The Peroxisome Proliferator-Activated Receptor (PPAR)-γ Antagonist 2-Chloro-5-Nitro-N-Phenylbenzamide (GW9662) Triggers Perilipin 2 Expression via PPARδ and Induces Lipogenesis and Triglyceride Accumulation in Human THP-1 Macrophages.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ SIGNIFICANCE STATEMENT: Peroxisome proliferator-activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.

A deep learning approach for successful big-bubble formation prediction in deep anterior lamellar keratoplasty.

The efficacy of deep learning in predicting successful big-bubble (SBB) formation during deep anterior lamellar keratoplasty (DALK) was evaluated. Medical records of patients undergoing DALK at the University of Cologne, Germany between March 2013 and July 2019 were retrospectively analyzed. Patients were divided into two groups: (1) SBB or (2) failed big-bubble (FBB). Preoperative images of anterior segment optical coherence tomography and corneal biometric values (corneal thickness, corneal curvature, and densitometry) were evaluated. A deep neural network model, Visual Geometry Group-16, was selected to test the validation data, evaluate the model, create a heat map image, and calculate the area under the curve (AUC). This pilot study included 46 patients overall (11 women, 35 men). SBBs were more common in keratoconus eyes (KC eyes) than in corneal opacifications of other etiologies (non KC eyes) (p = 0.006). The AUC was 0.746 (95% confidence interval [CI] 0.603-0.889). The determination success rate was 78.3% (18/23 eyes) (95% CI 56.3-92.5%) for SBB and 69.6% (16/23 eyes) (95% CI 47.1-86.8%) for FBB. This automated system demonstrates the potential of SBB prediction in DALK. Although KC eyes had a higher SBB rate, no other specific findings were found in the corneal biometric data.

Role of cAMP in the promotion of colorectal cancer cell growth by prostaglandin E2.

BACKGROUND: Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) reaction, stimulates the growth of colonic epithelial cells. It is inferred that the abrogation of prostaglandins' growth-promoting effects as a result of COX inhibition underlies the advantageous effects of non-steroidal anti-inflammatory drugs in colorectal carcinoma (CRC). Despite this appreciation, the underlying molecular mechanisms remain obscure since cell culture studies have yielded discrepant results regarding PGE2's mitogenicity. METHODS: We have employed several alternative approaches to score cell proliferation and apoptosis of 4 CRC cell lines exposed to PGE2 under various conditions. To investigate the role of cAMP in PGE2's functions, activation of the cAMP pathway was assessed at different levels (changes in cAMP levels and PKA activity) in cells subjected to specific manipulations including the use of specific inhibitors or prostanoid receptor-selective agonists/antagonists. RESULTS: Our data document that the dose-response curve to PGE2 is 'bell-shaped', with nano molar concentrations of PGE2 being more mitogenic than micro molar doses. Remarkably, mitogenicity inversely correlates with the ability of PGE2 doses to raise cAMP levels. Consistent with a major role for cAMP, cAMP raising agents and pertussis toxin revert the mitogenic response to PGE2. Accordingly, use of prostanoid receptor-selective agonists argues for the involvement of the EP3 receptor and serum deprivation of HT29 CRC cells specifically raises the levels of Gi-coupled EP3 splice variants. CONCLUSION: The present data indicate that the mitogenic action of low PGE2 doses in CRC cells is mediated via Gi-proteins, most likely through the EP3 receptor subtype, and is superimposed by a second, cAMP-dependent anti-proliferative effect at higher PGE2 doses. We discuss how these findings contribute to rationalize conflictive literature data on the proliferative action of PGE2.

Randomized placebo-controlled intervention with n-3 LC-PUFA-supplemented yoghurt: effects on circulating eicosanoids and cardiovascular risk factors.

BACKGROUND & AIMS: The study examined the value of n-3 LC-PUFA-enriched yogurt as means of improving cardiovascular health. DESIGN: Fifty three mildly hypertriacylglycerolemic subjects (TAG ≥ 1.7 mmol/L) participated in a randomized, placebo-controlled, double-blind, parallel designed study. The subjects consumed 1) control yoghurt; 2) yoghurt enriched with 0.8 g n-3 LC-PUFA/d; or 3) yoghurt enriched with 3 g n-3 LC-PUFA/d for a period of 10 wks. Blood samples were taken at the beginning and the end of the study period. RESULTS: Following daily intake of 3 g n-3 LC-PUFA for 10 weeks, n-3 LC-PUFA levels increased significantly in plasma and red blood cells (RBC) with concomitant increase in the EPA-derived mediators (PGE3, 12-, 15-, 18-HEPE) in plasma whilst cardiovascular risk factors such as HDL, TAG, AA/EPA ratio, and n-3 index were improved (P < 0.05); the decrease of TAG and increase in HDL were associated with the CD36 genotype. CONCLUSION: The observed increase of n-3 LC-PUFA in RBC and plasma lipids due to intake of n-3 LC-PUFA enriched yoghurt resulted in a reduction of cardiovascular risk factors and inflammatory mediators showing that daily consumption of n-3 PUFA enriched yoghurt can be an effective way of supplementing the daily diet and improving cardiovascular health.

Targeting PI3K in neuroblastoma.

PURPOSE: This work employs pharmacological targeting of phosphoinositide 3-kinases (PI3K) in selected neuroblastoma (NB) tumors with the inhibitor AS605240, which has been shown to express low toxicity and relative specificity for the PI3K species γ. METHODS: The expression pattern of PI3K isoforms in 7 NB cell lines and 14 tumor patient samples was determined by Western blotting and immunocytochemistry. The effect of AS605240 on the growth of four selected tumor cell lines was assessed. Two cell lines exhibiting (SK-N-LO) or lacking (SK-N-AS) PI3Kγ expression were chosen for further in vitro analysis, which involved propidium iodide (PI)-based cell cycle staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL-staining) of apoptotic cells and analysis of PI3K/Akt-related signaling pathways via Western blotting and translocation experiments. The action of AS605240 in vivo was addressed by xenograft experiments in severe combined immunodeficiency (SCID) mice, thereby comparing SK-N-LO and SK-N-AS derived tumors. Apoptosis induced in SK-N-LO tumors was shown by immunohistochemical TUNEL-staining. RESULTS: Significant expression of PI3Kγ in neuroblastoma patient biopsies and tumor cell lines was detected. AS605240 induced apoptosis in NB cell lines proportional to this expression and suppressed growth of PI3Kγ positive, but not negative, tumors in a xenograft mouse model. No adverse effects of the inhibitor treatment were observed. CONCLUSIONS: Our observations hint to an oncogenic function of PI3Kγ in distinct neuroblastoma entities and reveal PI3K targeting by AS605240 as a promising molecular therapy of these tumors.

Flow cytometric quantification of chlamydial infection in cell culture.

A flow cytometric method was developed, which allows fast and efficient analysis of cell cultures infected with chlamydiae. The proportion of positive cells increased with the infectious dose and correlated with chlamydia copy numbers calculated from real-time PCR. While retaining the advantages of single-cell analysis, flow cytometry allows handling of large sample numbers and counterstaining for additional marker proteins.

T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- bispecific single-chain antibody construct.

We have recently demonstrated that a recombinant single-chain bispecific Ab construct, bscCD19xCD3, in vitro induces rapid B lymphoma-directed cytotoxicity at picomolar concentrations with unstimulated peripheral T cells. In this study, we show that treatment of nonobese diabetic SCID mice with submicrogram doses of bscCD19xCD3 could prevent growth of s.c. human B lymphoma xenografts and essentially cured animals when given at an early tumor stage. The effect was dose dependent, dependent on E:T ratio and the time between tumor inoculation and administration of bscCD19xCD3. No therapeutic effect was seen in the presence of human lymphocytes alone, a vehicle control, or with a bispecific single-chain construct of identical T cell-binding activity but different target specificity. In a leukemic nonobese diabetic SCID mouse model, treatment with bscCD19xCD3 prolonged survival of mice in a dose-dependent fashion. The human lymphocytes used as effector cells in both animal models did not express detectable T cell activation markers at the time of coinoculation with tumor cells. The bispecific Ab therefore showed an in vivo activity comparable to that observed in cell culture with respect to high potency and T cell costimulus independence. These properties make bscCD19xCD3 superior to previously investigated CD19 bispecific Ab-based therapies.