Murine Inducible Nitric Oxide Synthase Expression Is Essential for Antifungal Defenses in Kidneys during Disseminated Cryptococcus deneoformans Infection.

Disseminated cryptococcosis has a nearly 70% mortality, mostly attributed to CNS infection, with lesser-known effects on other organs. Immune protection against Cryptococcus relies on Th1 immunity with M1 polarization, rendering macrophages fungicidal. The importance of M1-upregulated inducible NO synthase (iNOS) has been documented in pulmonary anticryptococcal defenses, whereas its role in disseminated cryptococcosis remains controversial. Here we examined the effect of iNOS deletion in disseminated (i.v.) C. deneoformans 52D infection, comparing wild-type (C57BL/6J) and iNOS-/- mice. iNOS-/- mice had significantly reduced survival and nearly 100-fold increase of the kidney fungal burden, without increases in the lungs, spleen, or brain. Histology revealed extensive lesions and almost complete destruction of the kidney cortical area with a loss of kidney function. The lack of fungal control was not due to a failure to recruit immune cells because iNOS-/- mice had increased kidney leukocytes. iNOS-/- mice also showed no defect in T cell polarization. We conclude that iNOS is critically required for local anticryptococcal defenses in the kidneys, whereas it appears to be dispensable in other organs during disseminated infection. This study exemplifies a unique phenotype of local immune defenses in the kidneys and the organ-specific importance of a single fungicidal pathway.

CCR2 Signaling Promotes Brain Infiltration of Inflammatory Monocytes and Contributes to Neuropathology during Cryptococcal Meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is a leading cause of central nervous system (CNS) infection-related mortality worldwide, with surviving patients often developing neurological deficiencies. While CNS inflammation has been implicated in the pathogenesis of CM, little is known about the relative contribution of the specific inflammatory/immune pathways to CNS pathology versus fungal clearance. Increased cerebrospinal fluid level of C-C chemokine receptor 2 (CCR2) ligand CCL2 is associated with disease deterioration in patients with CM. Using a murine model, we investigated the role of the CCR2 pathway in the development of CNS inflammation and pathology during CM. We found that CCR2-deficient mice exhibited improved 28-day survival and alleviated neurological disease scores despite a brain fungal burden higher than that of the WT mice. Reduced CM pathology in CCR2-deficient mice was accompanied by markedly decreased neuronal cell death around cryptococcal microcysts and restored expression of genes involved in neurotransmission, connectivity, and neuronal cell structure in the brains. Results show that CCR2 axis is the major pathway recruiting CD45hiCD11b+Ly6C+ inflammatory monocyte to the brain and indirectly modulates the accumulation of CD4+ T cells and CD8+ T cells. In particular, CCR2 axis promotes recruitment of interferon gamma (IFN-γ)-producing CD4+ T cells and classical activation of myeloid cells. In this context, CCR2 deletion limits the immune network dysregulation we see in CM and attenuates neuropathology. Thus, the CCR2 axis is a potential target for interventions aimed to limit inflammatory CNS pathology in CM patients. IMPORTANCE Cryptococcal meningoencephalitis (CM) causes nearly 200,000 deaths worldwide each year, and survivors frequently develop long-lasting neurological sequelae. The high rate of mortality and neurologic sequelae in CM patients indicate that antifungal therapies alone are often insufficient to control disease progression. Here, we reveal that CM disease progression in mice is accompanied by inflammatory monocytes infiltration at the periphery of the infected foci that overlap locally perturbed neuronal function and death. Importantly, we identified that CCR2 signaling is a critical pathway driving neuroinflammation, especially inflammatory monocyte recruitment, as well as CNS pathology and mortality in CM mice. Our results imply that targeting the CCR2 pathway may be beneficial as a therapy complementary to antifungal drug treatment, helping to reduce CNS damage and mortality in CM patients.

Extending Expectancy Theory to Food Intake: Effect of a Simulated Fast-Food Restaurant on Highly and Minimally Processed Food Expectancies.

Unhealthy diets are widespread and linked to a number of detrimental clinical outcomes. The current preregistered experiment extended Expectancy Theory into the study of food intake; specifically, we tested whether a fast-food restaurant affects food expectancies, or the emotions one expects to feel while eating highly (e.g., pizza) and minimally (e.g., carrots) processed foods. Participants (N = 200, M age = 18.79) entered a simulated fast-food restaurant or a neutral space, completed questionnaires, and engaged in a 'bogus' taste test. The simulated fast-food restaurant increased positive highly-processed food expectancies (d = 0.29). Palatable eating coping motives scores did not moderate the effect; however, this clinically-relevant pattern of eating behavior was associated with greater positive highly-processed food expectancies. In addition, there was an indirect effect of the fast-food restaurant on ad libitum food intake through positive highly-processed food expectancies. Reducing positive highly-processed food expectancies may improve diet, which may broadly impact health.

Chemokine receptor CXCR3 is required for lethal brain pathology but not pathogen clearance during cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is the major cause of infection-related neurological death, typically seen in immunocompromised patients. However, T cell-driven inflammatory response has been increasingly implicated in lethal central nervous system (CNS) immunopathology in human patients and murine models. Here, we report marked up-regulation of the chemokine receptor CXCR3 axis in human patients and mice with CM. CXCR3 deletion in mice improves survival, diminishes neurological deficits, and limits neuronal damage without suppressing fungal clearance. CD4+ T cell accumulation and TH1 skewing are reduced in the CNS but not spleens of infected CXCR3-/- mice. Adoptive transfer of WT, but not CXCR3-/- CD4+ T cells, into CXCR3-/- mice phenocopies the pathology of infected WT mice. Collectively, we found that CXCR3+CD4+ T cells drive lethal CNS pathology but are not required for fungal clearance during CM. The CXCR3 pathway shows potential as a therapeutic target or for biomarker discovery to limit CNS inflammatory damages.

Student observations of medication error reporting practices in community pharmacy settings.

BACKGROUND: Medication safety practices and methods for reporting errors in community pharmacies are relatively unknown. OBJECTIVE: (s): The primary objective of this study was to describe student-reported data on medication safety and error reporting practices in community pharmacies, and secondarily describe student learning from this assignment. METHODS: Second professional year pharmacy students enrolled at Purdue University College of Pharmacy in the United States observed and recorded medication safety and error reporting practices as part of an experiential assignment. Data were collected from 170 unique pharmacy settings between the years 2016-2018 and analyzed using descriptive statistics and a paired t-test to assess student learning. RESULTS: 51% of students reported documentation of 1-10 errors or near misses annually, with an additional 30% reporting 11-30. Near misses were only reported 26% of the time. Errors were most commonly reported to a pharmacy-specific reporting system (84%) and the Institute for Safe Medication Practices National Medication Errors Reporting Program (84%). The most frequently reported error types included wrong directions (34%), wrong drug (14%), wrong strength (13%), and wrong patient (12%). Pharmacists were observed to be interrupted approximately 19 times every hour. Anonymous error reporting was typically not allowed to the pharmacy's preferred error reporting system (71%). A policy requiring that the prescriber is contacted about errors was observed at 77% of the sites. The most common consequences of committing an error were education/training (72%) or progressive discipline (41%). Students reported a statistically significant increase in understanding of medication safety practices and methods for reporting errors in community pharmacies. (p < 0.01). CONCLUSION: This data supplements existing literature on medication safety practices and error reporting in community pharmacy settings, as well as highlights knowledge gaps outside the scope of this study.

Mechanisms underlying the anti-proliferative actions of adiponectin in human breast cancer cells, MCF7-dependency on the cAMP/protein kinase-A pathway.

Obesity is a risk factor for breast cancer, and low blood concentrations of adiponectin are associated with high incidence and poor prognosis of breast cancer. However, the molecular mechanisms underlying such inhibitory effects of adiponectin remain to be defined. By using MCF7 cells, we investigated the mechanisms underlying the inhibitory effects of adiponectin on breast cancer cells, particularly in the context of opposing IGF-1-induced proliferation. We found that adiponectin, at 20 and 40 µg/ml, reduced MCF7 cell growth in the absence and presence of IGF-1. These inhibitory effects were primarily the result of the significant increase of cells at G(1)/G(0) phase and concomitant decrease of cells at S phase. In addition, the percentage of apoptotic cells increased more than two-fold. Within 30-min of adiponectin addition, the phosphorylation of AMPKα was sharply elevated, and the level of IGF-1-activated Akt was decreased. Prolonged exposure to adiponectin resulted in reduction of cyclin D1 and cyclin E2 expression. Adiponectin also increased intracellular levels of cAMP and the activity of protein kinase-A (PKA). The inhibitors of PKA completely abolished the adiponectin's effects on cell growth. In conclusion, our studies revealed an important cellular mechanism underlying the relationship between reduced adiponectin concentrations and breast cancer development.

Human adiponectin inhibits cell growth and induces apoptosis in human endometrial carcinoma cells, HEC-1-A and RL95 2.

Obesity is one of the well-established risk factors for endometrial cancer. Recent clinical studies have demonstrated that circulating adiponectin concentrations are inversely correlated with the incidence of endometrial carcinoma. Such epidemiological findings are consistent with the paradoxical observations that adiponectin levels are reduced in obesity. This study investigated the direct effects of adiponectin on two endometrial carcinoma cell lines, HEC-1-A and RL95-2. These cell lines express both variants of adiponectin receptors, adipo-R1 and adipo-R2. Adiponectin treatment leads to suppression of cell proliferation in both cell types, which is primarily due to the significant increase of cell populations at G(1)/G(0)-phase and to the induction of apoptosis. The inhibition of growth in these two cell lines appears to be mediated by different signaling pathways. Although adiponectin treatment markedly increases the phosphorylation (Thr172) of AMP-activated protein kinase alpha in both HEC-1-A and RL95-2 within 30 min, prolonged exposure (48 h) leads to inactivation of Akt as well as reduction of cyclin D1 protein expression in HEC-1-A cells. In contrast, similar treatment of RL95-2 cells with adiponectin, while having no effects on Akt activity and cyclin D1 expression, causes a decrease in cyclin E2 expression and the activity of mitogen-activated kinase (p42/44). We conclude that adiponectin exerts direct anti-proliferative effects on HEC-1-A and RL95-2 cells by inducing cell cycle arrest and apoptosis. Depending on the genotypes of the endometrial cancer cells, the inhibitory effects of adiponectin are associated with the reduction of different pro-growth regulators of cell cycle and signaling proteins. Our study thus provides a cellular mechanism underlying the linkages between endometrial cancer and obesity.