Use of Transgenic Parasites and Host Reporters To Dissect Events That Promote Interleukin-12 Production during Toxoplasmosis

The intracellular parasite Toxoplasma gondii has multiple strategies to alter host cell function, including the injection of rhoptry proteins into the cytosol of host cells as well as bystander populations, but the consequence of these events is unclear. Here, a reporter system using fluorescent parasite strains that inject Cre recombinase with their rhoptry proteins (Toxoplasma-Cre) was combined with Ai6 Cre reporter mice to identify cells that have been productively infected, that have been rhoptry injected but lack the parasite, or that have phagocytosed T. gondii. The ability to distinguish these host-parasite interactions was then utilized to dissect the events that lead to the production of interleukin-12 p40 (IL-12p40), which is required for resistance to T. gondii. In vivo, the use of invasion-competent or invasion-inhibited (phagocytosed) parasites with IL-12p40 (YET40) reporter mice revealed that dendritic cell (DC) and macrophage populations that phagocytose the parasite or are infected can express IL-12p40 but are not the major source, as larger numbers of uninfected cells secrete this cytokine. Similarly, the use of Toxoplasma-Cre parasite strains indicated that dendritic cells and inflammatory monocytes untouched by the parasite and not cells injected by the parasite are the primary source of IL-12p40. These results imply that a soluble host or parasite factor is responsible for the bulk of IL-12p40 production in vivo, rather than cellular interactions with T. gondii that result in infection, infection and clearance, injection of rhoptry proteins, or phagocytosis of the parasite.     

Acute Kidney Injury After CABG Versus PCI : An Observational Study Using 2 Cohorts

Background

Acute kidney injury (AKI) is a known complication after coronary revascularization, but few studies have directly compared the incidence of AKI after coronary artery bypass surgery (CABG) or after percutaneous coronary intervention (PCI) in similar patients.

Objectives

The aim of this study was to investigate whether multivessel CABG compared with PCI as an initial revascularization strategy is associated with a higher risk for AKI.

Methods

A retrospective analysis of patients undergoing first documented coronary revascularization was conducted using 2 complementary cohorts: 1) Kaiser Permanente Northern California, a diverse, integrated health care delivery system; and 2) Medicare beneficiaries, a large, nationally representative older cohort. AKI was defined in the Kaiser Permanente Northern California cohort by an increase in serum creatinine of ≥0.3 mg/dl or ≥150% above baseline and in the Medicare cohort by discharge diagnosis codes and the use of dialysis.

Results

The incidence of AKI was 20.4% in the Kaiser Permanente Northern California cohort and 6.2% in the Medicare cohort. The incidence of AKI requiring dialysis was <1%. CABG was associated with a 2- to 3-fold significantly higher adjusted odds for developing AKI compared with PCI in both cohorts.

Conclusions

AKI is common after multivessel coronary revascularization and is more likely after CABG than after PCI. The risk for AKI should be considered when choosing a coronary revascularization strategy, and ways to prevent AKI after coronary revascularization are needed.     

The Impact of Research Participation on Adults with Severe Mental Illness

Experiences of adults with mental illness who participated in a 12-month managed care study are summarized. During exit interviews, participants were asked about consent procedures, study purpose, if questions were intrusive or anxiety producing, and concerns about information disclosure. Respondents rated their experience and likelihood of future participation. Almost 38% did not remember the consent procedures. Among those who did, 22.4% reported they lacked adequate detail about the scope of the study. Nearly 3% felt pressured into participating. Although most participants (96%) reported positive experiences, 8.8% became anxious, 16.8% were afraid responses would be disclosed, and 16.7% indicated questions were invasive. Age, race/ethnicity, and gender were not associated with adverse reactions. Symptomatology and perceived inadequacies in consent procedures were significantly, albeit weakly, associated with adverse reactions. Although most participants experienced no distress, rates of adverse responses among persons with mental illness exceeded those of community-based samples. Strategies for minimizing negative reactions are discussed.     

The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4

The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.     

Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and pHi

Summary The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), at nanomolar concentrations, induces rapid (t _1/2 approximately 30 s) protrusion of multiple petal-shaped lamellae by neutrophil leucocytes. Lamellae are richly endowed with actin filaments as determined by the localization of rhodamine-phalloidin, suggesting extensive assembly at the cell cortex. Direct measurement of the proportion of total cell actin which is polymerized, by using a deoxyribonuclease I inhibition assay, indicates that the proportion of polymerized actin approximately doubles, and that assembly initiated by 30nm TPA occurs with no obvious lag phase and with a t_1/2 of about 30 s. A half-maximal response was induced at 2nm TPA. Since both actin assembly and protrusion of lamellae are completely inhibited by 10^–6 m cytochalasin D, protrusion of lamellae is presumably dependent on actin filament assembly.To examine whether TPA induces actin assembly via changes in [Ca²⁺]_i or pH_i, these parameters were monitored in cells loaded with the fluorescent indicators quin2 and quene1 respectively. Addition of TPA caused no change in [Ca²⁺]_i but a biphasic change in pH_i. To examine further the potential role of ionic changes in regulation of actin assembly, the morphological responses of cells to TPA were monitored in severely Ca²⁺-depleted cells, or cells in which pH_i had been experimentally raised or lowered by simultaneous additions of a weak base (NH₄Cl) or weak acid (CH₃COONa) respectively. The protrusion of lamellae induced by TPA was completely unaffected by these experimental manipulations indicating that TPA can directly regulate actin assembly, and hence morphology, by mechanisms essentially independent of [Ca²⁺]_i and pH_i. Since TPA is believed to mimic diacylglycerol, which is putatively generated by cleavage of phosphatidylinositol-4, 5-bisphosphate in a number of cellular responses involving actin filament assembly, these results implicate diacylglycerol, either acting directly, or via protein kinase C-mediated phosphorylation, as a common step in the receptor-mediated regulation of cortical actin filament assembly in cells.     

Treatment seeking for depression: role of depressive symptoms and comorbid psychiatric diagnoses.

BACKGROUND: General population research has indicated that many persons with lifetime major depression report they have never sought medical help for their illness. We examined the role played by both depressive symptoms and comorbid psychiatric diagnoses in treatment seeking. METHODS: From standardized psychiatric interviews of 7214 Edmonton residents, we identified 1348 subjects with lifetime depressive illness; 866 subjects (64%) had sought medical treatment. RESULTS: Psychomotor retardation (odds ratio, OR = 1.4), suicidal ideation (OR = 1.4), comorbid mania (OR = 9.5), comorbid panic disorder (OR = 3.0), and comorbid drug abuse/dependence (OR = 0.6) were significantly associated with treatment seeking in multivariate analysis. CONCLUSION: The finding that only a few depressive symptoms were associated with treatment seeking underlines the importance of public health education to address the undertreatment of depression.