β-arrestin 2 quenches TLR signaling to facilitate the immune evasion of EPEC

ABSTRACT

The protein translocated intimin receptor (Tir) from enteropathogenic Escherichia coli shares sequence similarity with the host cellular immunoreceptor tyrosine-based inhibition motifs (ITIMs). The ITIMs of Tir are required for Tir-mediated immune inhibition and evasion of host immune responses. However, the underlying molecular mechanism by which Tir regulates immune inhibition remains unclear. Here we demonstrated that β-arrestin 2, which is involved in the G-protein-coupled receptor (GPCR) signal pathway, interacted with Tir in an ITIM-dependent manner. For the molecular mechanism, we found that β-arrestin 2 enhanced the recruitment of SHP-1 to Tir. The recruited SHP-1 inhibited K63-linked ubiquitination of TRAF6 by dephosphorylating TRAF6 at Tyr288, and inhibited K63-linked ubiquitination and phosphorylation of TAK1 by dephosphorylating TAK1 at Tyr206, which cut off the downstream signal transduction and subsequent cytokine production. Moreover, the inhibitory effect of Tir on immune responses was diminished in β-arrestin 2-deficient mice and macrophages. These findings suggest that β-arrestin 2 is a key regulator in Tir-mediated immune evasion, which could serve as a new therapeutic target for bacterial infectious diseases.     

Shikonin-mediated PD-L1 degradation suppresses immune evasion in pancreatic cancer by inhibiting NF-κB/STAT3 and NF-κB/CSN5 signaling pathways

BackgroundPancreatic cancer (PC) is a highly fatal malignancy with few effective therapies currently available. Recent studies have shown that PD-L1 inhibitors could be potential therapeutic targets for the treatment of PC. The present study aims to investigate the effect of Shikonin on immune evasion in PC with the involvement of the PD-L1 degradation.MethodsInitially, the expression patterns of PD-L1 and NF-κB in PC were predicted in-silico using the GEPIA database, and were subsequently validated using PC tissues. Thereafter, the correlation of NF-κB with STAT3, CSN5 and PD-L1 was examined. PC cells were treated with Shikonin, NF-κB inhibitor, STAT3 activator, and CSN5 overexpression plasmid to investigate effects on PD-L1 glycosylation and immune evasion in PC. Finally, in vivo tumor formation was induced in C57BL/6J mice, in order to verify the in vitro results.ResultsPD-L1, NF-κB, NF-κB p65, STAT3, and CSN5 were highly expressed in PC samples, and NF-κB was positively correlated with STAT3/CSN5/PD-L1. Inhibition of NF-κB decreased PD-L1 glycosylation and increased PD-L1 degradation, whereas activated STAT3 and overexpressed CSN5 reversed these trends. Shikonin blocked immune evasion in PC, and lowered the expression of PD-L1, NF-κB, NF-κB p65, STAT3 and CSN5 in vivo and in vitro.ConclusionThe findings indicated Shikonin inhibited immune evasion in PC by inhibiting PD-L1 glycosylation and activating the NF-κB/STAT3 and NF-κB/CSN5 signaling pathways. These effects of Shikonin on PC cells may bear important potential therapeutic implications for the treatment of PC.     

TRAF3IP2 gene is associated with cutaneous extraintestinal manifestations in Inflammatory Bowel Disease

Background and aimsGenome-wide association (GWA) studies recently identified a novel gene, TRAF3IP2, involved in the susceptibility to psoriasis. Common immune-mediated mechanisms involving the skin or the gut have been suggested. We therefore aimed to assess the role of TRAF3IP2 gene in IBD, with particular regard to the development of cutaneous extraintestinal manifestations (pyoderma gangrenosum, erythema nodosum). The association with psoriasis was also assessed in a secondary analysis.MethodsThe analysis included 267 Crohn's disease (CD), 200 ulcerative colitis (UC) patients and 278 healthy controls. Three TRAF3IP2 SNPs were genotyped by allelic discrimination assays. A case/control association study and a genotype/phenotype correlation analysis have been performed.ResultsAll three SNPs conferred a high risk to develop cutaneous manifestations in IBD. A higher risk of pyoderma gangrenosum and erythema nodosum was observed in CD patients carrying the Rs33980500 variant (OR 3.03; P = 0.026). In UC, a significantly increased risk was observed for both the Rs13190932 and the Rs13196377 SNPs (OR 5.05; P = 0.02 and OR 4.1; P = 0.049). Moreover, association of TRAF3IP2 variants with ileal (OR = 1.92), fibrostricturing (OR = 1.91) and perianal CD (OR = 2.03) was observed.ConclusionsThis is the first preliminary report indicating that TRAF3IP2 variants increase the risk of cutaneous extraintestinal manifestations in IBD suggesting that the analysis of the TRAF3IP2 variants may be useful for identifying IBD patients at risk to develop these manifestations.     

Protective effects of edaravone on experimental chronic pancreatitis induced by dibutyltin dichloride in rats

Background/aimsTo investigate the effects of edaravone, a potent free radical scavenger, on dibutyltin dichloride (DBTC)-induced chronic pancreatitis (CP) and pancreatic fibrosis.MethodsMale Sprague–Dawley rats were randomly divided into four groups (n = 16 each): control, DBTC, DBTC + edaravone, and control + edaravone. Edaravone or normal saline at a daily dose of 6 mg/kg body weight was given intraperitoneally from day 5 to day 28 after DBTC administration. On days 14 and 28, the rats were evaluated morphologically and biochemically. The expression of cytokines in pancreas TGF-β, IL-6 and TNF<alpha> was detected using RT-PCR. The activation of nuclear factor (NF)-κB in pancreatic tissue was evaluated by immunostaining and western-blot for NF-κB p65. α-smooth muscle actin (α-SMA) expression was also evaluated by immunostaining and western-blot to investigate the activation of pancreatic stellate cells (PSCs).ResultEdaravone treatment improved the rats' body weight (p < 0.01) and feed intake levels (p < 0.05), improved the histological scores and alleviated the fibrosis of pancreas samples (p < 0.05), as well as markedly increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) concentrations in pancreatic tissue (p < 0.01 for both). The expression of cytokines TGF-β, IL-6 and TNF<alpha> in pancreas of DBTC group was also down-regulated by edaravone after treatment. Edaravone inhibited the activation of NF-κB and PSCs and exhibited protective effects on pancreatic tissue damage in CP.ConclusionsThis antioxidant may be a promising therapeutic intervention for human CP.     

Anti-inflammatory effects of fatty acids isolated from Chromolaena odorata

ObjectiveTo identify inhibitors of nitric oxide production and NF-κB activity from Chromolaena odorata (C. odorata).MethodsThe compounds isolated from the aerial parts of C. odorata by bioassay-guided fractionation were investigated for their inhibitory effects on the NO production and NF-κB activity in LPS-stimulated RAW264.7 cells.ResultsSix fatty acids (S)-coriolic acid (1), (S)-coriolic acid methyl ester (2), (S)-15,16-didehydrocoriolic acid (3), (S)-15,16-didehydrocoriolic acid methyl ester (4), linoleamide (5) and linolenamide (6) were isolated. All compounds inhibited the NO production at concentrations consistent with those required for NF-κB inhibition. Compound 2 was the most active with the IC₅₀ values of 5.22 and 5.73 μM. The addition of a double bond in the fatty chain decreased the inhibitory effects while the methyl esterification increased the activities.ConclusionsThe fatty acid components in C. odorata with NF-κB inhibitory activity could explain the anti-inflammation property of this plant in traditional medicine. This study could also contribute to the better use of C. odorata for human health care.     

Specific expression of k63-linked ubiquitination of calmodulin-like protein 5 in breast cancer of premenopausal patients

Purpose

Posttranslational modifications such as ubiquitination regulate many functions of proteins by affecting their interaction with other molecules, their activity, and their subcellular localization. In cancer biology, the ubiquitin network has gained major interest. K63-linked ubiquitination has emerged as a posttranslational modification with functional consequences, as it acts in several processes such as protein trafficking, DNA repair, and inflammation. Moreover, k63-linked ubiquitination is involved in the regulation of carcinogenesis. Based on previous findings, the aim of this study was to evaluate the ubiquitination of CALML5 in breast cancer patients.

Patients and methods

The breast cancer cell lines SkBr3, MCF7, HCC1937, and BT474 as well as 23 tumor samples of patients with primary breast cancer and the normal adjacent breast tissue were analyzed by one-dimensional immunoblot.

Results

Using specific antibodies against CALML5 and k63-linked ubiquitin, we demonstrate a k63-linked ubiquitination in the nuclear fraction of premenopausal breast cancer patients. K63-linked ubiquitination of CALML5 was found in breast cancer tissue, but not found in surrounding healthy tissue.

Conclusion

Our findings support the concept that ubiquitination of CALML5 in the nucleus is involved in the carcinogenesis of breast cancer in premenopausal women.     

Emergence of the A20/ABIN-mediated inhibition of NF-κB signaling via modifying the ubiquitinated proteins in a basal chordate

In the past decade, ubiquitination has been well documented to have multifaceted roles in regulating NF-κB activation in mammals. However, its function, especially how deubiquitinating enzymes balance the NF-κB activation, remains largely elusive in invertebrates. Investigating bbtA20 and its binding proteins, bbt A20-binding inhibitor of NF-κB (bbtABIN1) and bbtABIN2, in Chinese amphioxus Branchiostoma belcheri tsingtauense, we found that bbtABIN2 can colocalize and compete with bbt TNF receptor-associated factor 6 to connect the K63-linked polyubiquitin chains, whereas bbtABIN1 physically links bbtA20 to bbt NF-κB essential modulator (bbtNEMO) to facilitate the K48-linked ubiquitination of bbtNEMO. Similar to human A20, bbtA20 is a dual enzyme that removes the K63-linked polyubiquitin chains and builds the K48-linked polyubiquitin chains on bbt receptor-interacting serine/threonine protein kinase 1b, leading to the inhibition of NF-κB signaling. Our study not only suggests that ubiquitination is an ancient strategy in regulating NF-κB activation but also provides the first evidence, to our knowledge, for ABINs/A20-mediated inhibition of NF-κB via modifying the ubiquitinated proteins in a basal chordate, adding information on the stepwise development of vertebrate innate immune signaling.Studies emerging in the past decade have revealed multifaceted roles of ubiquitin in receptor trafficking, signal transduction, and DNA damage response (1). In particular, it has been reported to be instrumental in regulating several steps in NF-κB signaling (2). For instance, when Toll-like receptor (TLR) signaling is triggered, the ubiquitin ligase activity of TNF receptor-associated factor 6 (TRAF6) was activated, leading to K63-linked polyubiquitination of targets including NF-κB essential modulator (NEMO) and TRAF6 itself (2). Because ubiquitination is a reversible posttranslational modification of proteins, NF-κB signaling is generally attenuated by deubiquitinating enzymes (DUBs), which “trim” ubiquitin chains from specific substrate proteins (3).In mammals, one of the best-known DUBs involved in NF-κB signaling is A20 (also known as TNFAIP3), which belongs to the ovarian tumor (OTU) DUB family (4). A20 can restrict NF-κB signaling downstream of TNF receptor 1 (TNFR1), CD40, TLRs, NOD-like receptors, and the IL-1 receptor (5–8). Indeed, A20 is not only able to deubiquitinate substrates modified by K63-linked polyubiquitins, but can also induce their K48-linked polyubiquitination and degradation through its E3 activity. For example, A20 may regulate NF-κB activation by cleaving K63-linked polyubiquitin chains from receptor-interacting serine/threonine protein kinase 1 (RIP1), TRAF6, and/or NEMO through its deubiquitination activity dependent on its OTU domain (6, 7). Meanwhile, it also builds K48-linked polyubiquitin chains on RIP1 (7). Moreover, A20 can regulate NF-κB activation by supporting the degradation of the E2 enzymes UbcH5 and Ubc13, thereby inhibiting E3 activity that is dependent on these enzymes (8). Therefore, A20 interfaces with, and modifies, ubiquitinated protein substrates in multiple ways and acts through a variety of biochemical mechanisms to regulate NF-κB signals.Although A20 can interact with TRAF2 and NEMO, some studies have shown that these interactions are not sufficient for the inhibition of NF-κB, leading to the identification of a gene family called A20-binding inhibitor of NF-κB (ABIN) (9). The ABIN family comprises three members, ABIN-1, ABIN-2, and ABIN-3. All of them share a conserved ubiquitin-binding domain (UBD) and are characterized as negative regulators of NF-κB signaling when overexpressed (10). For example, overexpressed ABIN-1 physically links A20 to NEMO to facilitate deubiquitination of NEMO (11). ABIN-2 blocks the interaction between RIP1 and NEMO, thus resulting in inhibition of NF-κB (12). ABIN-3 negatively regulates the LPS-induced NF-κB activation downstream of TRAF6 and upstream of the IκB kinase (IKK) (13).Several lines of evidence have suggested that the K63-linked polyubiquitination is also crucial for the activation of Drosophila Relish (14). The ubiquitin chains in Drosophila immune deficiency (IMD) and caspase 8 homolog DREDD serve as scaffolds for the recruitment of TGF-β–activated kinase 1 (dTAK1) and dIKK complex, allowing DREDD-mediated proteolysis of Relish and the expression of Relish-dependent antimicrobial peptide genes (15, 16). Although homologs of cylindromatosis and ubiquitin-specific protease 36, two other important DUBs in mammalian NF-κB signaling, have been found to deubiquitinate dTRAF2 and dIMD, likely serving as a switch to deactivate the IMD pathway (17, 18), no A20 or ABINs have been reported in Drosophila and other invertebrates. Therefore, identifying the A20 and ABIN homologs and characterizing their roles in ubiquitination in the basal chordate amphioxus will help us not only to understand when and in what ways the ABINs and A20 appeared in classical NF-κB signaling, but also to characterize the inactivation of NF-κB by DUBs in invertebrates.     

Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis

Abstract: We explored anti‐inflammatory potential of melatonin against the lipopolysaccharide (LPS)‐induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment with melatonin or vehicle at various time intervals. In a mouse model of meningitis induced by LPS, melatonin (5 mg/kg) or vehicle was intravenously injected at 30 min postinsult. The activity of matrix metalloproteinase‐2 (MMP‐2) and metalloproteinase‐9 (MMP‐9) was determined by gelatin zymography. Nuclear factor‐kappa B (NFκB) translocation and binding activity were determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA). Our results showed that either pretreatment or cotreatment with melatonin at 50–500 μm effectively inhibited the LPS‐induced proMMP‐9 activation in the RAW 264.7 and BV2 cells, respectively (P < 0.05). This melatonin‐induced proMMP‐9 inhibition remained effective when treatment was delayed up to 2 and 6 hr postinsult for RAW 264.7 and BV2 cells, respectively (P < 0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP‐9 activity, respectively (P < 0.05) and reduced the loss of body weight (P < 0.05) in mice with meningitis. Moreover, melatonin (50 μm ) effectively inhibited nuclear factor‐kappa B (NFκB) translocation and binding activity in the LPS‐treated RAW 264.7 and BV2 cells, respectively (P < 0.05). These results demonstrate direct inhibitory actions of melatonin against postinflammatory NFκB translocation and MMP‐9 activation and highlight its ability to inhibit systemic and cerebral MMP‐9 activation following brain inflammation.     

Fisetin mitigates hepatic ischemia-reperfusion injury by regulating GSK3β/AMPK/NLRP3 inflammasome pathway

Background: Hepatic ischemia-reperfusion(I/R) injury(IRI) represents a crucial challenge in liver transplantation. Fisetin has anti-inflammatory, anti-aging and anti-oxidative properties. This study aimed to examine whether fisetin mitigates hepatic IRI and examine its underlying mechanisms. Methods: Sham or warm hepatic I/R operated mice were pretreated with fisetin(5, 10 or 20 mg/kg). Hepatic histological assessments, TUNEL assays and serum aminotransferase measurements were performed. An in vitro hypoxia/reoxygenation(H/R) model using RAW264.7 macrophages pretreated with fisetin(2.5, 5 or 10 μmol/L) was also used. Serum and cell supernatant concentrations of interleukin-1 β(IL-1 β), IL-18 and tumor necrosis factor-α(TNF-α) were determined by enzyme-linked immunosorbent assay(ELISA). Protein levels of p-GSK3 β, p-AMPK and NLR family pyrin domain-containing 3(NLRP3)-associated proteins were detected by Western blotting. Results: Compared with the I/R group, fisetin pretreatment reduced pathological liver damage, serum aminotransferase levels, serum concentrations of IL-1 β, IL-18 and TNF-α in the murine IRI model. Fisetin also reduced the expression of NLRP3 inflammasome-associated proteins(NLRP3, cleaved caspase-1, IL-1 β and IL-18) in I/R-operated liver. The experiments in vitro showed that fisetin decreased the release of IL-1 β, IL-18 and TNF-α, and reduced the expression of NLRP3 inflammasome-associated proteins in H/R-treated RAW264.7 cells. Moreover, fisetin increased the expressions of p-GSK3 β and p-AMPK in both models, indicating that its anti-inflammatory effects were dependent on GSK3 β/AMPK signaling. The antiinflammatory effects of fisetin were partially inhibited by the AMPK specific inhibitor compound C. Conclusions: Fisetin showed protective effects against hepatic IRI, countering inflammatory responses through mediating the GSK3 β/AMPK/NLRP3 inflammasome pathway.     

Melatonin attenuates hepatic ischemia-reperfusion injury in rats by inhibiting NF-κB signaling pathway

BackgroundThe sterile inflammatory response is one of the key mechanisms leading to hepatic ischemia-reperfusion injury. Melatonin has been shown to prevent organ injuries, but its roles in the inflammatory response after hepatic ischemia-reperfusion injury have not been fully explored, especially in late ischemia-reperfusion injury. The present study aimed to investigate the roles and possible mechanisms of melatonin in the inflammatory response after hepatic ischemia-reperfusion injury.MethodsSixty Sprague-Dawley rats were randomly divided into a sham group, ischemia-reperfusion injury group (I/R group), and melatonin-treated group (M + I/R group). The rats in the I/R group were subjected to 70% hepatic ischemia for 45 min, followed by 5 or 24 h of reperfusion. The rats in the M + I/R group were injected with melatonin (10 mg/kg, intravenous injection) 15 min prior to ischemia and immediately before reperfusion. Serum and samples of ischemic liver lobes were harvested for future analysis, and the 7-day survival rate was assessed after hepatic ischemia-reperfusion surgery.ResultsIn comparison with the I/R group, the M + I/R group showed markedly decreased expression levels of inflammatory cytokines (IL-6 and TNF-α) and numbers of apoptotic hepatocytes (P < 0.05). Immunoblotting showed that the expression levels of IL-6, p-NF-κBp65/t-NF-κBp65 and p-IκB-α/t-IκB-α in the M + I/R group were significantly lower than those in the I/R group, and immunofluorescence staining showed that the expression level of p-NF-κBp65 in the M + I/R group was lower than that in the I/R group (P < 0.05). The 7-day survival rates were 20% in the I/R group and 50% in the M + I/R group (P < 0.05).ConclusionsMelatonin downregulated the activity of the NF-κB signaling pathway in the early and late stages of hepatic ischemia-reperfusion injury, alleviated the inflammatory response, protected the liver from ischemia-reperfusion injury, and increased the survival rate.     

Cocoa consumption reduces NF-κB activation in peripheral blood mononuclear cells in humans

Background and aimsEpidemiological studies have demonstrated an association between high-polyphenol intake and reduced incidence of atherosclerosis. The healthy effects of cocoa-polyphenols may be due to their antioxidant and anti-inflammatory actions, although the exact mechanisms are unknown and depend on the matrix in which cocoa-polyphenols are delivered. Nuclear factor κB (NF-κB) is a key molecule in the pathophysiology of atherosclerosis involved in the regulation of adhesion molecules(AM) and cytokine expression and its activation is the first step in triggering the inflammatory process. The aim of this study was to evaluate the effect of acute cocoa consumption in different matrices related to the bioavailability of cocoa-polyphenols in NF-κB activation and the expression of AM.Methods and resultsEighteen healthy volunteers randomly received 3 interventions: 40g of cocoa powder with milk (CM), with water (CW), and only milk (M). NF-κB activation in leukocytes and AM (sICAM, sVCAM, E-selectin) were measured before and 6h after each intervention. Consumption of CW significantly decreased NF-κB activation compared to baseline and to CM (P < 0.05, both), did not change after CM intervention, and significantly increased after M intervention (P = 0.014). sICAM-1 concentrations significantly decreased after 6h of CW and CM interventions (P ≤ 0.026; both) and E-selectin only decreased after CW intervention (P = 0.028). No significant changes were observed in sVCAM-1 concentrations.ConclusionsThe anti-inflammatory effect of cocoa intake may depend on the bioavailability of bioactive compounds and may be mediated at least in part by the modulation of NF-κB activation and downstream molecules reinforcing the link between cocoa intake and health.     

Prevalence and characteristics of anti-HCV positivity and chronic hepatitis C virus infection in HFE p.C282Y homozygotes

Introduction and aimObservations of hepatitis C virus (HCV) infection in adults with hemochromatosis are limited.Materials and methodsWe determined associations of serum ferritin (SF) with anti-HCV in non-Hispanic white North American adults in a post-screening examination. Cases included p.C282Y homozygotes (regardless of screening transferrin saturation (TS) and SF) and participants (regardless of HFE genotype) with high screening TS/SF. Controls included participants without p.C282Y or p.H63D who had normal screening TS/SF. Participants with elevated alanine aminotransferase underwent anti-HCV testing. We determined prevalence of chronic HCV infection in consecutive Alabama and Ontario referred adults with HFE p.C282Y homozygosity.ResultsIn post-screening participants, anti-HCV prevalence was 0.3% [95% CI: 0.02, 2.2] in 294 p.C282Y homozygotes, 9.5% [7.2, 12.3] in 560 Cases without p.C282Y homozygosity, and 0.7% [0.2, 2.3] in 403 Controls. Anti-HCV was detected in 7.2% of 745 participants with and 0.8% of 512 participants without elevated SF (odds ratio 9.9 [3.6, 27.6]; p < 0.0001). Chronic HCV infection prevalence in 961 referred patients was 1.0% (10/961) [95% confidence interval (CI): 0.5, 2.0]. Ten patients with chronic HCV infection had median age 45 y (range 29–67) and median SF 1163 μg/L (range 303–2001). Five of eight (62.5%) patients had biopsy-proven cirrhosis.ConclusionsOdds ratio of anti-HCV was increased in post-screening participants with elevated SF. Prevalence of anti-HCV in post-screening participants with HFE p.C282Y homozygosity and chronic HCV infection in referred adults with HFE p.C282Y homozygosity in North America is similar to that of Control participants with HFE wt/wt and normal screening TS/SF.     

Patients with ulcerative colitis responding to steroid treatment up-regulate glucocorticoid receptor levels in colorectal mucosa

Background and aimsGlucocorticosteroid treatment (GCS) is effective for attacks of ulcerative colitis (UC). However, 25–30% of patients fails to respond and may be considered steroid resistant. Glucocorticoid receptors (GR) mediate the effects of GCS. Colorectal mucosa levels of GR and NF-κB were analysed before, during and after treatment with GCS-compounds.MethodsPatients with moderate–severe attacks of ulcerative colitis were included. Patients undergoing colonoscopy with normal finding served as controls. GR and NF-κB levels in colorectal mucosa were analysed by Western Blotting and the DNA-binding activity of NF-κB by EMSA.ResultsTwenty-eight patients and seven controls were included. Ten patients were judged clinically steroid resistant.Responders had significantly higher levels of GR in colorectal mucosa after one week of treatment than non-responders (P = 0.039) and significantly higher levels of GR were found in responders in remission as compared to before treatment (P = 0.013). NF-κB levels did not differ between the groups at first visit. Increasing levels were found only in responders as remission was obtained (P = 0.031). EMSA detected 20% lower DNA-binding of NF-κB in responders in remission as compared to first visit (P = 0.021).ConclusionGR levels increase in UC-patients responding to GCS-therapy but not in steroid resistant patients and may be the reason for the lack of steroid-efficacy. Increasing NF-κB levels were found in responders attaining remission, possibly reflecting a lower turnover. A decrease in DNA-binding of NF-κB was found in these patients, perhaps because of the increased GR levels counteracting NF-κB activity.     

Prospective blinded Evaluation of the smartphone-based AliveCor Kardia ECG monitor for Atrial Fibrillation detection: The PEAK-AF study

IntroductionThe AliveCor Kardia ECG monitor (ACK) offers a smartphone-based one-lead ECG recording for the detection of atrial fibrillation. We compared ACK lead I recordings with the 12-lead ECG and introduce a novel parasternal lead (NPL).MethodsConsecutive cardiac inpatients were recruited. In all patients a 12-lead ECG, ACK lead I and NPL were obtained. Two experienced electrophysiologists were blinded and separately evaluated all ECG. We calculated sensitivity, specificity, and predictive values of the ACK ECG compared to the 12-lead ECG.Results296 ECG from 99 patients (38 female, age 64 ± 15 years, BMI 27.8 ± 5.1 kg/m²) were analyzed. 20% of ACK lead I recordings contained a critical amount of artifact. The electrophysiologists’ interpretation of the ACK recordings yielded a sensitivity of 100% and specificity of 94% for atrial fibrillation or flutter in lead I (κ = 0.90) and a sensitivity of 96% and specificity of 97% in the NPL (κ = 0.92). The ACK diagnostic algorithm displayed a significantly lower sensitivity (55–70%), specificity (60–69%), and accuracy (κ = 0.4–0.53) but a high negative predictive value (100%). Patients with atrial flutter (n = 5) and with ventricular stimulation (n = 12) had a high likelihood of being misclassified by the algorithm.ConclusionThe AliveCor Kardia ECG monitor allows a highly accurate detection of atrial fibrillation by an interpreting electrophysiologist both in the standard lead I and a novel parasternal lead. The diagnostic algorithm offered by the system may be useful in screening recordings for further review. Diagnostic challenges present in atrial flutter and ventricular pacemaker stimulation.     

Th2 immune response by the iron-regulated protein HupB of Mycobacterium tuberculosis

BackgroundHupB is an iron-regulated protein essential for the growth of Mycobacterium tuberculosis inside macrophages. To investigate if HupB induced a dominant Th2 type immune response, we studied the effect of rHupB on PBMCs from TB patients and by infecting mouse macrophages with wild type and hupB KO mutants.MethodsPBMCs from pulmonary TB (n = 60), extra pulmonary TB (n = 23) and healthy controls (n = 30) were stimulated with purified HupB and the cytokines secreted were assayed. The sera were screened for anti-HupB antibodies by ELISA. Mouse macrophages cell line (RAW 264.7) was infected with wild type, hupB KO and hupB-complemented strains of M. tuberculosis grown in high and low iron medium and the expression of cytokines was assayed by qRT-PCR.ResultsMurine macrophages infected with the hupB KO strain produced low levels of the pro-inflammatory cytokines IFN-γ, TNF-α, IL-1, and IL-18 and high levels of IL-10. HupB induced IL-6 and IL-10 production in PBMCs of TB patients and down-regulated IFN-γ and TNF-α production. The influence of HupB was remarkable in the EPTB group.ConclusionHupB shifted the immune response to the Th2 type. Low IFN-γ and elevated IL-10 in EPTB patients is noteworthy.     

Prospective comparative study between two first-line regimens for Helicobacter pylori eradication: Non-bismuth quadruple versus bismuth quadruple therapy

BackgroundThe Maastricht V Consensus recommends quadruple therapies as first-line Helicobacter pylori treatment in high clarithromycin (CLA) resistance areas.AimsTo compare efficacy, side effects and compliance between quadruple concomitant non-bismuth vs bismuth quadruple therapy.MethodProspective study enrolling H. pylori-positive patients. Omeprazol and a three-in-one formulation of bismuth–metronidazol–tetracycline (OBMT-3/1) for 10 days, or combination of omeprazol–clarithromycin–amoxicillin–metronidazol (OCAM) for 14 days, were prescribed. Eradication outcome was assessed by urea breath test or histology. Side effects and compliance were recorded during the treatment period with specific questionnaires.Results404 patients were recruited (median age 53 years; 62.87% women). In 382 (183 with OCAM, 199 with OBMT-3/1) the post-treatment test result was available. The eradication rates were 85.94% (CI95%: 80.20–90.52) with OCAM and 88.21% (CI95%: 83.09–92.22) with OBMT-3/1 (p = 0.595) in intention-to-treat analysis, whilst in per protocol analysis they were 91.12% (CI95%: 85.78–94.95) and 96.17% (CI95%: 92.28–98.45) respectively (p = 0.083). Compliance over 90% was 91.35% with OCAM and 92.04% with OBMT-3/1 (p = 0.951). Some side effect was present in 94.02% with OCAM and in 88.89% with OBMT-3/1 (p = 0.109), being longer (12 vs 7 days, p < 0.0001) and more severe (p < 0.0001) with OCAM.ConclusionsIn a high CLA-resistance area, there are no differences between OBMT-3/1 and OCAM in H. pylori eradication and compliance rates, but OBMT-3/1 achieves a higher safety profile.     

Inflammatory mediator release by Brugia malayi from macrophages of susceptible host Mastomys coucha and THP-1 and RAW 264.7 cell lines

ObjectiveTo investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.MethodsThe human macrophage/monocyte cell line THP-1, the mouse macrophage cell line RAW 264.7 and naive peritoneal macrophages (PM) from the rodent host Mastomys coucha (M. coucha) were incubated at 37 °C in 5% CO₂ atmosphere with extracts of microfilariae (Mf), third stage infective larvae (L₃) and adult worms (Ad) of Brugia malayi. After 48 hr post exposure, IL-1β, IL-6, TNF-α, IL-10 and nitric oxide (NO) in cell-free supernatants were estimated.ResultsExtracts of all the life stages of the parasite were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines in both the cell lines and peritoneal macrophages of M. coucha. Mf was the strongest stimulator of pro-inflammatory cytokines followed by L₃ and Ad; however, Ad was a strong stimulator of IL-10 release. Mf was found to have potential to modulate LPS-induced NO release in RAW cells. Ad-induced NO release was concentration dependent with maximum at 20 μg/mL in both RAW and PMs.ConclusionsThe results show that parasites at all life stages were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines and NO release from macrophages of susceptible host M. coucha, human and mouse macrophage cell lines. Mf can suppress the LPS-induced NO release in RAW cells. The findings also show that the two cell lines may provide a convenient in vitro system for assaying parasite-induced inflammatory mediator release.     

Long-term follow-up of pepsinogen I/II ratio after eradication of Helicobacter pylori in patients who underwent endoscopic mucosal resection for gastric cancer

BackgroundAlthough the pepsinogen I/II (PGI/II) ratio after Helicobacter pylori eradication is recovered at short-term follow-up, long-term follow-up studies of PGI/II are rare.MethodsA total of 773 patients with gastric cancer who underwent endoscopic resection and pepsinogen and H. pylori tests were enrolled. H. pylori was eradicated in these patients. Endoscopic and pepsinogen tests were performed every year. A low PGI/II ratio was defined as ≤3.ResultsThe PGI/II ratio was higher in non-infected patients (n = 275, 4.99) than infected patients (n = 498, 3.53). After H. pylori eradication, the PGI/II ratio increased to 5.81 and 5.63 after 1 and 2 years (each p < 0.05). The PGI/II ratio in the non-eradication group decreased to 3.94 and 2.75 after 1 and 2 years. The PGI/II ratio in the H. pylori eradication group became similar to that of the H. pylori-negative group at 3 (4.48 vs. 4.34), 4 (4.88 vs. 4.34), and 5 years (4.89 vs. 4.23). The adjusted odds ratios for a lower PG I/II ratio in the non-eradication group compared to the eradication group were 4.78 (95% CI 2.15–10.67) after 1 year and 8.13 (95% CI 2.56–25.83) after 2 years.ConclusionsAfter H. pylori eradication, the PGI/II ratio increased and was similar to that of H. pylori-negative controls for up to 5 years of follow-up.