Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

Background

T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.

Methods/ Findings

Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4⁺ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice.

Conclusions

The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.     

The effect of high dose digoxin on cytokines in healthy dogs

BACKGROUND: Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are pro-inflammatory cytokines, causing myocardial dysfunction and a negative inotropic effect. The drugs used to treat heart failure affect the production of cytokines. Digoxin, on which this study was focused, is one of the drugs for the treatment of heart failure. AIM: The present study was designed to examine the early effects of high doses of digoxin on the production of cytokines in healthy dogs. METHODS: Digoxin was given parenterally to dogs at 0.15 mg/kg. IL-1beta and TNF-alpha production and levels of digoxin in the serum were measured 0, 12, 24, 48, and 72 h following administration of digoxin. RESULTS: As the levels of serum digoxin taken at 12, 24, 48, and 72 h of administration were considered significantly high compared with preceding values (p < 0.001), no notable change in serum IL-1beta and TNF-alpha levels was observed. CONCLUSIONS: These results suggest that high doses of digoxin do not cause a significant cytokine production in heart muscle in the early phase.     

Takayasu's arteritis is associated with HLA-B*52, but not with HLA-B*51, in Turkey

Introduction

HLA-B*51 and HLA-B*52 are two close human leukocyte antigen (HLA) allele groups with minor amino acid differences. However, they are associated with two different vasculitides (HLA-B*51 in Behçet's disease and HLA-B*52 in Takayasu's arteritis (TAK)) and with major clinical and immunological differences. In this study, we aimed to screen a large cohort of TAK patients from Turkey for the presence of HLA-B*51 and HLA-B*52 as susceptibility and severity factors.

Methods

TAK patients (n = 330) followed at a total of 15 centers were included in the study. The mean age of the patients was 37.8 years, and 86% were women. DNA samples from the patients and healthy controls (HC; n = 210) were isolated, and the presence of HLA-B*51 or HLA-B*52 was screened for by using PCR with sequence-specific primers.

Results

We found a significant association of HLA-B*52 with TAK (20.9% vs HC = 6.7%, P = 0.000, OR = 3.7, 95% CI = 2.02 to 6.77). The distribution of HLA-B*51 did not differ between TAK patients and HCs (22.7% vs 24.8%, OR = 0.9, 95% CI = 0.60 to 1.34). The presence of HLA-B*52 decreased in late-onset patients (> 40 years of age; 12.0%, P = 0.024, OR = 0.43, 95% CI = 0.20 to 0.91). Patients with angiographic type I disease with limited aortic involvement also had a lower presence of HLA-B*52 compared to those with all other disease subtypes (13.1% vs 26%, P = 0.005, OR = 0.43, 95% CI = 0.23 to 0.78).

Conclusions

In this study, the previously reported association of TAK with HLA-B*52 in other populations was confirmed in patients from Turkey. The functional relevance of HLA-B*52 in TAK pathogenesis needs to be explored further.     

The a2b adenosine receptor modulates glucose homeostasis and obesity

Background

High fat diet and its induced changes in glucose homeostasis, inflammation and obesity continue to be an epidemic in developed countries. The A2b adenosine receptor (A2bAR) is known to regulate inflammation. We used a diet-induced obesity murine knockout model to investigate the role of this receptor in mediating metabolic homeostasis, and correlated our findings in obese patient samples.

Methodology/Principal Findings

Administration of high fat, high cholesterol diet (HFD) for sixteen weeks vastly upregulated the expression of the A2bAR in control mice, while A2bAR knockout (KO) mice under this diet developed greater obesity and hallmarks of type 2 diabetes (T2D), assessed by delayed glucose clearance and augmented insulin levels compared to matching control mice. We identified a novel link between the expression of A2bAR, insulin receptor substrate 2 (IRS-2), and insulin signaling, determined by Western blotting for IRS-2 and tissue Akt phosphorylation. The latter is impaired in tissues of A2bAR KO mice, along with a greater inflammatory state. Additional mechanisms involved include A2bAR regulation of SREBP-1 expression, a repressor of IRS-2. Importantly, pharmacological activation of the A2bAR by injection of the A2bAR ligand BAY 60-6583 for four weeks post HFD restores IRS-2 levels, and ameliorates T2D. Finally, in obese human subjects A2bAR expression correlates strongly with IRS-2 expression.

Conclusions/Significance

Our study identified the A2bAR as a significant regulator of HFD-induced hallmarks of T2D, thereby pointing to its therapeutic potential.     

Inducible Toll-like receptor and NF-kappaB regulatory pathway expression in human adipose tissue

Objective: Inflammatory activity in fat tissue has recently been implicated in mechanisms of insulin resistance and obesity‐related metabolic dysfunction. Toll‐like receptors (TLRs) play a key role in innate immune responses and recent studies implicate the TLR pathway in mechanisms of inflammation and atherosclerosis. The aim of this study was to examine differential TLR expression and function in human adipose tissue. Methods and Procedures: We biopsied subcutaneous abdominal fat from 16 obese subjects (age 39 ± 11 years, BMI 49 ± 14 kg/m²) and characterized TLR expression using quantitative real‐time PCR and confocal immunofluorescence imaging. In tissue culture, we stimulated isolated human adipocytes with Pam3CSK4 and lipopolysaccharide (LPS) (TLR2 and TLR4 agonists, respectively) and quantified TLR activity, interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) production, and nuclear factor‐κB (NF‐κB) p65 nuclear activation using real‐time PCR, enzyme‐linked immunosorbent assay (ELISA), and immunofluorescence. Results: TLR1, 2, and 4 protein colocalized with adiponectin in human adipocytes with TLR4 exhibiting the highest immunohistochemical expression. Using real‐time PCR, we confirmed higher level of gene expression for TLR4 as compared to other members of the TLR family (TLR1, 2, 7, 8) in human adipose depots (P < 0.001). In tissue culture, adipocyte TLR2/TLR4 mRNA expression and protein increased significantly following Pam3CSK4 and LPS (P < 0.001). TLR2/TLR4 stimulation was associated with NF‐κB p65 nuclear translocation and proinflammatory cytokine production. Discussion: The findings demonstrate that TLRs are inducible in adipose tissue and linked with downstream NF‐κB activation and cytokine release. Adipose stores may play a dynamic role in the regulation of inflammation and innate immunity in human subjects via modulation of the TLR/NF‐κB regulatory pathway.     

Middle eastern origin model forHomo sapiens (Moderns & Neanderthals), language and modern behaviour

A new model may resolve the problem of when and where did appear anatomically modern humans. According to this model, Neanderthals were probably neither our ancestor nor different species.Homo sapiens appeared probably in the Middle East, approximately 150 ka ago and differentiated to anatomically modern humans and Neanderthals because of the genetic programme. The fossils older than 150 ka are probably not Neanderthal such as Zuttiyeh and Biache-Saint-Vaast specimens. Cultural capacities of Neanderthals were probably equivalent to Moderns. Most of pre-Homo sapiens populations may be extinct without replacement byHomo sapiens. Language and modern behaviour should have arisen with our own species.     

NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation

AimsThe aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI).Main methodsNinety adult male Sprague–Dawley rats were divided into 3 groups (n = 30, each): control (laminectomy), SCI (hemisection at T12–T13 segments) and SCI + ENSC. Each group was further divided into sub-groups (n = 5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; l-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2 h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall–Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI.Key findingsTail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic l-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute l-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic l-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in l-arginine groups with and without ENSC transplantation.SignificanceThis study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.     

p27 Protein Protects Metabolically Stressed Cardiomyocytes from Apoptosis by Promoting Autophagy

p27^Kip1 (p27), a key regulator of cell division, has been implicated in autophagy of cancer cells. However, its role in autophagy, the evolutionarily conserved catabolic process that enables cells to remove unwanted proteins and damaged organelles, had not been examined in the heart. Here we report that ectopic delivery of a p27 fusion protein (TAT-p27) was sufficient to induce autophagy in neonatal rat ventricular cardiomyocytes in vitro, under basal conditions and after glucose deprivation. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels and suppressed basal and glucose-deprived levels of autophagy in cardiomyocytes in vitro. Glucose deprivation mimics myocardial ischemia and induces apoptosis in cardiomyocytes. During glucose deprivation, TAT-p27 inhibited apoptosis, whereas down-regulation of p27 decreased survival of cardiomyocytes. However, inhibition of autophagy by pharmacological (3-methyladenine, chloroquine, or bafilomycin A1) or genetic approaches (siRNA-mediated knockdown of Atg5) sensitized cardiomyocytes to glucose deprivation-induced apoptosis, even in the presence of TAT-p27. TAT-p27 was also able to provoke greater levels of autophagy in resting and fasting cardiomyocytes in vivo. Further, TAT-p27 enhanced autophagy and repressed cardiomyocytes apoptosis, improved cardiac function, and reduced infarct size following myocardial infarction. Again, these effects were lost when cardiac autophagy in vivo was blocked by chloroquine. Taken together, these data show that p27 positively regulates cardiac autophagy in vitro and in vivo, at rest and after metabolic stress, and that TAT-p27 inhibits apoptosis by promoting autophagy in glucose-deprived cardiomyocytes in vitro and in post-myocardial infarction hearts in vivo.     

An Adenosine Receptor-Krüppel-like Factor 4 Protein Axis Inhibits Adipogenesis

Adipogenesis represents a key process in adipose tissue development and remodeling, including during obesity. Exploring the regulation of adipogenesis by extracellular ligands is fundamental to our understanding of this process. Adenosine, an extracellular nucleoside signaling molecule found in adipose tissue depots, acts on adenosine receptors. Here we report that, among these receptors, the A2b adenosine receptor (A2bAR) is highly expressed in adipocyte progenitors. Activation of the A2bAR potently inhibits differentiation of mouse stromal vascular cells into adipocytes, whereas A2bAR knockdown stimulates adipogenesis. The A2bAR inhibits differentiation through a novel signaling cascade involving sustained expression of Krüppel-like factor 4 (KLF4), a regulator of stem cell maintenance. Knockdown of KLF4 ablates the ability of the A2bAR to inhibit differentiation. A2bAR activation also inhibits adipogenesis in a human primary preadipocyte culture system. We analyzed the A2bAR-KLF4 axis in adipose tissue of obese subjects and, intriguingly, found a strong correlation between A2bAR and KLF4 expression in both subcutaneous and visceral human fat. Hence, our study implicates the A2bAR as a regulator of adipocyte differentiation and the A2bAR-KLF4 axis as a potentially significant modulator of adipose biology.