Decreased Histone Deacetylase 2 (HDAC2) in Peripheral Blood Monocytes (PBMCs) of COPD Patients

Background

Histone deacetylase 2 (HDAC2) is a class I histone deacetylase family member that plays a critical role in suppressing inflammatory gene expression in the airways, lung parenchyma, and alveolar macrophages in patients with chronic obstructive pulmonary disease (COPD). However, the expression of HDAC2 in peripheral blood monocytes (PBMCs), nuclear factor kappa B (NF-κB) p65, and serum inflammatory cytokine levels in COPD patients, smokers, and non-smokers remains unclear.

Methods

PBMCs were obtained from COPD patients, healthy smokers, and healthy nonsmokers. The HDAC2 and NF-κB p65 expression were quantified by Western Blot. HDAC activity was assessed by an HDAC fluorometric immunoprecipitation activity assay kit. Serum tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) levels were measured by ELISA.

Results

HDAC2 expression and HDAC activity were decreased in PBMCs in COPD patients compared with smokers and non-smokers. Increased NF-κB p65 expression, serum TNF-α and IL-8 levels were observed in COPD patients compared with nonsmokers. The FEV₁%pred was positively correlated with HDAC2 expression and HDAC activity in COPD patients. Smokers had decreased HDAC activity, increased NF-κB p65 expression and serum TNF-α compared with nonsmokers.

Conclusions

HDAC2 expression was decreased in PBMCs of COPD patients and was correlated with disease severity. The reduction of HDAC2 expression not only directly enhances the expression of inflammatory genes, but may account for the activation of NF-κB mediated inflammation. Decreased HDAC2 may serve as a potential biomarker of COPD and predict the decline of lung function.     

Tim-4 Inhibits NO Generation by Murine Macrophages

Objective

T cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation.

Methods

Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot.

Results

Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages.

Conclusion

These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.     

Catecholaminergic Gene Polymorphisms Are Associated with GI Symptoms and Morphological Brain Changes in Irritable Bowel Syndrome

Background

Genetic and environmental factors contribute to the pathophysiology of irritable bowel syndrome (IBS). In particular, early adverse life events (EALs) and the catecholaminergic system have been implicated.

Aims

To investigate whether catecholaminergic SNPs with or without interacting with EALs are associated with: 1) a diagnosis of IBS, 2) IBS symptoms and 3) morphological alterations in brain regions associated with somatosensory, viscerosensory, and interoceptive processes.

Methods

In 277 IBS and 382 healthy control subjects (HCs), 11 SNPs in genes of the catecholaminergic signaling pathway were genotyped. A subset (121 IBS, 209 HCs) underwent structural brain imaging (magnetic resonance imaging [MRI]). Logistic and linear regressions evaluated each SNP separately and their interactions with EALs in predicting IBS and GI symptom severity, respectively. General linear models determined grey matter (GM) alterations from the SNPs and EALs that were predictive of IBS.

Results

1) Diagnosis: There were no statistically significant associations between the SNPs and IBS status with or without the interaction with EAL after adjusting for multiple comparisons. 2) Symptoms: GI symptom severity was associated with ADRA1D rs1556832 (P = 0.010). 3) Brain morphometry: In IBS, the homozygous genotype of the major ADRA1D allele was associated with GM increases in somatosensory regions (FDR q = 0.022), left precentral gyrus (q = 0.045), and right hippocampus (q = 0.009). In individuals with increasing sexual abuse scores, the ADRAβ2 SNP was associated with GM changes in the left posterior insula (q = 0.004) and left putamen volume (q = 0.029).

Conclusion

In IBS, catecholaminergic SNPs are associated with symptom severity and morphological changes in brain regions concerned with sensory processing and modulation and affect regulation. Thus, certain adrenergic receptor genes may facilitate or worsen IBS symptoms.     

Differentially Expressed in Chondrocytes 2 (DEC2) Increases the Expression of IL-1β and Is Abundantly Present in Synovial Membrane in Rheumatoid Arthritis

Objective

Patients with rheumatoid arthritis (RA) have altered circadian rhythm of circulating serum cortisol, melatonin and IL-6, as well as disturbance in the expression of clock genes ARNTL2 and NPAS2. In humans, TNFα increases the expression ARNTL2 and NPAS2 but paradoxically suppresses clock output genes DPB and PER3. Our objective was to investigate the expression of direct clock suppressors DEC1 and DEC2 (BHLHE 40 and 41 proteins) in response to TNFα and investigate their role during inflammation.

Methods

Cultured primary fibroblasts were stimulated with TNFα. Effects on DEC2 were studied using RT-qPCR and immunofluorescence staining. The role of NF-κB in DEC2 increase was analyzed using IKK-2 specific inhibitor IMD-0354. Cloned DEC2 was transfected into HEK293 cells to study its effects on gene expression. Transfections into primary human fibroblasts were used to confirm the results. The presence of DEC2 was analyzed in (RA) and osteoarthritis (OA) synovial membranes by immunohistochemistry.

Results

TNFα increased DEC2 mRNA and DEC2 was mainly detected at nuclei after the stimulus. The effects of TNFα on DEC2 expression were mediated via NF-κB. Overexpression, siRNA and promoter activity studies disclosed that DEC2 directly regulates IL-1β, in both HEK293 cells and primary human fibroblasts. DEC2 was increased in synovial membrane in RA compared to OA.

Conclusion

Not only ARNTL2 and NPAS2 but also DEC2 is regulated by TNFα in human fibroblasts. NF-κB mediates the effect on DEC2, which upregulates IL-1β. Circadian clock has a direct effect on inflammation in human fibroblasts.     

Irritable Bowel Syndrome Is Associated with an Increased Risk of Dementia: A Nationwide Population-Based Study

Purpose

Abnormal interaction in the brain–gut axis has emerged as one of the relevant pathophysiological mechanisms for the development of irritable bowel syndrome (IBS). Moreover, the brain–gut axis has recently been demonstrated to be crucial for the maintenance of cognitive performance. Therefore, we assessed the risk of dementia following diagnosis of IBS.

Methods

Using the Taiwan National Health Insurance Research Database (NHIRD) to obtain medical claims data from 2000 to 2011, we employed a random sampling method to enroll32 298 adult patients with IBS and frequency-matched them according to sex, age, and baseline year with 129 192 patients without IBS.

Results

The patients with IBS exhibited an increased risk of dementia [adjusted hazard ratio (aHR) = 1.26, 95% confidence interval (CI) = 1.17–1.35]after adjustment for age, sex, diabetes, hypertension, stroke, coronary artery disease (CAD), head injury, depression, and epilepsy, and the overall incidence of dementia for the cohorts with and without IBS was 4.86 and 3.41 per 1000 person-years, respectively. IBS was associated with an increased risk of dementia in patients older than 50 years in both male and female, and in those with comorbidity or without comorbidity. After adjustment for age, sex, and comorbidity, patients with IBS were also more likely to develop either non- Alzheimer’s disease (AD) dementia (aHR = 1.24, 95% CI = 1.15–1.33) or AD (aHR = 1.76, 95% CI = 1.28–2.43).

Conclusions

IBS is associated with an increased risk of dementia, and this effect is obvious only in patients who are ≥50 years old.     

Modulation of NF-κB/miR-21/PTEN Pathway Sensitizes Non-Small Cell Lung Cancer to Cisplatin

Background

Platinum-based chemotherapy is a standard strategy for non-small cell lung cancer (NSCLC), while chemoresistance remains a major therapeutic challenge in current clinical practice. Our present study was aimed to determine whether inhibition of the NF-κB/miR-21/PTEN pathway could increase the sensitivity of NSCLC to cisplatin.

Methods

The expression of miR-21 in NSCLC tissues was determined using in situ hybridization. Next, the effect of miR-21 on the sensitivity of A549 cells to cisplatin was determined in vitro. Whether miR-21 regulated PTEN expression was assessed by luciferase assay. Furthermore, whether NF-κB targeted its binding elements in the miR-21 gene promoter was determined by luciferase and ChIP assay. Finally, we measured the cell viability and apoptosis under cisplatin treatment when NF-κB was inhibited.

Results

An elevated level of miR-21 was observed in NSCLC lung tissues and was related to a short survival time. Exogenous miR-21 promoted cell survival when exposed to cisplatin, while miR-21 inhibition could reverse this process. The RNA and protein levels of PTEN were significantly decreased by exogenous miR-21, and the 3′-untranslated region of PTEN was shown to be a target of miR-21. The expression of miR-21 was regulated by NF-κB binding to its element in the promoter, a finding that was verified by luciferase and ChIP assay. Hence, inhibition of NF-κB by RNA silencing protects cells against cisplatin via decreasing miR-21 expression.

Conclusion

Modulation of the NF-κB/miR-21/PTEN pathway in NSCLC showed that inhibition of this pathway may increase cisplatin sensitivity.     

A20 Mutation Is Not a Prognostic Marker for Activated B-Cell-Like Diffuse Large B-Cell Lymphoma

Background

Constitutive activation of nuclear factor κB (NF-κB) is a hallmark of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Mutations in the A20 gene activate NF-κB, but the prognostic value of A20 mutations in ABC-DLBLC is unclear.

Purpose

To investigate the prognostic value of A20 mutation in ABC-DLBCL patients.

Methods

The somatic mutation of A20 was investigated in 68 de novo ABC-DLBCLs by PCR/sequencing. The Kaplan-Meier method was used to estimate median overall survival (OS) and progression-free survival (PFS).

Results

The A20 mutation rate in ABC-DLBCL patients was 29.4%. Complete remission rates were 35% and 45.8% in patients with and without A20 mutations, respectively (P = 0.410). In patients with and without A20 mutations, the median OS was 24.0 and 30.6 months, respectively (P = 0.58), and the median PFS was 15 and 17.4 months, respectively (P = 0.52). None of the differences between the patient groups were significant.

Conclusions

Our findings suggested that the A20 mutation is a frequent event in ABC-DLBCLs. However, there was no significant difference in PFS and OS in patients with or without A20 mutations. Further study is required to completely exclude A20 somatic mutation as a prognostic marker in the ABC subtype of DLBLC.     

Differential Relevance of NF-κB and JNK in the Pathophysiology of Hemorrhage/Resususcitation-Induced Liver Injury after Chronic Ethanol Feeding

Background

Chronic ethanol (EtOH) abuse worsens pathophysiological derangements after hemorrhagic shock and resuscitation (H/R) that induce hepatic injury and strong inflammatory changes via JNK and NF-κB activation. Inhibiting JNK with a cell-penetrating, protease-resistant peptide D-JNKI-1 after H/R in mice with healthy livers ameliorated these effects. Here, we studied if JNK inhibition by D-JNKI-1 in chronically EtOH-fed mice after hemorrhagic shock prior to the onset of resuscitation also confers protection.

Methods

Male mice were fed a Lieber-DeCarli diet containing EtOH or an isocaloric control (ctrl) diet for 4 weeks. Animals were hemorrhaged for 90 min (32 ± 2 mm Hg) and randomly received either D-JNKI-1 (11 mg/kg, intraperitoneally, i. p.) or sterile saline as vehicle (veh) immediately before the onset of resuscitation. Sham animals underwent surgical procedures without H/R and were either D-JNKI-1 or veh treated. Two hours after resuscitation, blood samples and liver tissue were harvested.

Results

H/R induced hepatic injury with increased systemic interleukin (IL)-6 levels, and enhanced local gene expression of NF-κB-controlled genes such as intercellular adhesion molecule (ICAM)-1 and matrix metallopeptidase (MMP)9. c-Jun and NF-κB phosphorylation were increased after H/R. These effects were further increased in EtOH-fed mice after H/R. D-JNKI-1 application inhibited the proinflammatory changes and reduced significantly hepatic injury after H/R in ctrl-fed mice. Moreover, D-JNKI-1 reduces in ctrl-fed mice the H/R-induced c-Jun and NF-κB phosphorylation. However, in chronically EtOH-fed mice, JNK inhibition did not prevent the H/R-induced hepatic damage and proinflammatory changes nor c-Jun and NF-κB phosphorylation after H/R.

Conclusions

These results indicate, that JNK inhibition is protective only in not pre-harmed liver after H/R. In contrast, the pronounced H/R-induced liver damage in mice being chronically fed with ethanol cannot be prevented by JNK inhibition after H/R and seems to be under the control of NF-κB.     

Rapamycin Inhibits Oxidized Low Density Lipoprotein Uptake in Human Umbilical Vein Endothelial Cells via mTOR/NF-κB/LOX-1 Pathway

Background

Lectin-like oxidized low-density lipoprotein-1 (LOX-1) is the major receptor for oxidized low density lipoprotein (ox-LDL) uptake in human umbilical vein endothelial cells (HUVECs). Previously, we found that rapamycin inhibited ox-LDL accumulation in HUVECs, and this effect was related to its role in increasing the activity of autophagy-lysosome pathway. In this study, we determined whether rapamycin could also reduce ox-LDL uptake in HUVECs and investigated the underlying signaling mechanisms.

Results

Flow cytometry and live cell imaging showed that rapamycin reduced Dil-ox-LDL accumulation in HUVECs. Furthermore, rapamycin reduced the ox-LDL-induced increase in LOX-1 mRNA and protein levels. Western blotting showed that rapamycin inhibited mechanistic target of rapamycin (mTOR), p70s6k and IκBα phosphorylation triggered by ox-LDL. Flow cytometry implied that mTOR, NF-κB knockdown and NF-κB inhibitors significantly reduced Dil-ox-LDL uptake. Moreover, immunofluorescent staining showed that rapamycin reduced the accumulation of p65 in the nucleus after ox-LDL treatment for 30 h. mTOR knockdown decreased LOX-1 protein production and IκBα phosphorylation induced by ox-LDL. NF-κB knockdown and NF-κB inhibitors reduced LOX-1 protein production, but did not inhibit mTOR phosphorylation stimulated by ox-LDL.

Conclusions

These findings demonstrate that rapamycin reduce mTOR phosphorylation and subsequently inhibit NF-κB activation and suppresses LOX-1, resulting in a reduction in ox-LDL uptake in HUVECs.     

Meta-Analysis Identifies NF-κB as a Therapeutic Target in Renal Cancer

Objective

To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes.

Methods

Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC.

Results

A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an ‘interferon signature’ may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis.

Conclusions

These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently-incurable malignancy.     

A Role for NF-κB Activity in Skin Hyperplasia and the Development of Keratoacanthomata in Mice

Background

Previous studies have implicated NF-κB signaling in both cutaneous development and oncogenesis. However, these studies have been limited in part by the lethality that results from extreme over- or under-expression of NF-κB in available mouse models. Even cre-driven tissue specific expression of transgenes, or targeted deletion of NF-κB can cause cell death. Therefore, the present study was undertaken to evaluate a novel mouse model of enhanced NF-κB activity in the skin.

Methods

A knock-in homologous recombination technique was utilized to develop a mouse model (referred to as PD mice) with increased NF-κB activity.

Results

The data show that increased NF-κB activity leads to hyperproliferation and dysplasia of the mouse epidermis. Chemical carcinogenesis in the context of enhanced NF-κB activity promotes the development of keratoacanthomata.

Conclusion

Our findings support an important role for NF-κB in keratinocyte dysplasia. We have found that enhanced NF-κB activity renders keratinocytes susceptible to hyperproliferation and keratoacanthoma (KA) development but is not sufficient for transformation and SCC development. We therefore propose that NF-κB activation in the absence of additional oncogenic events can promote TNF-dependent, actinic keratosis-like dysplasia and TNF-independent, KAs upon chemical carcinogensis. These studies suggest that resolution of KA cannot occur when NF-κB activation is constitutively enforced.     

Therapeutic Effects of Tangshen Formula on Diabetic Nephropathy in Rats

Objective

Inflammation and fibrosis are essential promoters in the pathogenesis of diabetic nephropathy (DN) in type 2 diabetes. The present study examined the anti-inflammation and anti-fibrosis effect of Tangshen Formula (TSF), a traditional Chinese medicine, on DN.

Research Design and Methods

Protective role of TSF in DN was examined in a rat model of type 2 DN that was established by high-fat diet-fed and low-dose-streptozotocin injection. TSF was suspended in 0.5% CMC-Na solution and delivered by oral gavage at a dosage of 1.67g/Kg body weight/day. The therapeutic effects and mechanisms of TSF on diabetic kidney injury were examined.

Results

We found that TSF treatment for 20 weeks attenuated DN by significantly inhibiting urinary excretion of albumin and renal histological injuries. These beneficial effects were associated with an inactivation of NF-κB signaling, thereby blocking the upregulation of pro-inflammatory cytokines (IL-1β, TNFα), chemokine (MCP-1), and macrophage infiltration in the TSF-treated rats with type 2 DN. In addition, TSF treatment also inactivated TGF-β/Smad3 signaling and therefore suppressed renal fibrosis including expressions of fibronectin, collagen I, and collagen IV. Further studies revealed that the inhibitory effect of TSF on TGF-β/Smad3 and NF-κB signaling in DN was associated with inhibition of Smurf2-dependent ubiquitin degradation of Smad7.

Conclusions

The present study reveals that TSF has therapeutic potential for type 2 DN in rats. Blockade of NF-κB-driven renal inflammation and TGF-β/Smad3-mediated renal fibrosis by preventing the Smurf2-mediated Smad7 degradation pathway may be mechanisms through which TSF inhibits type 2 DN.     

Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells

Background

Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine’s effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects.

Methods

Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis.

Results

The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-κB activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-κB inhibitor, Bay 11–7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11–7082 pretreatment reduced their expression.

Conclusions

Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-κB signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.     

Staple Foods Consumption and Irritable Bowel Syndrome in Japanese Adults: A Cross-Sectional Study

Background

Carbohydrates can cause gastrointestinal symptoms due to incomplete absorption in the small bowel. Thus, high-carbohydrate diets may induce symptoms of irritable bowel syndrome (IBS).

Objective

This observational and cross-sectional study assessed the association between consumption of several carbohydrate-enriched staple foods, such as rice, Japanese wheat noodles, Chinese noodles, bread, pasta, and buckwheat noodles, and the prevalence of IBS in Japanese adults.

Subjects and Methods

One thousand and eighty-two (837 men) Japanese adult employees aged 19-85 were included in this cross-sectional study conducted in 2011. IBS diagnosis was based on the Rome III criteria. Consumption of staple foods was assessed using a brief self-administered diet history questionnaire, and divided into three categories (low, middle, high) depending on their distribution.

Results

In the multivariate analysis, daily consumption of rice (odds ratios [ORs] and [95% confidence interval (CI)]: middle, 1.36 [0.93–1.99]; high, 1.67 [1.12–2.49]; P for trend = 0.01), bread (middle, 1.88 [1.28–2.75]; high, 1.63 [1.10–2.41]; P for trend = 0.01), pasta (middle, 1.47 [1.01–2.15]; high, 1.68 [1.12–2.52]; P for trend = 0.01), and buckwheat noodles (middle, 1.76 [1.18–2.61]; high, 1.98 [1.31–3.00]; P for trend = 0.001) were associated with higher prevalence of IBS after adjustment for socio-demographic, anthropometric, and lifestyle-related factors. Buckwheat noodles, but not other staple foods, retained an association with the prevalence of IBS even after adjustment for daily intake of carbohydrates or plant proteins.

Conclusions

This cross-sectional study demonstrated that the consumption of staple foods, such as rice, bread, pasta, and buckwheat noodles is associated with the prevalence of IBS. Of these, the consumption of buckwheat noodles, but not other staple foods, is associated with IBS independent of carbohydrate or plant protein contents.     

Extracts of Feijoa Inhibit Toll-Like Receptor 2 Signaling and Activate Autophagy Implicating a Role in Dietary Control of IBD

Background

Inflammatory bowel disease (IBD) is a heterogeneous chronic inflammatory disease affecting the gut with limited treatment success for its sufferers. This suggests the need for better understanding of the different subtypes of the disease as well as nutritional interventions to compliment current treatments. In this study we assess the ability of a hydrophilic feijoa fraction (F3) to modulate autophagy a process known to regulate inflammation, via TLR2 using IBD cell lines.

Method

Mouse embryonic fibroblasts (MEF) deleted for ATG5, and two intestinal epithelial cells HCT15 and HCT116, were used to test the anti-inflammatory effect of F3 after stimulating the cells with a TLR2 specific ligand PAM3CSK4.

Results

F3 was able to reduce TLR2 specific inflammation and stimulate autophagy in MEFs and HCT15 cells but not in HCT116 cells. The anti-inflammatory effect was reduced in the MEF cells deleted for ATG5. In addition, the activation of autophagy by F3 was enhanced by PAM3CSK4.

Conclusion

F3 of feijoa can interact with cells via a TLR2 specific mechanism and reduce Nuclear factor kappa B (NF-κB) activation in part due to stimulation of autophagy. These results suggest that there is potential benefit in using feijoa extracts as part of dietary interventions to manage IBD in patients.     

Cyclophilin A (CypA) Interacts with NF-κB Subunit,p65/RelA,and Contributes to NF-κB Activation Signaling

Background

Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects.

Methodology

Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes.

Results

GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-κB subunit p65 at the N-terminal 170–176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity.

Conclusions

Our findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant activation of NF-κB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases.     

Physical Activity Characteristics across GOLD Quadrants Depend on the Questionnaire Used

Background

The GOLD multidimensional classification of COPD severity combines the exacerbation risk with the symptom experience, for which 3 different questionnaires are permitted. This study investigated differences in physical activity (PA) in the different GOLD quadrants and patient’s distribution in relation to the questionnaire used.

Methods

136 COPD patients (58±21% FEV₁ predicted, 34F/102M) completed COPD assessment test (CAT), clinical COPD questionnaire (CCQ) and modified Medical Research Council (mMRC) questionnaire. Exacerbation history, spirometry and 6MWD were collected. PA was objectively measured for 2 periods of 1 week, 6 months apart, in 5 European centres; to minimise seasonal and clinical variation the average of these two periods was used for analysis.

Results

GOLD quadrants C+D had reduced PA compared with A+B (3824 [2976] vs. 5508 [4671] steps.d-1, p<0.0001). The choice of questionnaire yielded different patient distributions (agreement mMRC-CAT κ = 0.57; CCQ-mMRC κ = 0.71; CCQ-CAT κ = 0.72) with different clinical characteristics. PA was notably lower in patients with an mMRC score ≥2 (3430 [2537] vs. 5443 [3776] steps.d^-1, p <0.001) in both the low and high risk quadrants.

Conclusions

Using different questionnaires changes the patient distribution and results in different clinical characteristics. Therefore, standardization of the questionnaire used for classification is critical to allow comparison of different studies using this as an entry criterion.

Clinical Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01388218","term_id":"NCT01388218"}}NCT01388218     

Aberrant Expression of NF-κB in Liver Fluke Associated Cholangiocarcinoma: Implications for Targeted Therapy

Background

Up-regulation and association of nuclear factor kappa B (NF-κB) with carcinogenesis and tumor progression has been reported in several malignancies. In the current study, expression of NF-κB in cholangiocarcinoma (CCA) patient tissues and its clinical significance were determined. The possibility of using NF-κB as the therapeutic target of CCA was demonstrated.

Methodology

Expression of NF-κB in CCA patient tissues was determined using immunohistochemistry. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific NF-κB inhibitor, was used to inhibit NF-κB action. Cell growth was determined using an MTT assay, and cell apoptosis was shown by DNA fragmentation, flow cytometry and immunocytofluorescent staining. Effects of DHMEQ on growth and apoptosis were demonstrated in CCA cell lines and CCA-inoculated mice. DHMEQ-induced apoptosis in patient tissues using a histoculture drug response assay was quantified by TUNEL assay.

Principal Findings

Normal bile duct epithelia rarely expressed NF-κB (subunits p50, p52 and p65), whereas all CCA patient tissues (n  =  48) over-expressed all NF-κB subunits. Inhibiting NF-κB action by DHMEQ significantly inhibited growth of human CCA cell lines in a dose- and time-dependent manner. DHMEQ increased cell apoptosis by decreasing the anti-apoptotic protein expressions–Bcl-2, XIAP–and activating caspase pathway. DHMEQ effectively reduced tumor size in CCA-inoculated mice and induced cell apoptosis in primary histocultures of CCA patient tissues.

Conclusions

NF-κB was over-expressed in CCA tissues. Inhibition of NF-κB action significantly reduced cell growth and enhanced cell apoptosis. This study highlights NF-κB as a molecular target for CCA therapy.