaust particles on T cell differentiation and autoimmune disease.

BACKGROUND: Exposure to particulate matter (PM) has been associated with increased incidence and severity of autoimmune disease. Diesel PM is primarily composed of an elemental carbon core and adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and contributes up to 40% of atmospheric PM. The organic fraction (OF) of PM excludes all metals and inorganics and retains most organic compounds, such as PAHs. Both PM and OF increase inflammation in vitro and aggravate autoimmune disease in humans. PAHs are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and effector function in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify whether the total mass or active components of PM are responsible for activating pathways associated with exposure to PM and autoimmune disease. This study tests the hypothesis that active components present in diesel PM and their OF enhance effector T cell differentiation and aggravate autoimmune disease. RESULTS: Two different diesel samples, each characterized for their components, were tested for their effects on autoimmunity. Both diesel PM enhanced effector T cell differentiation in an AHR-dose-dependent manner and suppressed regulatory T cell differentiation in vitro. Both diesel PM aggravated EAE in vivo. Fractionated diesel OFs exhibited the same effects as PM in vitro, but unlike PM, only one diesel OF aggravated EAE. Additionally, both synthetic PAH mixtures that represent specific PAHs found in the two diesel PM samples enhanced Th17 differentiation, however one lost this effect after metabolism and only one required the AHR. CONCLUSIONS: These findings suggest that active components of PM and not total mass are driving T cell responses in vitro, but in vivo the PM matrix and complex mixtures adsorbed to the particles, not just the OF, are contributing to the observed EAE effects. This implies that examining OF alone may not be sufficient in vivo. These data further suggest that bioavailability and metabolism of organics, especially PAHs, may have an important role in vivo.

Evaluation of the criterion and predictive validity of the Alcohol Reduction Strategies-Current Confidence (ARS-CC) in a natural drinking environment.

To evaluate several psychometric properties of a questionnaire designed to assess young people's self-efficacy to employ 31 alcohol reduction strategies, we assessed breath alcohol concentration, self-reported drinking, current strategy self-efficacy, and recent past use of these strategies in 100 young adults as they walked to and from the local bar district of a Midwestern college town. In support of criterion and predictive validity of the questionnaire, we found that lower self-efficacy at the initial assessment was significantly correlated with higher scores on a screening measure of consumption, with having engaged in more heavy drinking episodes in the past 30 days, and with higher levels of intoxication and use of fewer reduction strategies later that evening. Frequency counts indicate that 10 of the alcohol reduction strategies had been used by at least half of the sample to reduce their drinking earlier that evening.

Development of a questionnaire to assess university students' intentions to use behavioral alcohol-reduction strategies.

OBJECTIVE: To evaluate the psychometric properties of a new self-report questionnaire designed to assess college students' intentions to employ 31 specific alcohol-reduction strategies. METHOD: Students attending a large public university were recruited to complete alcohol-reduction, drinking history, and personality questionnaires online. RESULTS: Based on item-total correlations and principal components analysis, the authors eliminated 3 items and calculated average intention ratings across the remaining 28 items. The resulting scale had appropriate unidimensionality and excellent internal consistency. Correlations of intention questionnaire scores with measures of drinking history, alcohol outcome expectancies, sensation seeking, and impression management provided some support for criterion and discriminant validity of the questionnaire. CONCLUSION: This questionnaire could be employed as an outcome measure to evaluate prevention programs and as a clinical tool to identify clients who have little intention to employ drinking reduction strategies in heavy drinking situations.

Assessing self-efficacy to reduce one's drinking: further evaluation of the Alcohol Reduction Strategies-Current Confidence questionnaire.

AIMS: To evaluate the psychometric properties of a previously published questionnaire designed to assess young drinkers' self-efficacy to employ 31 cognitive-behavioral alcohol reduction strategies. METHODS: Undergraduates (n = 353) recruited from a large Midwestern university completed the previously published Alcohol Reduction Strategies-Current Confidence questionnaire (and other measures) for a self-selected heavy drinking setting. RESULTS: Item loadings from a principal components analysis, a high internal consistency reliability coefficient, and a moderate mean inter-item correlation suggested that all 31 items comprised a single scale. Correlations of questionnaire scores with selected aspects of drinking history and personality provided support for criterion and discriminant validity, respectively. Women reported higher current confidence to use these strategies than did men, but current confidence did not vary as a function of recent binge status. CONCLUSION: Given this further demonstration of its psychometric qualities, this questionnaire holds promise as a clinical tool to identify clients who lack confidence in their ability to employ cognitive-behavioral coping strategies to reduce their drinking.

Quantitative and qualitative assessment of university students' definitions of binge drinking.

This study was designed to assess undergraduates' (N = 424) definitions of binge drinking and to evaluate whether the number of drinks they said comprise a binge varied as a function of beverage type, respondent gender, and respondent binge drinking status. When asked to designate the specific number of drinks that comprise a binge for each of four beverage types, students reported that the number of beers constituting a binge was significantly larger than the number of glasses of wine, shots of hard liquor, and servings of any combination of alcoholic beverage types; men reported that a larger number of drinks constitute a binge than did women; and those who had engaged in 3 or more binges in the past 2 weeks reported that more drinks comprise a binge than those who had binged less often. Responses to an open-ended question asking their definition of a binge revealed that students sometimes characterize a binge in terms of motivations for and unhealthy consequences of drinking, in addition to defining a binge as comprising consumption of a large amount of alcohol in a limited (though often unspecified) time period. Furthermore, students attributed their open-ended definitions of binge drinking to informal sources of information and observation of others' drinking almost as often as they did to school-based or media-based sources. This suggests that educators might look for innovative ways to use both formal and informal social networking, and video illustrations of restrained drinking, as ways to influence young people's views of binge drinking.

Assessing university students' self-efficacy to employ alcohol-related harm reduction strategies.

OBJECTIVE: Develop and evaluate key psychometric properties of a self-report questionnaire specifically designed to assess student drinkers' self-confidence to employ a variety of strategies intended to reduce unhealthy consequences of high-risk drinking. METHODS: Four hundred ninety-eight participants rated their confidence (from "not at all confident" to "completely confident") to employ 17 harm reduction strategies when drinking. RESULTS: Factor analysis and internal consistency reliability analyses indicated that the 17 items constitute a single scale with good test-retest reliability. Consistent with other research examining previous use of such strategies, women in our sample reported significantly higher harm reduction self-efficacy than did men. Harm reduction self-efficacy was also associated with reported number of high-risk drinking episodes in the previous 2 weeks. CONCLUSION: This brief and easily administered questionnaire holds promise as a clinical tool to identify individuals with low harm reduction self-efficacy and as an outcome measure for health promotion and educational interventions.

Family ties in tough times: how young adults and their parents view the U.S. economic crisis.

The present intergenerational study examined the perceived impact of the recent U.S. economic crisis on a sample of 68 young adult-parent dyads. The relative contribution of perceived economic pressure, reports of adult child-parent relationship quality, and concerns about the economic future in accounting for variation in self-reports of psychological distress for adult children and their middle-aged parents were examined. Parents' concerns about their children's economic future accounted for variation in their reports of anxiety and depressed mood above and beyond that of perceived economic pressures and their views of the parent-child relationship. In contrast, for young adults, reports of personal economic pressure were generally related to self-reported anxiety and depressed mood. Implications of findings for research and practice are discussed.

Measuring university students' self-efficacy to use drinking self-control strategies.

Using a Web-based, self-administered questionnaire, we assessed 498 university-student drinkers' self-efficacy to use 31 different behavioral strategies to reduce excessive drinking in each of three different locations (bar, party, own dorm/apartment). Averaging all 31 items within each drinking situation to create a single scale score revealed high internal consistency reliabilities and moderate inter-item correlations. Testing the association of self-efficacy with drinking location, sex, and frequency of recent binge drinking, we found that respondents reported higher self-efficacy to use these strategies when drinking in their own dorm/apartment than when drinking in bars and at parties; women reported higher mean self-efficacy than men; and drinkers who engaged in 3-or-more binges in the previous 2 weeks reported lower self-efficacy than those who reported either 0 or 1-or-2 binges in the same time period. This questionnaire could be used to identify self-efficacy deficits among clients with drinking problems and as an outcome measure to assess the degree to which interventions influence reported confidence to use specific drinking-reduction strategies in high-risk drinking situations.