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Low levels of IgM antibodies recognizing oxidation-specific epitopes are associated with human non-alcoholic fatty liver disease
Tim Hendrikx1,2,3,
Martin L. Watzenböck2,3,
Sofie M. A. Walenbergh1,
Shahzada Amir2,3,
Sabrina Gruber2,3,
Maria Ozsvar Kozma2,3,
Heike I. Grabsch1,
Ger H. Koek1,
Marieke J. Pierik1,
Katharina Staufer2,
Michael Trauner2,
Satish C. Kalhan4,
Daisy Jonkers1,
Marten H. Hofker5,
Christoph J. Binder2,3 &
Ronit Shiri-Sverdlov1
Lipid oxidation of membrane phospholipids is accompanied by the formation of oxidation-specific epitopes (OSE). These epitopes are recognized by specific antibodies and represent danger-associated molecular patterns that are generated during chronic inflammatory processes. In a murine model for hepatic inflammation during non-alcoholic fatty liver disease (NAFLD), increased antibody levels targeting OSE were found to be protective. Here, our aim was to determine an association between OSE-specific antibody titers and NAFLD in humans.
IgM and IgG levels with specificity for various OSE were assessed in the plasma of patients with NAFLD (n = 71) and healthy controls (n = 68). Antibody titers were comprehensively analyzed in patients with NAFLD after classification by histological evaluation of liver biopsies. Statistical analysis was performed to determine significant correlations and odds ratios. To study the specificity for NAFLD, plasma antibody titers were measured in patients with hepatitis C (n = 40) and inflammatory bowel disease (n = 62).
IgM titers against OSE were lower in patients with NAFLD compared to controls. Further biopsy-based classification of patients with NAFLD did not show any difference in IgM levels. Plasma IgM titers towards the P1 mimotope demonstrated an inverse correlation with markers for obesity, systemic inflammation, and liver damage. In contrast, hepatitis C and increased disease activity during inflammatory bowel disease was not associated with reduced IgM titers.
Our data highlight the importance of immune recognition of OSE by IgM antibodies in the pathophysiology of NAFLD.
Non-alcoholic fatty liver disease (NAFLD) constitutes a spectrum of liver diseases characterized by hepatic lipid accumulation (steatosis), which when combined with hepatic inflammation is known as non-alcoholic steatohepatitis (NASH). Steatosis is generally considered to be a benign and reversible condition that is present in about two thirds of patients with the metabolic syndrome [1]. The presence of inflammation in a fatty liver is thought to drive disease progression, including the development of fibrosis, cirrhosis, or hepatocellular carcinoma, ultimately requiring liver transplantation [2]. The mechanisms leading to hepatic inflammation are currently not fully understood. Hence, therapy options are limited and accurate, early, non-invasive diagnostic tools are lacking [3].
Immune reactions triggered by oxidative stress have been shown to be involved during the progression of NAFLD to fibrosis [4]. Moreover, oxidized low-density lipoproteins (OxLDL) are known to play a major role in atherosclerosis and various other related metabolic disturbances, such as NASH [5]. The oxidation of LDL is accompanied by the formation of various epitopes on the surface of OxLDL, called oxidation-specific epitopes (OSE) [6]. These epitopes include protein adducts with lipid peroxidation breakdown products, such as malondialdehyde (MDA), malondialdehyde-acetaldehyde (MAA), and 4-hydroxynonenal, as well as with phosphocholine (PC)-containing oxidized phospholipids [6]. OSE on the surface of OxLDL are recognized by both innate and acquired humoral immunity, including specific antibodies that are found in the plasma of humans and animal models [6, 7]. Evidence for a protective role of OSE-specific immunoglobulin M (IgM) antibodies in NASH is based on our findings that increased IgM levels protect from hepatic inflammation in hyperlipidemic mice [8]. To summarize, immunized Ldlr−/− mice with heat-inactivated pneumococci, which induce the production of anti-OxLDL IgM due to molecular mimicry with OxLDL, had less hepatic inflammation than control mice after a Western diet. Moreover, we recently showed that increased levels of IgM antibodies as a result of Siglec-G deficiency inhibits diet-induced hepatic inflammation and atherosclerosis in Ldlr−/− mice [9]. However, the relationship between plasma antibody levels targeting OSE and hepatic inflammation during NAFLD has not been investigated in humans so far.
Here, we aimed to determine an association between plasma antibodies targeting OSE and NAFLD in humans. For this purpose, IgM and IgG antibody titers against different model epitopes of oxidized lipids were measured in the plasma of patients with NAFLD and compared to those in control subjects. Liver biopsies from patients with biopsy-proven NASH were examined for the presence of MDA epitopes by immunohistochemistry. Additionally, to test the specificity of our findings for NAFLD, antibody levels were determined in two cohorts consisting of patients with hepatitis C and inflammatory bowel disease (IBD). Our data highlight the importance of immune recognition of OSE by IgM antibodies in NAFLD and suggest that low IgM levels against end products of lipid oxidation during fatty liver disease are a consequence of obesity.
Antigens and antibodies
Human MDA-LDL, MAA-LDL, CuOx-LDL, and PC-BSA were prepared as previously described [10]. MDA2 (a kind gift from Dr J. L. Witztum, San Diego, CA, USA) is a murine IgG monoclonal antibody against MDA-lysine epitopes. Linear peptide P1 (HSWTNSWMATFL) was purchased from Peptide 2.0 Inc. (Chantilly, VA, USA).
Immunohistochemistry for MDA using the monoclonal antibody MDA2 was performed on 10 liver biopsies diagnosed as NASH, originating from the Maastricht Pathology Tissue Collection. Collection, storage, and use of tissue and patient data were performed in agreement with the "Code for Proper Secondary Use of Human Tissue in the Netherlands" (http://www.federa.org). The staining was performed as described previously (antibody dilution 1:2000) and biopsies were evaluated for the presence of MDA [10].
Chemiluminescent ELISA
Chemiluminescent ELISA was performed as previously described [10]. In brief, purified anti-human IgM (BD Pharmingen, San Jose, CA, USA) and antigens (MDA-LDL, MAA-LDL, CuOx-LDL, PC-BSA) at concentrations of 5 μg/ml in 50 μl phosphate-buffered saline (PBS)-EDTA were added to each well of a 96-well white, round-bottom microtitration plate (MicrofluorII roundbottom; Thermo, Rochester, New York, USA) and incubated overnight at 4 °C. P1 peptide (Peptide 2.0 Inc.) was coated at 10 μg/ml in NaHCO3 (pH 8.5) coating buffer and incubated overnight at 4 °C. After washing and blocking with Tris-buffered saline (TBS) with or without EDTA (pH 7.4, containing 1 % bovine serum albumin (BSA), 30 min at room temperature), the plate was incubated with plasma samples in their respective dilutions (total IgM: 1:40,000; MDA-LDL, MAA-LDL, P-1, Cu-OxLDL, PC-BSA IgM/IgG: 1:200) in 1 % BSA in TBS with EDTA (pH 7.4) for 2 h at room temperature or overnight at 4 °C. Alkaline phosphatase (AP)-labeled goat anti-human IgM (μ-chain specific; Sigma-Aldrich, Vienna, Austria; 1:50,000 in TBS-BSA) or AP-labeled goat anti-human IgG (γ-chain specific; Sigma-Aldrich, Vienna, Austria; 1:50,000 in TBS-BSA) was used for detection. AP-conjugated secondary reagents were detected using Lumi-Phos (Lumigen, Southfield, Michigan, USA; 33 % solution in water) and a Synergy 2 Luminometer (BioTek, Winooski, Vermont, USA). Washing steps were performed on an ELx405 Select Deep Well Microplate Washer (BioTek, Winooski, Vermont, USA) with PBS or PBS-EDTA. Internal controls were included on each microtiter plate to detect potential variations between microtiter plates. The intra-assay coefficients of variation for all assays were 5–15 %.
NAFLD cohort for antibody measurements
Healthy controls (n = 68) were recruited by advertisement and patients classified as NAFLD (n = 53) were recruited from the metabolic clinics of the Cleveland Clinic and MetroHealth Medical Center in Cleveland, OH, USA. The study protocol was approved by the Institutional Review Boards at the Cleveland Clinic and MetroHealth Medical Center. A detailed clinical history was obtained for each participant, followed by a physical examination. Hepatic ultrasonography was performed on all participants by the same investigator (S. Dasarathy) in order to confirm the absence of steatosis in controls. Moreover, plasma transaminase levels were determined in all participants. Where steatosis and increased plasma transaminase levels were detected, a liver biopsy was taken for further characterization of NAFLD. Liver biopsies from patients were reviewed in a blinded manner by the same pathologist and NASH was defined as an NAFLD activity score ≥4 and a histologic diagnosis of NASH by the pathologist. Participants with other forms of chronic liver disease were excluded by screening for autoimmune liver disease (autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis), for metabolic liver disease (hemochromatosis, Wilson's disease), for alpha-1 antitrypsin deficiency, and for viral hepatitis (hepatitis B and hepatitis C). Participants with a history of daily alcohol intake (women >20 g, men >30 g), intravenous drug use, or a history of bowel surgery were also excluded. None of the participants were on any medications known to cause hepatic steatosis or taking vitamin supplements. Written informed consent was obtained from all participants after having the procedure fully explained. Blood and liver sampling was done as described before [11–13]. Plasma samples were used to perform the antibody determinations. Relevant clinical data, including gender, age, weight, and height, together with inflammatory and liver blood test results, were collected at the time of liver biopsy and are represented in Table 1. The body mass index (BMI) was calculated using the formula: weight (kg)/[height (m)]2. Further clinical and biochemical data for all patients are summarized in Table 1 (Additional file 1).
Table 1 Clinical and biological characteristics of patients with non-alcoholic steatohepatitis for antibody measurements
Hepatitis C cohort
Healthy controls (n = 20) were recruited by advertisement and patients with chronic hepatitis C (n = 20) were recruited from the Department of Internal Medicine III, Division of Gastroenterology and Hepatology, Medical University of Vienna, Austria. The study protocol was approved by the Institutional Review Boards at the Medical University of Vienna. Hepatic ultrasonography and liver biopsy was performed on all participants prior to scheduled antiviral therapy. Moreover, plasma transaminase levels were determined in all participants. Liver biopsies of patients were reviewed in a blinded manner by two independent pathologists. Participants with other forms of chronic liver disease or with a history of daily alcohol intake were excluded by screening. Written informed consent was obtained from all participants after having the procedure fully explained. Further clinical data for the patients are summarized in Table 2 (Additional file 2).
Table 2 Clinical characteristics of patients with hepatitis C
Patients with inflammatory bowel disease
Between September 2009 and December 2010, consecutive patients with IBD were recruited during routine follow-up at the Gastroenterology Outpatient Clinic of the MUMC+ and participated in a prospective 1-year follow-up study [14]. All patients had an established diagnosis of IBD based on clinical, endoscopic, histological, and/or radiological criteria. Demographic and clinical data were obtained using the MUMC+ computer-based medical registration database. Disease phenotype at the time of diagnosis was determined by the Montreal classification [15]. The study was approved by the Medical Ethics Committee of MUMC+ and written informed consent was obtained prior to participation. A subset of patients who underwent an endoscopy for clinical reasons was included for the present study. Disease activity was assessed during endoscopy by an experienced gastroenterologist (MP) as inactive (0), mild (1), moderate (2), or severe (3) (Additional file 3).
Continuous variables are presented as means and standard deviations or as medians and interquartile ranges for skewed distributions. Categorical data are presented as absolute frequencies and percentages. Student's t-test or the Mann–Whitney U test (for non-normally distributed data) was used to compare a variable between two subgroups. Multivariate logistic regression models were used to assess the association between P1-specific IgM levels and NAFLD in the specific cohort after adjusting for age, gender, BMI, and total IgM levels. All reported p-values are two-tailed and considered significant at p < 0.05. Statistical procedures were carried out using Graphad Prism 6 or IBM SPSS 20.
MDA epitopes are present in livers of patients with NASH
To determine whether OSE are generated during the spectrum of NAFLD, immunohistochemical staining for MDA epitopes with the monoclonal antibody MDA2 was performed on liver biopsies of patients with biopsy-proven NASH. As illustrated in Fig. 1a–c, small clusters of MDA2-positive staining were detected between swollen hepatocytes of liver biopsies from patients with NASH, indicating the presence of MDA epitopes.
Malondialdehyde (MDA) epitopes in livers of patients with non-alcoholic steatohepatitis (NASH) and relationship between plasma immunoglobulin M (IgM) titers and non-alcoholic fatty liver disease (NAFLD). Immunohistochemical detection of MDA epitopes in liver sections (magnification 20×) of a a negative control and b two patients with NASH – arrows indicate localization of MDA2-positive staining in the liver tissue. Plasma IgM antibody titers against c MDA-low-density lipoprotein (LDL), d malondialdehyde-acetaldehyde (MAA)-LDL, e P1 mimotope, f CuOx-LDL, g PC-BSA, and h total IgM levels in patients with NAFLD and healthy controls. i Ratio of anti-P1 IgM titers to total IgM levels in patients with NAFLD and controls. Data are expressed in log10 relative light units (RLU)/100 ms. *p < 0.05, **p < 0.01, ***p < 0.001
IgM antibody titers towards different OSE are lower in patients with NAFLD
To investigate the potential relationship between antibodies specifically targeting epitopes of OxLDL and NAFLD, IgM and IgG antibody titers against MDA, MAA (an immunodominant advanced MDA-lysine adduct), P1 (a highly specific peptide mimotope of MAA [10]), Cu-OxLDL, and PC-BSA were measured in the plasma of patients with NAFLD (n = 71) and control participants with confirmed absence of steatosis (n = 68). The values of the different measurements were tested for normality with Shapiro–Wilks test and the results indicated that data were not normally distributed for most of the measurements in either of the groups (Additional file 4: Table S1). Therefore, all data were logarithmically transformed (log10) and represented using a log10 scale in Fig. 1. The observed relative lights units can be found in Additional file 4: Table S2.
In line with our expectations, patients with NAFLD had significantly lower IgM titers than controls against MDA-LDL (4.755 versus 4.819 log10 RLU/100 ms, p = 0.0019; Fig. 1c, Additional file 4: Table S2), MAA-LDL (4.801 versus 4.868 log10 RLU/100 ms, p = 0.0026; Fig. 1d, Additional file 4: Table S2), P1 (3.877 versus 4.08 log10 RLU/100 ms, p < 0.0001; Fig. 1e, Additional file 4: Table S2), CuOx-LDL (4.46 versus 4.612 log10 RLU/100 ms, p = 0.0004; Fig. 1f, Additional file 4: Table S2), and PC-BSA (4.255 versus 4.383 log10 RLU/100 ms, p = 0.0096; Fig. 1g, Additional file 4: Table S2). No differences were found in specific IgG antibody titers towards the different epitopes of OxLDL in patients with NAFLD compared to controls (Additional file 4: Table S3).
Interestingly, total IgM antibodies were also significantly lower in patients with NAFLD compared to healthy participants (3.773 versus 3.712 log10 RLU/100 ms, p = 0.0044; Fig. 1h, Additional file 4: Table S2). Notably, IgM titers towards the specific P1 mimotope, which showed the strongest reduction in patients with NAFLD compared to controls, remained significant after adjusting for total IgM levels (0.3074 versus 0.1658 (ratio), p = 0.0002, Fig. 1i). These data point towards a negative association between IgM antibodies and oxidized lipids and fatty liver disease, and indicate an important role for lipid oxidation in NAFLD.
Plasma anti-P1 IgM levels are reduced in the early phase of NAFLD and are not influenced by inflammation or fibrosis
Because anti-P1 IgM levels showed the strongest reduction in patients with NAFLD and due to its high clinical reproducibility, further analyses were conducted using the anti-P1 IgM measurements. To determine in which phase in the disease spectrum of NAFLD that plasma IgM titers towards P1 are reduced, participants were divided according to their ALT levels (<25, 25–50, >50 IU/L) because a normal ALT level is expected to be <50 IU/L. Interestingly, P1-IgM levels were reduced in participants with moderately elevated ALT levels (25–50 IU/L) or high ALT levels (>50 IU/L) when compared with the P1-IgM levels of participants with ALT levels <25 IU/L (Fig. 2a). Moreover, more than 90 % of control participants were classified as having low ALT levels. These data suggest that IgM titers are already lower in early phases of NAFLD, before liver damage is detected based on ALT levels.
Relationship between plasma P1-immunoglobulin (IgM) levels and non-alcoholic fatty liver disease (NAFLD) upon further characterization of liver biopsies. a IgM titers towards P1 in controls and patients with NAFLD grouped based on their ALT levels. b Plasma IgM antibody titers against the P1 mimotope in patients with NAFLD in which the liver biopsy was evaluated and scored as simple steatosis versus non-alcoholic steatohepatitis (NASH). Anti-P1 IgM titers after scoring liver biopsies for c steatosis, d inflammation, e hepatocyte ballooning, f applying the NAFLD activity score (NAS), and g scoring fibrosis. Data are expressed in relative light units (RLU)/100 ms and presented as means. **p < 0.01
Next, we evaluated the relationship between IgM to P1 and different classifications based on histological scoring of liver biopsies taken from patients with NAFLD. First, the potential of anti-P1 IgM to distinguish simple steatosis from NASH was tested. Whereas IgM titers were lower in NAFLD compared with controls, no difference was observed in specific IgM levels between patients with steatosis and NASH (Fig. 2b). Moreover, no differences were observed in plasma P1-IgM titers after classifying the patients according to a score for steatosis (Fig. 2c), inflammation (Fig. 2d), hepatocyte ballooning (Fig. 2e), the NAFLD activity score (NAS) (Fig. 2f), or for fibrosis (Fig. 2g). These data further suggest that antibodies recognizing oxidized lipids are involved during early processes in the development of NAFLD.
Low IgM titers against the P1 mimotope have a high predictive value for the presence of NAFLD
To determine the power of low plasma IgM titers against the P1 mimotope to predict the presence of fatty liver disease, age, gender, BMI, total IgM levels, and anti-P1 IgM levels were combined in a single logistic regression model. To account for skewness in their distribution, base-2 logarithms of P1-specific IgM levels were used in the model. Thus, adjusted odds ratios (OR) for this variable reflect the change in odds for an increase of one log2 (the equivalent of a doubling of the value) in the measure. Without adjustment for other risk factors, age (OR = 1.206; confidence interval = 1.039–1.4), BMI (OR = 1.353; confidence interval = 1.228–1.491), total IgM (OR = 0.619; confidence interval = 0.431–0.888), and P1-IgM (OR = 0.428; confidence interval = 0.282–0.65) were all significant predictors for the presence of NAFLD, and female gender was not (Additional file 4: Table S4). Importantly, P1-IgM levels (OR = 0.419; confidence interval = 0.216–0.813) were still a significant predictor for the presence of NAFLD after adjustment for age, gender, BMI, and total IgM (Fig. 3a, Additional file 4: Table S5). Moreover, adjusting for waist circumference instead of BMI in our model did not change the significance for anti-P1 IgM levels (OR = 0.432; confidence interval = 0.261–0.813) in predicting the presence of NAFLD (Additional file 4: Table S6 and Additional file 5: Figure S1). In line with our expectations, BMI (OR = 1.343; confidence interval = 1.211–1.49) and waist circumference (OR = 1.666; confidence interval = 1.354–2.049) were still significant predictors for NAFLD after adjustment, whereas other variables lost their significance (Additional file 4: Tables S5 and S6).
Single logistic regression model, odds ratio determination, and receiver operating characteristic (ROC) curve analysis for P1-specific IgM levels in non-alcoholic fatty liver disease (NAFLD). a Odds ratios for the predictive power of anti-P1 immunoglobulin (IgM) titers (log2 scale) for fatty liver disease after adjustment for age, gender, body mass index (BMI), and total IgM. b ROC curve analysis for the diagnosis of NAFLD with P1-IgM. c IgM titers towards P1 were divided into quartiles (black: controls; red: NAFLD) and d the odds ratios P1-IgM (log2 scale) for NAFLD were computed across these quartiles after adjusting for age, gender, BMI, and total IgM levels. CI confidence interval
A receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic value of anti-P1 IgM titers for NAFLD. An area under the curve (AUC) of 0.703 (confidence interval: 0.617–0.790) for the diagnosis of NAFLD using IgM titers towards P1 was found (Fig. 3b). In addition, the measurements of IgM against P1 were divided into quartiles and the OR for NAFLD were computed across these quartiles after adjusting for age, gender, BMI, and total IgM levels (Fig. 3c, Table 3). Our results indicate that participants in quartile 1 (lowest P1-IgM levels) had an odds ratio of 5.8 (p = 0.01, confidence interval: 1.209–27.818), indicating an increased likelihood of NAFLD with decreasing P1-IgM levels, independent of age, gender, BMI, and total IgM levels (Fig. 3d, Table 3). Taken together, these data indicate that P1-specific IgM titers have a high predictive value for the presence of NAFLD, independent of total IgM levels.
Table 3 Odds ratios of P1-specific immunoglobulin M levels for non-alcoholic fatty liver disease
Anti-P1 IgM titers are not correlated with lipid levels and negatively correlate with markers for liver damage and systemic markers of inflammation
First, the correlation between P1-IgM and plasma lipid levels in all participants was tested. No correlation was found between IgM levels towards P1 and plasma triglycerides (Pearson R: −0.11, Fig. 4a), cholesterol (Pearson R: −0.09, Fig. 4b), or LDL (Pearson R: −0.03, Fig. 4c), well-known indicators for steatosis and risk factors for NAFLD. Next, we assessed the potential correlation with markers for obesity: BMI, waist circumference, and leptin concentration. A strong inverse correlation was observed between P1-IgM and BMI (Pearson R: −0.28, p = 0.0009, Fig. 4d), waist circumference (Pearson R: −0.22, p = 0.02, Fig. 4e), and leptin concentration (Pearson R: −0.32, p = 0.0026, Fig. 4f). These data are in line with a previously reported study showing elevated serum leptin levels during NAFLD [16]. Moreover, obesity, which is a known risk factor for NAFLD, has been associated with increased leptin levels [17]. Additionally, we tested the correlation between P1-IgM and circulatory markers for liver damage, inflammation, and adipokines. Interestingly, a strong inverse correlation was found between P1-IgM levels and monocyte chemoattractant protein-1 (MCP-1; Pearson R: −0.39, p = 0.0006, Fig. 4g), IL-6 (Pearson R: −0.24, p = 0.041, Fig. 4h), and cathepsin D (Pearson R −0.25, p = 0.0033, Fig. 4I), which we recently validated as a marker for pediatric NASH. Moreover, anti-P1-IgM titers were inversely correlated with alanine aminotransferase (ALT; Pearson R: −0.17, p = 0.047, Additional file 6: Figure S2A) and aspartate aminotransferase (AST; Pearson R: −0.18, p = 0.037, Additional file 6: Figure S2B) and a trend was observed with gamma-glutamyltransferase (γGT; Pearson R: −0.11, Additional file 6: Figure S2C). These data indicate that increased liver damage, assessed by AST and ALT levels, is associated with reduced levels of IgM antibody titers towards OSE. No correlation was found with IL-1β (Pearson R: 0.16, Additional file 6: Figure S2D) or IL-8 (Pearson R: −0.10, Additional file 6: Figure S2E). Taken together, these data suggest that immune recognition of oxidized lipids is affected during obesity and plays an important role during related diseases, such as the early stages of hepatic inflammation in NAFLD. These results also suggest an indicative role of low P1-IgM levels in regard to systemic markers of inflammation and liver damage.
Correlation between plasma P1-immunoglobulin M (IgM) levels and plasma lipids and systemic markers of inflammation. Shown are the Pearson R correlation between plasma IgM titers towards P1 and a plasma triglycerides (TG), b cholesterol, low-density lipoprotein (LDL), d body mass index (BMI), e waist circumference, f leptin, and the markers for inflammation g monocyte chemoattractant protein 1 (MCP1), h interleukin 6 (IL-6), and i cathepsin D. RLU relative light unit
Low OSE-specific IgM levels are not associated with hepatitis C or IBD-dependent inflammation
To determine the association between plasma P1-IgM levels and other chronic inflammatory diseases, a separate analysis was conducted on two cohorts consisting of patients with hepatitis C or autoimmune IBD. Total and OSE-specific IgM and IgG antibody titers were assessed in both cohorts. Total IgM titers were unchanged between patients with hepatitis C and controls (167 versus 117 mg/dL, Fig. 5a, Additional file 4: Table S7) whereas total IgG levels were higher in patients with hepatitis C than in controls (1506 versus 1091 mg/dL, Additional file 4: Table S7). Importantly, in contrast to our findings in NAFLD, IgM titers towards P1 were higher in patients with hepatitis C than in controls (99,785 versus 62,828 RLU/100 ms, p < 0.0001, Fig. 5b, Additional file 4: Table S7). Adjusting for total IgM antibody levels resulted in no significant differences between the two groups (686 versus 644 (ratio), Fig. 5c). OSE-specific IgG levels were higher in patients with hepatitis C than in controls (Additional file 4: Table S8). These data further suggest that low levels of IgM antibodies targeting oxidized lipids are specific for obesity-related diseases such as NAFLD.
Plasma immunoglobulin M (IgM) titers in cohorts consisting of patients with hepatitis C or patients with inflammatory bowel disease (IBD). a Total, b anti-P1 IgM titers, and c P1/total IgM ratio in healthy controls and patients with hepatitis C. Relationship between the activity score during IBD and d total IgM, e P1-specific IgM, and f P1/total IgM ratio. Inactive disease was given score 0, mild to moderate disease activity was given a score of 1–2, and severe activity was given a score of 3. Data are expressed in mg/dl or relative light units (RLU)/100 ms and presented as means and standard deviations. *p < 0.05, ***p < 0.001
In the IBD cohort (n = 62; mean male age 49.4 years and female age 54.8 years), 46 had Crohn's disease (8 ileal, 17 colonic, and 21 ileocolonic), 15 ulcerative colitis (1 procolitis, 10 distal, and 4 pancolitis), and one patient had indeterminate colitis. IBD is characterized by fluctuations in the inflammatory state of the gut [18]. The patients were classified during endoscopy according to the activity state of inflammation at the time of blood sampling with a score ranging from 0 to 3, in which 0 means inactive (n = 23), 1–2 refers to mild to moderate activity (n = 10), and 3 indicates severely active disease (n = 29). In contrast to our measurements in patients with NAFLD, total IgM levels were not influenced by the inflammatory activity state in patients with IBD (36,025 versus 37,013 versus 40,280 RLU/100 ms, Fig. 5d). Next, IgM antibody titers towards the P1 mimotope were determined in these patients, reflecting the IgM titers against OSE. In contrast to the decreased levels found in the plasma of patients with NAFLD, slightly higher P1-specific IgM titers were found in the plasma of patients classified with high activity IBD (score 3) compared to patients with inactive disease (score 0) (31,052 versus 21,121 RLU/100 ms, p = 0.0383, Fig. 5e). We could not detect a difference in P1-specific IgM in patients scored as 0 and 1–2 (21,121 versus 23,060 RLU/100 ms, Fig. 5e), nor between patients scored as 1–2 and 3 (23,060 versus 31,052 RLU/100 ms, Fig. 5e), respectively. Additionally, no difference was observed between the groups after normalization for total IgM levels (0.628 versus 0.608 versus 0.755 (ratio), Fig. 5f). These data suggest that decreased IgM antibody titers towards MDA-type epitopes are more specifically associated with lipid-induced inflammation, as present in obesity-related diseases like atherosclerosis and in the spectrum of NAFLD.
The multifactorial mechanisms responsible for the worsening of NAFLD are still poorly understood. Although inflammation is believed to set the stage for further progression into fibrosis and cirrhosis, relevant insights into underlying pathways are missing. Hence, therapy options are only partially effective and accurate, non-invasive diagnostic tools to detect early signs of fatty liver disease are lacking. In the present study, we assessed the relationship between antibodies specific for model epitopes of oxidized lipids and human NAFLD. Our data indicate an inverse association between OSE-specific IgM titers and NAFLD, while no association was observed with hepatitis C or IBD.
Elevated IgM antibody levels to epitopes of OxLDL due to immunization strategies have been reported in mouse models of atherosclerosis with distinct athero-protective functions [19–22]. It has also been demonstrated that immunization with autologous MDA-LDL reduces atherosclerotic lesion formation in rabbits and mice [23, 24]. MDA epitopes were found to be highly expressed in atherosclerotic lesions [10] and antibodies recognizing immunogenic lipid peroxidation end products have also been intensively studied in patients with cardiovascular disease. IgM titers were found to be inversely associated with CVD events and low levels of OSE-specific IgM were shown to be associated with an increased risk for myocardial infarction [25, 26]. Also, during NASH, lipid peroxidation as a result of steatosis and the consequential formation of OSE such as MDA have been implicated. In addition, IgM antibodies targeting these OSE have been proposed to be protective against hepatic inflammation [8]. In line with our current data, we have previously shown that immunization of hyperlipidemic Ldlr−/− mice leads to reduced hepatic inflammation upon high-cholesterol diet due to increased IgM titers towards PC, another OSE [8]. We have recently shown that deficiency of Siglec-G, a negative regulator of B-cells that results in increased B-1 cells and OSE-specific IgM antibodies, inhibits diet-induced hepatic inflammation and atherosclerosis [9]. Taken together, our current findings further support the idea that IgM targeting OxLDL and/or specific OSE play an important role during chronic fatty liver disease.
Immune reactions triggered by oxidative stress have been shown to be involved during the progression of NAFLD to fibrosis [4]. In line with data from patients with alcoholic liver disease [27], increased IgG levels against antigens derived from lipid peroxidation, such as proteins adducted with MDA, have been found in patients with established fibrosis [4]. An explanation for this association may be the potential dual effect of MDA adducts on hepatic stellate cells. It was shown that MDA is specifically recognized by hepatic stellate cells via an interaction with CD36 scavenger receptors [28], thereby potentially stimulating the production of collagen and fibronectin, and promoting an immune response towards MDA as human hepatic stellate cells can promote lymphocyte proliferation [29]. Another study demonstrated that serum IgA levels are elevated in patients with late-stage NASH in comparison with those with early-stage NASH [30]. More recently, a study by McPherson et al. indicated that serum IgA levels are frequently elevated in patients with NAFLD and could be useful to predict advanced fibrosis [31]. These findings were confirmed in a study by Maleki et al., showing that IgA levels are useful to evaluate the severity of liver fibrosis [32]. In both studies, total IgM levels were determined in addition to IgA levels. In line with our data, no differences were observed in the levels of total IgM antibodies in patients with late-stage NASH compared to patients with early-stage NASH, or between patients with different stages of fibrosis [30, 31]. Nevertheless, because IgM antibody titers targeting OSE were not determined in these studies it is not possible to conclude any association between levels of OSE-specific IgM to NASH development in these cohorts. Additional investigations of IgM antibodies towards different OSE, and in particular the P1 mimotope, in multiple larger cohorts are necessary to confirm the inverse correlation with NAFLD. It will be particularly interesting to follow up anti-OSE-IgM levels in patients with NAFLD and NASH who undergo treatment or lifestyle changes.
Recently, Tsiantoulas et al. showed that MDA epitopes are present on circulating microparticles and are recognized by specific IgM antibodies, thereby potentially dampening their pro-inflammatory effects [33]. Microparticles are part of a group of extracellular vesicles (0.1–1 μm in diameter) produced and released by budding from the plasma membrane upon activation or apoptosis of different cell types, including monocytes [34]. Microparticles have been found to play an important role in NAFLD and related diseases (reviewed in [35]). Patients with NASH were shown to have increased numbers of circulating microparticles from invariant natural killer T cells and macrophages/monocytes (CD14+), dominant cell types in mediating the pathogenesis of NASH [36]. Moreover, the level of microparticles, which was shown to correlate positively with ALT and disease severity, has been proposed as a new biomarker for NASH [36]. Another study has recently demonstrated that lipids induce microparticle release from hepatocytes, which activate macrophages and lead to an inflammatory response in NASH [37]. In line with our described findings, one may hypothesize that increased levels of microparticles due to lipid overload during the onset of NAFLD and NASH result in a consumption of OSE-specific IgM. Further studies are needed to address the role and interplay between microparticles and OSE-specific antibodies in the pathogenesis of fatty liver disease.
The similarities between atherosclerosis and NAFLD, and our observations regarding the correlations with BMI and waist circumference allow us to hypothesize that low IgM titers towards oxidized lipids are a consequence of obesity and are specifically associated with related diseases. In contrast to lipid-induced inflammation during obesity, IBD is believed to result from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host [18]. Altered serum IgG levels have previously been found in patients with IBD [38, 39]: serum IgG subclass IgG1 and IgG3 levels were elevated in ulcerative colitis, and IgG2 and IgG4 levels were increased in patients with Crohn's disease. In line with our findings, no significant differences were found in total IgM concentration, whereas anti-OSE-IgM titers were not determined [38]. Further, different serum immunoglobulins were recently reported in patients with IBD in a cross-sectional study [40]. Similar to our observation, no correlation was found between disease activity during IBD and IgM levels [40].
Importantly, increased serum levels of MDA and lipid oxidation were found to be present in individuals with chronic hepatitis C [41], which might be reflected in a different host immune response towards epitopes of lipid oxidation, thereby limiting the specificity for our findings towards NAFLD. In line with previous reported findings, total and OSE-specific IgG antibodies were increased in patients with hepatitis C compared to healthy controls [42–45], indicative of an increased immune response towards oxidative lipids in hepatitis C. Moreover, in contrast to our findings in NAFLD, IgM titers towards P1 are increased in patients with hepatitis C compared to controls. However, adjusting for total IgM antibody levels indicates no differences between the two groups. Besides our findings in patients with IBD, these data indicate that reduced levels of IgM antibodies targeting oxidation products of lipids are not specific to fatty liver disease but rather associated with obesity-related diseases such as atherosclerosis and NAFLD/NASH. Nevertheless, additional validation in other cohorts with different chronic liver diseases is needed to support the specificity of low OSE-specific IgM titers in NAFLD.
So far, the reason behind the lower OSE-specific IgM levels in comparison to healthy participants is not clear. Similar to the view in CVD [26], one possible explanation is that, during NAFLD, there is increased consumption of IgM antibodies due to increased levels of OSE and ultimately increased clearance and/or uptake in the vessel wall. Alternatively, it is possible that genetic variation leading to lower basal IgM levels are the underlying cause for patients being at higher risk for developing lipid-induced related diseases such as atherosclerosis and NAFLD/NASH. In the latter case, low OSE-specific IgM levels should be considered a risk factor for developing fatty liver disease. Further research is needed to understand why basal differences in OSE-specific IgM levels exist and whether this can be seen as a risk factor for NAFLD.
In the current manuscript we point out that IgM titers to epitopes on oxidized lipids are negatively associated with NAFLD using the P1 mimotope, which serves as a highly reproducible antigen to assess antibody titers in patients. While the link between anti-P1 IgM measurements and NAFLD, which is independent of classical risk factors for NAFLD, provides critical insights into the pathophysiology of chronic fatty liver disease, plasma levels of anti-P1 IgM are not suitable as a biomarker for the progression of NAFLD. This conclusion is based on the inability of anti-P1 IgM titers to distinguish steatosis from NASH, the correlation between P1 IgM titers to BMI, and the large overlap between NAFLD and control participants for the different measurements. Of note, our findings suggest that obesity/hyperlipidemia is a causal factor for reduced IgM levels, rendering people more prone to develop fatty liver disease and related disorders such as atherosclerosis. Additional studies assessing IgM antibodies towards different OSE, and in particular the P1 mimotope, in multiple larger cohorts are necessary to confirm the inverse correlation with NAFLD and to address the potential causal link with obesity.
ALT, alanine aminotransferase; AST, aspartate aminotransferase; AP, alkaline phosphatase; AUC, area under curve; BMI, body mass index; BSA, bovine serum albumin; CuOx-LDL, Copper oxidized low-density lipoprotein; HDL, high-density lipoprotein; IBD, inflammatory bowel disease; Ig, immunoglobulin; IL-1β, interleukin 1 beta; IL-6, interleukin 6; LDL, low-density lipoprotein; MAA, malondialdehyde-acetaldehyde; MCP-1, monocyte chemoattractant protein 1; MDA, malondialdehyde; NALFD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; OR, odds ratio; OSE, oxidation-specific epitope; OxLDL, oxidized low-density lipoprotein; PC, phosphocholine; RLU, relative light units; ROC, receiver operating characteristic (curve); TBS, Tris-buffered saline; γGT, gamma-glutamyltransferase.
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Bieghs V, van Gorp PJ, Walenbergh SM, Gijbels MJ, Verheyen F, Buurman WA, et al. Specific immunization strategies against oxidized low-density lipoprotein: a novel way to reduce nonalcoholic steatohepatitis in mice. Hepatology. 2012;56(3):894–903.
Gruber S, Hendrikx T, Tsiantoulas D, Ozsvar-Kozma M, Goderle L, Mallat Z, Witztum JL, Shiri-Sverdlov R, Nitschke L, Binder CJ. Sialic acid-binding immunoglobulin-like lectin G promotes atherosclerosis and liver inflammation by suppressing the protective functions of B-1 cells. Cell Rep. 2016;14(10):2348–61.
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Kasumov T, Edmison JM, Dasarathy S, Bennett C, Lopez R, Kalhan SC. Plasma levels of asymmetric dimethylarginine in patients with biopsy-proven nonalcoholic fatty liver disease. Metabolism. 2011;60(6):776–81.
Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313–21.
Bodelier AG, Pierik MJ, van den Heuvel T, Bovee-Oudenhoven IM, de Boer E, Hameeteman W, et al. Pancreatitis-associated protein has no additional value as a marker of disease activity in a real-life cohort of IBD patients. Eur J Gastroenterol Hepatol. 2014;26(8):902–9.
Satsangi J, Silverberg MS, Vermeire S, Colombel JF. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut. 2006;55(6):749–53.
Chitturi S, Farrell G, Frost L, Kriketos A, Lin R, Fung C, et al. Serum leptin in NASH correlates with hepatic steatosis but not fibrosis: a manifestation of lipotoxicity? Hepatology. 2002;36(2):403–9.
Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med. 1996;334(5):292–5.
Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med. 2009;361(21):2066–78.
Binder CJ, Horkko S, Dewan A, Chang MK, Kieu EP, Goodyear CS, et al. Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med. 2003;9(6):736–43.
Cesena FH, Dimayuga PC, Yano J, Zhao X, Kirzner J, Zhou J, et al. Immune-modulation by polyclonal IgM treatment reduces atherosclerosis in hypercholesterolemic apoE-/- mice. Atherosclerosis. 2012;220(1):59–65.
Kyaw T, Tay C, Krishnamurthi S, Kanellakis P, Agrotis A, Tipping P, et al. B1a B lymphocytes are atheroprotective by secreting natural IgM that increases IgM deposits and reduces necrotic cores in atherosclerotic lesions. Circ Res. 2011;109(8):830–40.
Lewis MJ, Malik TH, Ehrenstein MR, Boyle JJ, Botto M, Haskard DO. Immunoglobulin M is required for protection against atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation. 2009;120(5):417–26.
Freigang S, Horkko S, Miller E, Witztum JL, Palinski W. Immunization of LDL receptor-deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes. Arterioscler Thromb Vasc Biol. 1998;18(12):1972–82.
Palinski W, Miller E, Witztum JL. Immunization of low density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad Sci U S A. 1995;92(3):821–5.
Ravandi A, Boekholdt SM, Mallat Z, Talmud PJ, Kastelein JJ, Wareham NJ, et al. Relationship of IgG and IgM autoantibodies and immune complexes to oxidized LDL with markers of oxidation and inflammation and cardiovascular events: results from the EPIC-Norfolk Study. J Lipid Res. 2011;52(10):1829–36.
Tsimikas S, Brilakis ES, Lennon RJ, Miller ER, Witztum JL, McConnell JP, et al. Relationship of IgG and IgM autoantibodies to oxidized low density lipoprotein with coronary artery disease and cardiovascular events. J Lipid Res. 2007;48(2):425–33.
Rolla R, Vay D, Mottaran E, Parodi M, Vidali M, Sartori M, et al. Antiphospholipid antibodies associated with alcoholic liver disease specifically recognise oxidised phospholipids. Gut. 2001;49(6):852–9.
Schneiderhan W, Schmid-Kotsas A, Zhao J, Grunert A, Nussler A, Weidenbach H, et al. Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and stimulate extracellular matrix synthesis. Hepatology. 2001;34(4 Pt 1):729–37.
Vinas O, Bataller R, Sancho-Bru P, Gines P, Berenguer C, Enrich C, et al. Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation. Hepatology. 2003;38(4):919–29.
Tomita K, Teratani T, Yokoyama H, Suzuki T, Irie R, Ebinuma H, et al. Serum immunoglobulin a concentration is an independent predictor of liver fibrosis in nonalcoholic steatohepatitis before the cirrhotic stage. Dig Dis Sci. 2011;56(12):3648–54.
McPherson S, Henderson E, Burt AD, Day CP, Anstee QM. Serum immunoglobulin levels predict fibrosis in patients with non-alcoholic fatty liver disease. J Hepatol. 2014;60(5):1055–62.
Maleki I, Aminafshari MR, Taghvaei T, Hosseini V, Rafiei A, Torabizadeh Z, et al. Serum immunoglobulin A concentration is a reliable biomarker for liver fibrosis in non-alcoholic fatty liver disease. World J Gastroenterol. 2014;20(35):12566–73.
Tsiantoulas D, Perkmann T, Afonyushkin T, Mangold A, Prohaska TA, Papac-Milicevic N, et al. Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies. J Lipid Res. 2015;56(2):440–8.
Kornek M, Schuppan D. Microparticles: Modulators and biomarkers of liver disease. J Hepatol. 2012;57(5):1144–6.
Hirsova P, Ibrahim SH, Gores GJ, Malhi H. Lipotoxic lethal and sublethal stress signaling in hepatocytes: relevance to NASH pathogenesis. J Lipid Res. 2016. [Epub ahead of print].
Kornek M, Lynch M, Mehta SH, Lai M, Exley M, Afdhal NH, et al. Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis. Gastroenterology. 2012;143(2):448–58.
Hirsova P, Ibrahim SH, Krishnan A, Verma VK, Bronk SF, Werneburg NW, Charlton MR, Shah VH, Malhi H, Gores GJ. Lipid-induced signaling causes release of inflammatory extracellular vesicles from hepatocytes. Gastroenterology. 2016;150(4):956–67.
Gouni-Berthold I, Baumeister B, Berthold HK, Schmidt C. Immunoglobulins and IgG subclasses in patients with inflammatory bowel disease. Hepatogastroenterology. 1999;46(27):1720–3.
MacDermott RP, Nash GS, Auer IO, Shlien R, Lewis BS, Madassery J, et al. Alterations in serum immunoglobulin G subclasses in patients with ulcerative colitis and Crohn's disease. Gastroenterology. 1989;96(3):764–8.
Rai T, Wu X, Shen B. Frequency and risk factors of low immunoglobulin levels in patients with inflammatory bowel disease. Gastroenterol Rep (Oxf). 2015;3(2):115–21.
De Maria N, Colantoni A, Fagiuoli S, Liu GJ, Rogers BK, Farinati F, et al. Association between reactive oxygen species and disease activity in chronic hepatitis C. Free Radic Biol Med. 1996;21(3):291–5.
Vidali M, Occhino G, Ivaldi A, Rigamonti C, Sartori M, Albano E. Combination of oxidative stress and steatosis is a risk factor for fibrosis in alcohol-drinking patients with chronic hepatitis C. Am J Gastroenterol. 2008;103(1):147–53.
Ortarik Z, Toyran A, Sen S, Mart Komurcu SZ, Guvener E. [Evaluation of serum IgG, IgA and IgM levels as indicators of hepatic fibrosis in patients with chronic hepatitis C infection]. Mikrobiyol Bul. 2011;45(2):296–305.
Gonzalez-Quintela A, Alende MR, Gamallo R, Gonzalez-Gil P, Lopez-Ben S, Tome S, et al. Serum immunoglobulins (IgG, IgA, IgM) in chronic hepatitis C. A comparison with non-cirrhotic alcoholic liver disease. Hepatogastroenterology. 2003;50(54):2121–6.
Watt K, Uhanova J, Gong Y, Kaita K, Doucette K, Pettigrew N, et al. Serum immunoglobulins predict the extent of hepatic fibrosis in patients with chronic hepatitis C virus infection. J Viral Hepat. 2004;11(3):251–6.
This research was supported by Cardiovascular Research the Netherlands (CVON 2012-03, IN-CONTROL), the Netherlands Organisation for Scientific Research (NWO) (Vidi 016.126.327), and by the SFB Lipotox F30 of the Austrian Science Fund. TH was supported by a Boehringer Ingelheim Fonds travel grant.
TH: study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript for important intellectual content; statistical analysis; obtained funding. MW: critical revision of the manuscript; analysis and interpretation of data; statistical analysis. CJB, RS: study concept and design; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript; obtained funding; administrative, technical, or material support; study supervision. SW, SA, SG, MOK, SK, HG, GK, MP, DJ, KS, MT, MH: critical revision of the manuscript; technical or material support. All authors read and approved the final manuscript.
Departments of Molecular Genetics, Pathology, and Internal Medicine, Division of Gastroenterology and Hepatology, School of Nutrition and Translational Research in Metabolism (NUTRIM) and School of Oncology and Developmental Biology (GROW), Maastricht University and Maastricht University Medical Center (MUMC), PO Box 616, 6200 MD, Maastricht, The Netherlands
Tim Hendrikx
, Sofie M. A. Walenbergh
, Heike I. Grabsch
, Ger H. Koek
, Marieke J. Pierik
, Daisy Jonkers
& Ronit Shiri-Sverdlov
Departments of Laboratory Medicine, Surgery, and Internal Medicine III, Division of Gastroenterology And Hepatology, Medical University of Vienna, Vienna, Austria
, Martin L. Watzenböck
, Shahzada Amir
, Sabrina Gruber
, Maria Ozsvar Kozma
, Katharina Staufer
, Michael Trauner
& Christoph J. Binder
Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences, Vienna, Austria
Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH, USA
Satish C. Kalhan
Department of Pathology & Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
Marten H. Hofker
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Correspondence to Ronit Shiri-Sverdlov.
Data NAFLD cohort. (XLSX 1353 kb)
Data HepC cohort. (XLSX 28 kb)
Data IBD cohort. (XLS 44 kb)
Shapiro–Wilk test of normality for the OSE-IgM measurements. Table S2 Plasma total IgM and OSE-specific IgM titers in NAFLD patients and control subjects without steatosis. Table S3 Plasma OSE-specific IgG titers in NAFLD patients and control subjects without steatosis. Table S4 Odds ratios for the predictive power for NAFLD for different variables without adjustment. Table S5 Odds ratios for the predictive power for NAFLD for different variables with adjustment for age, gender, BMI and total IgM levels. Table S6 Odds ratios for the predictive power for NAFLD for different variables with adjustment for age, gender, BMI and total IgM levels. Table S7 Plasma total and OSE-specific IgM titers in hepatitis C patients and control subjects. Table S8 Plasma total and OSE-specific IgG titers in hepatitis C patients and control subjects. (DOCX 26 kb)
Single logistic regression model and odds ratio determination for P1-specific IgM levels in NAFLD. Odds ratios for the predictive power of anti-P1 IgM titers (log2 scale) for fatty liver disease after adjustment for age, gender, waist circumference, and total IgM. (TIF 68 kb)
Correlation between plasma P1-IgM levels and systemic markers of liver damage, inflammation, and adipokines. Pearson R correlation between plasma IgM titers towards P1 and plasma ALT (A), AST (B), γ-GT (C), IL-1β (D), and IL-8 (E), respectively. (TIF 110 kb)
Hendrikx, T., Watzenböck, M.L., Walenbergh, S.M.A. et al. Low levels of IgM antibodies recognizing oxidation-specific epitopes are associated with human non-alcoholic fatty liver disease. BMC Med 14, 107 (2016) doi:10.1186/s12916-016-0652-0
Non-alcoholic steatohepatitis
Lipid oxidation
Adaptive immune response


text:
#ifndef PACKETCONTAINERTREE_H
#define PACKETCONTAINERTREE_H

#include "config.h"
#include "PacketContainer.h"

#include <map>

/**
 *   Class for holding a sorted tree of PacketContainers.
 *
 */
class PacketContainerTree {
public:
      /**
       * Destructor.
       */
      ~PacketContainerTree();

      /**
       * Adds a new PacketContaner to the tree.
       *
       * @param cont The PacketContaner to add.
       */
      inline void add( PacketContainer* cont );

      /**
       * Removes a PacketContaner from the tree.
       *
       * @param cont The PacketContaner to remove.
       */
      inline void remove( PacketContainer* cont );

      /**
       *   Get the packetcontainer with the lowest absolute
       *   timeout time.
       *
       *   @return The minimun PacketContainer.
       */
      inline PacketContainer* getMin();

      /**
       *   Get the packetcontainer with the lowest absolute
       *   timeout time.
       *   @return The minimun PacketContainer.
       */
      inline PacketContainer* extractMin();

      /**
       *   Returns packet container with the supplied ids or NULL.
       */
      PacketContainer* linearGetWithRequestAndPackID( uint32 reqID,
                                                      uint32 packID );

      /**
       * The number of PacketContainers in tree.
       *
       * @return The number of PacketContainers.
       */
      inline uint32 getCardinal() const;

      /**
       *   Returns true if the tree is empty.
       */
      inline bool empty() const;
private:

   /// Type of map of packetcontainers.
   typedef multimap<uint32, PacketContainer*> packetMap_t;

   /// Packet containers are kept here.
   packetMap_t m_packets;
};

// =======================================================================
//                                 Implementation of the inlined methods =

void
PacketContainerTree::add( PacketContainer* cont )
{
   m_packets.insert( make_pair( cont->getTimeoutAbsTime(),
                                cont ) );
}

void
PacketContainerTree::remove( PacketContainer* cont )
{
   // You could possible find the start it with lower_bound
   // on the time, but not if the time has changed in the cont.
   for( packetMap_t::iterator it = m_packets.begin();
        it != m_packets.end();
        ++it ) {
      if ( it->second == cont ) {
         m_packets.erase(it);
         return;
      }
   }
}

PacketContainer*
PacketContainerTree::getMin()
{
   if ( ! m_packets.empty() ) {
      return m_packets.begin()->second;
   } else {
      return NULL;
   }
}

PacketContainer*
PacketContainerTree::extractMin()
{
   PacketContainer* tmp = getMin();
   if ( tmp != NULL ) {
      remove(tmp);
   }
   return tmp;
}

uint32
PacketContainerTree::getCardinal() const
{
   return m_packets.size();
}

bool
PacketContainerTree::empty() const
{
   return m_packets.empty();
}

#endif // PACKETCONTAINERTREE_H

redpajama_set_name: RedPajamaGithub


Opcare is a private company that works in partnership with the NHS to provide Orthotic & Prosthetic Services to NHS patients.
We are currently seeking an experienced Prosthetist to join our service in Manchester. The role sits within a large team of Prosthetists providing clinical care to NHS patients in Greater Manchester. The patient portfolio of the service is varied, with challenging levels and presentations for both Lower and Upper limb amputees, including paediatrics. The service has first class on site facilities and is situated in a purpose built facility, with access to a large MDT team, including Consultants, Physios and Occupational Health.
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Even tough there are a lot of sticky footers out there, there is always a downside. It's not responsive, not supported by older browsers or it requires you to copy and paste a lot of code you don't want to maintain afterwards. The jQuery.stickerFooter plugin is designed to take care of these problems.
It's a simple jQuery plugin that can be used with just one line of code. It's maintained on GitHub and you can install and update the plugin with Bower.io. Check out GitHub for the code and documentation.


text:
Aitmatov Deserved a Nobel
Jun. 17 2008 00:00
My father and I spent the long Russia Day holiday weekend at our dacha, where we reminisced about Chingiz Aitmatov, the great writer who died last week and was buried on Saturday in Bishkek.
My father became friends with Aitmatov back in the early 1960s, and he recalled many dramatic, touching and curious moments from that friendship.
In 1970, my father -- who was then the editor-in-chief of Komsomolskaya Pravda -- wrote a review of "The White Steamship," possibly Aitmatov's best work. My father had wanted to run the review on the same day the work was published, but since Aitmatov's novel did not fit in at all with the canon of Soviet socialist realism, the review was rejected by the first secretary of the Komsomol's Central Committee.
Not long after "The White Steamship" was published, Aitmatov and my father found themselves in the Komsomol first secretary's office. Aitmatov began the conversation by saying, "Oh, I see you have passages from the Koran!" and pointing to the Arabic words woven into the secretary's decorative carpet, probably a gift from a Communist youth organization in a Middle East country. After the first secretary realized that he could have been pinned with "ideological sedition" for the religious passages in the carpet, he became very concerned and quickly gave permission for my father's review to be published. The review's publication in one of the country's leading papers meant that "The White Steamship" acquired official state recognition and approval. This, in turn, opened up new possibilities for other writers like Aitmatov.
My father and I discussed how illogical and absurd life was back then. We also discussed how arbitrary the distinction was between a person who was given official recognition and the person who was stigmatized as a political dissident. And we were surprised to note how many first-rate writers, poets, playwrights, film directors and artists could be found in the former Soviet republics at that time, despite the absurd political system.
The period from the 1960s to the 1980s were years marked by political stagnation, but it also turned out to be the golden age for those republics. It was a period that defined their national cultures. Strangely enough, the subsequent 17 years of freedom since the collapse of the Soviet Union have not produced anything even remotely comparable in quality to the works of Kyrgyzstan's Aitmatov, Russia's Vasily Shukshin and Fyodor Abramov, Lithuania's Juozas Baltusis, Georgia's Chabua Amirejibi, Kazakhstan's Abdizhamil Nurpeisov and Abish Kekilbayev, or Belarus' Vasily Bykov, to name only a few.
My father and I discussed how sad it was that Aitmatov had not been awarded the Nobel Prize in literature. And we regretted this not so much for Aitmatov's sake, but for the Islamic world into which he was born and which shaped his cultural heritage. One of the possible reasons for Islam's radicalization has been the need to define itself and to reconcile its great past with the fact that it lags behind Western civilization. Any recognition the West gives to the Islamic society's contribution to world culture increases its sense of self-respect and dignity.
For the people in the secularized Islamic former Soviet republics of Central Asia who are searching for their identity while feeling pressure from radical Islam, international recognition such as the Nobel Prize would be extremely important. And if Aitmatov received such a prestigious award, it would have been justly earned in its own right and by no means be construed as a patronizing or politically correct gesture by Western institutions.
But it is not too late to take positive action. In Kazakhstan, for example, Nurpeisov continues to create fine works of literature. If Western countries give him, and others like him, due recognition, they would do the world a great service.
Alexei Pankin is the editor of IFRA-GIPP Magazine for publishing business professionals.

redpajama_set_name: RedPajamaCommonCrawl


A new week is going to start, bringing you new motivation and new invitations for the wedding. Wedding season is on and you need to get yourselves prepared for it. Here are some health tips just in case you plan to reduce your weight or belly fat . If you are wearing a saree or lehenga, your tummy lurking out would ruin your entire look. If you have got belly fat, it makes your outfit doesn't suit you. In order to look amazing, you could go for some fitness tips.
Go for a diet low on sugar and low on carbs(carbohydrates). Since eating a lot of carbohydrates and even salt could cause blotting in your stomach and make you fat. It doesn't mean that you totally quit eating salt because in summers you really need intake of salt. Just minimise the amount. Also, don't quit carbs, just make the quantity less. You would see results very soon.
A regular exercise could do wonders to your body. If you are not exercising and sitting on a chair eating all the day and even if you are sitting on a chair but eating healthy , it would not give your results. For good results, you need to start working out. Working out makes you feel good and helps you reduce stress also. You must consume more healthy things. Go for fruits, coconut water, watermelons to keep yourself hydrated. Drink more water and green tea to burn fat. You could also go for low sugar ice creams and low fat yoghurt if you want to eat something delicious. You could go to some health cafes and have amazing food there. This all would keep you results very soon.


I raved to you about my love for the mini silicone baking cups which pretty much eliminate the need for disposable baking cups and help reduce the need for cooking spray or oil. The set I purchased also came with these adorable mini loaf pans, and I'd like to give a few tips for using these.
If you have a metal pan which is divided into rectangular sections to make mini cakes or loaves, you can line the sections with these and eliminate the need for cooking spray. However, I just put them on a baking sheet lined with a Velesco silicone baking liner to catch any drips when I was pouring the batter into them and baked them at 325 Fahrenheit for 20 minutes.
I would suggest not filling the mini loaf cups more than between half and two-thirds of the way full. I ended up with a bit of overhang filling them 3/4 full. This is more a cosmetic issue than anything else, and if you are going to frost them, you can always trim the excess.
These mini loaves or cakes would be wonderful for parties, either for adults or children. For a kids' party, you could even let the youngsters decorate their own mini cake, providing frosting and fun things like sprinkles or gummy bears. This might be best in summer months where you could set up a picnic table outdoors and let the tykes get as messy as they wanna be.
You could decorate the cakes to look like little cars for auto enthusiasts, either adult or juvenile. You could make a train with the mini cakes. The possibilities are endless.
I wasn't feeling quite that inspired when I tried out my mini cake/loaf pans. I just had a box of spice cake mix that I wanted to use up!
Just like their round counterparts, these mini loaf cups get the hat trick. They are inexpensive, they cut down on overall expenses over time by reducing the need for cooking spray and cupcake liners, and they easy (and fun) to use. They definitely earn the Seal of Approval!


There are lots of sites telling you what to do in order to find the right home but knowing what not to do can be just as important. After finding the right home and dealing with all of the paperwork, many buyers, quite understandably, start making plans to move in and put their personal touches on the home.
In today's tenuous lending environment, little things can derail the process which isn't over until the papers are signed at the closing and your new deed is recorded. When you first applied for your loan, your lender verified your income, assets and job to determine if you qualify for the mortgage. Right before the closing, your lender will check these verifications again (sometimes 24 hours before the closing) to make sure there haven't been any material changes to your credit or income that might disqualify you from getting your loan.
It's understandable to be excited about one of the biggest decisions you'll make and that you feel you need to be getting ready for the move.
Planning is smart but don't do anything that would affect your credit or income while you're waiting to close on your new home.


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Legion of Leia
Posted on Published: December 8, 2020 Categories Movies
By: Author Mirko Parlevliet
Viggo Mortensen's Falling to Release in February 2021
Home » Viggo Mortensen's Falling to Release in February 2021
Perceval Pictures and Quiver Distribution announced today that they will be partnering on the U.S. release of Academy Award nominee Viggo Mortensen's (Captain Fantastic, Eastern Promises) directorial debut Falling.
The drama stars Viggo Mortensen, who also penned the script, alongside Academy Award nominee and Golden Globe winner Laura Linney (Ozark, The Savages), Golden Globe nominee Lance Henriksen (Aliens, Millennium), Terry Chen (Jessica Jones, House of Cards), Sverrir Gudnason (The Girl in the Spider's Web, Borg vs. McEnroe) and Hannah Gross (Mindhunter, Joker).
Following its festival run this year at Sundance, Cannes, Toronto and San Sebastian Film Festivals, Falling will be released in theatres, On Digital and On Demand February 5, 2021, in time for 2020 awards consideration. Henriksen just won the International Film Festival and Awards Macao (IFFAM) Competition award for Best Actor for his performance in Falling.
Mortensen, who composed the score, also produced the film alongside Scythia Films' Daniel Bekerman and Zephyr Films' Chris Curling. The Executive Producers include Danielle Virtue, Brian Hayes Currie, Peter Touche, Stephen Dailey, Peter Hampden and Norman Merry.
The Director of Photography is Marcel Zyskind, the Production Designer is Carol Spier, the Editor is Ronald Sanders, and the Costume Designer is Anne Dixon.
"Viggo Mortensen has fashioned a complex tale of love lost and regained that explores generational tensions," said Quiver Co-Presidents Berry Meyerowitz and Jeff Sackman.
"This is a stunning directorial debut from Viggo with a stand-out performance from Lance Henriksen, and we are excited to see this one through the awards season."
Mortensen said: "While not strictly autobiographical, some events and conversations in Falling are inspired by the childhood I shared with my two brothers, Charles and Walter, to whom the movie is dedicated. It is a complex story, layered throughout with the subjective, imperfect memories of its principal characters.
"Most people, it seems to me, yearn to be seen and heard, and thrive on empathy. It took me a long time to accept my own father as he was, with as little judgment as possible, but I'm glad that I eventually did so. And he returned the gesture, for which I'll always be grateful."
He added: "Falling is, in large part, about the importance of accepting and forgiving, of learning to live with others and with oneself. I feel very fortunate to be collaborating with Quiver in order to bring this story of hard-won grace and compassion to U.S. audiences."
Falling follows John (Mortensen) who lives with his partner, Eric (Chen), and their daughter, Mónica (Gabby Velis), in California, far from the traditional rural life he left behind years ago. John's father, Willis (Henriksen), a headstrong man from a bygone era, lives alone on the isolated farm where John grew up.
Willis is in the early stages of dementia, making running the farm on his own increasingly difficult, so John brings him to stay at his California home so that he and his sister Sarah (Linney) might help him find a place near them to relocate to.
Unfortunately, their best intentions ultimately run up against Willis's adamant refusal to change his way of life in the slightest.
FallingLance HenriksenLaura LinneyPerceval PicturesQuiver DistributionViggo Mortensen
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Science beyond medicine
Sunday, 11 February 2018, 15:16
(Representational Image)
"For wellness, the manifest and unmanifest state must be equilibrium"
New Delhi, February 11, 2018:
All recognised systems of medicine work at different levels. While modern 'particle-based' medicine treats the disease, the 'wave'-based medicine seeks the origin of the disease. However, ultimately, all systems of medicine work towards one common end point, which is, healing.
The duality of the human body exists at the level of photon and symbolizes the concept of Purusha the unmanifest (consciousness) and Prakriti (matter) or the manifest. For wellness, the manifest and the unmanifest state should be in equilibrium.
Matter is made up of atoms, which can be broken down to subatomic particles (protons, neutrons and electrons), photons, quantum and finally to wave. A photon is both a wave and a particle, which has been termed as the wave particle duality. Vedic knowledge says that we are both particle and a wave at the same time. The manifest is the disturbed state of mind and the visible body. Unmanifest is the state where there is no matter. It is the beginning of all things and this state can be described as "potential" or "shunya". This is the extreme parasympathetic state of the body.
Delivering a guest lecture during the World Congress of Holistic Medicine organized by Tamil Nadu Dr MGR Medical University Padma Shri Awardee Dr K K Aggarwal, President Heart Care Foundation of India (HCFI) and Immediate Past National President Indian Medical Association (IMA), said, "Modern medicine mainly takes care of the particle part of the body. The manifest part of the mind is covered in the subject of psychiatry taught over only a few classes during MBBS. The unmanifest soul consciousness us usually silent in modern medicine.
Nature is made up of five elements – air, space, water, fire and earth. These five elements must be in a balanced state. Any disturbance of these five elements will result in natural disasters such as volcanic eruptions, hurricanes, earthquakes, landslides, floods, forest fire, storms etc. Naturopathy or nature cure uses these five elements in wellness. It balances these five elements and using them as a part of therapy. This includes sunbath for vitamin D deficiency and daily wash for personal hygiene."
Imbalance of the three Doshas of humors : movement, metabolism and structure is responsible for any disease. Ayurveda is the science of balancing body humors, the three Doshas and the seven dhatus, which can be described as a type of primordial prevention. Homeopathy works at the level of nanotechnology (potentization) and at the level of quantum energy (trituration) on the principle that the action of a drug potentiates till 6X and 6C levels but reverses beyond that.
Adding further, Dr Aggarwal, who is also Group Editor of IJCP, said, "Just as photon is the smallest unit of light and quantum is the smallest unit of energy, Qualia is the smallest unit of thought. Quantum teleportation is the transportation of quantum information via the penomenon of entanglement. At the level of quantum, consciousness is the extreme parasympathetic state of mind or Samadhi."
Modrn medicine works on the manifest at the level of tissues and organs and is the science of acute emergencies, advanced surgical strikes and advanced diagnostics.
When two photons particles interact so that they act similar even when separated by huge distances, this is called quantum entanglement and it occurs at a speed much faster than the speed of light. In this entangled state, the two photons share an existence or characteristics (quantum information measured as quantum bits 'qubits') and are interdependent. When one is altered in any way, the other too is altered even at a distance. Believing this phenomenon to be impossible, Albert Einstein called this connection as "spooky action at a distance".
Jaypee Hospital organized the 2nd edition of 'Noida Grand Marathon'.
Quackery must be tackled in an urgent manner


We're are back in Kuala Lumpur! We are extremely delighted once again to partner with Malaysia Digital Association (MDA) this year! Kicking off this year in KL, we are taking our recent Programmatic and Mobile Training curated by our specialist Committees in collaboration with the MDA.
We're offering full day training and will be followed by an industry meet and greet so make sure you let your regional offices know. Also, MDA Members get IAB Membership rates to our partnership events as we are one, big, happy family.
You are no stranger to these acronyms: DMP, DSP, SSP, DCO, SEO, SEM …. but you want to know more and adopt a hands-on approach to your own ad tech needs.
Hear from those who have taken the automated ad buying and selling journey and find out how they steered through the digital fog and created blueprints suited to their needs.
MDA & IAB Singapore are collaborating once again this year to bring the experts in the Programmatic field together to share their knowledge with you. This will be followed by our interactive breakout sessions during which you can have all of your questions answered.
09:30am – What are the options for a Brand? Go for it alone or partner for success?
Mobile is eating the internet and taking over the world! Brands are already embracing the vast opportunities it offers in the region for audience targeting and relevant consumer messaging at scale. Get beyond the hype and learn how to use Mobile to your advantage.
After our successful collaboration in 2015, the MDA & IAB Singapore are hosting this training workshop where Mobile experts from agencies, brands and their partners will share their secrets and then converge to share knowledge and experience in six key areas of Mobile solutions.
Don't miss your opportunity to learn how to harness the power of mobile.


Please read the instructions below to register your child for camp using our system, CampDoc.
The only option it should give you for registration is "2019 >> Campers 2019". Check that off and press "continue" (bottom right corner).
It will then take you to the protection plan information. DECLINE THE PROTECTION PLAN. (this is something we do not use for our camp), then press "continue".
This will then take you to the application.
For returning campers, it should allow you to update and confirm information, and it will ask you to answer a few new questions as you go along.
For new campers, you will have to complete each section.
Your application is complete when each section on the right-hand side of the page has a green check mark next to it ü, rather than a red dot l. Those with a red dot still need questions answered. To further ensure that your application is complete, the bottom of the application it gives you a percentage of completion, and you should receive an e-mail when the application needs to be completed or is completed. The application will also tell you the last time it was saved (it saves as you go).
If you register a camper for camp but later find out the camper is unable to attend, please e-mail me to let me know and I can delete the camper's application.
If you have any questions or need any help, please do not hesitate to reach out!


text:
package jp.fieldnotes.hatunatu.dao.impl.dao;

import jp.fieldnotes.hatunatu.dao.impl.bean.Department2;

public interface Department2AutoDao {

    public Department2 getDepartment(int deptno);

    public void insert(Department2 department);
}


text:
Loma is a city in Cavalier County, North Dakota, United States. The population was 10 at the 2020 census.  Loma was founded in 1905.

Geography
Loma is located at  (48.637896, -98.525406).

According to the United States Census Bureau, the city has a total area of , of which  is land and  is water.

Despite the small number of residents, Loma and nearby Alsen each cover approximately as much land within their corporate boundaries as Minot.

Demographics

2010 census
As of the census of 2010, there were 16 people, 7 households, and 3 families residing in the city. The population density was . There were 13 housing units at an average density of . The racial makeup of the city was 100.0% White.

There were 7 households, of which 28.6% had children under the age of 18 living with them, 28.6% were married couples living together, 14.3% had a female householder with no husband present, and 57.1% were non-families. 57.1% of all households were made up of individuals, and 28.6% had someone living alone who was 65 years of age or older. The average household size was 2.29 and the average family size was 4.00.

The median age in the city was 43 years. 25% of residents were under the age of 18; 12.6% were between the ages of 18 and 24; 12.5% were from 25 to 44; 31.3% were from 45 to 64; and 18.8% were 65 years of age or older. The gender makeup of the city was 56.3% male and 43.8% female.

2000 census
As of the census of 2000, there were 21 people, 9 households, and 7 families residing in the city. The population density was 0.8 people per square mile (0.3/km2). There were 10 housing units at an average density of 0.4 per square mile (0.1/km2). The racial makeup of the city was 100.00% White.

There were 9 households, out of which 11.1% had children under the age of 18 living with them, 55.6% were married couples living together, 11.1% had a female householder with no husband present, and 22.2% were non-families. 22.2% of all households were made up of individuals, and none had someone living alone who was 65 years of age or older. The average household size was 2.33 and the average family size was 2.71.

In the city, the population was spread out, with 19.0% under the age of 18, 4.8% from 18 to 24, 19.0% from 25 to 44, 19.0% from 45 to 64, and 38.1% who were 65 years of age or older. The median age was 58 years. For every 100 females, there were 110.0 males. For every 100 females age 18 and over, there were 112.5 males.

The median income for a household in the city was $63,750, and the median income for a family was $63,750. Males had a median income of $43,750 versus $18,750 for females. The per capita income for the city was $18,245. None of the population and none of the families were below the poverty line.

References

Cities in Cavalier County, North Dakota
Cities in North Dakota
Populated places established in 1905
1905 establishments in North Dakota

redpajama_set_name: RedPajamaWikipedia


text:
La Vie De Mahomet
The French satirical newspaper Charlie Hebdo has published the second part of its comic biography of Mohammed, La Vie De Mahomet, by Stephane Charbonnier and Zineb el Rhazoui. The comic depicts Mohammed's adult life, and is subtitled Le Prophete De L'Islam. The first part, which dealt with Mohammed's childhood, was published earlier this year.
Charlie Hebdo first published a Mohammed cartoon appeared in 2002, followed by a front-page Mohammed caricature in 2006. This was followed by a Charia Hebdo edition 'guest-edited' by Mohammed in 2011, and a naked Mohammed caricature last September (criticising the protests against Innocence Of Muslims).
Mohammed cartoons first caused controversy when a dozen of them were published by Jyllands-Posten in 2005. Since then, many other newspapers and magazines have also printed Mohammed caricatures: Weekendavisen, France Soir, The Guardian, Philadelphia Daily News, Le Monde, Liberation, Het Nieuwsblad, The Daily Tar Heel, Akron Beacon Journal, The Strand, Nana, Gorodskiye Vesti, Misselijke Grappen, HP/De Tijd, Dagbladet, Adresseavisen, Uke-Adressa, Harper's, and the International Herald Tribune (in 2006 and 2012).
Categories: blasphemy, censorship, media


Just like any online transaction, betting too carries the risk of your finances getting misappropriated by hackers searching for weak points in a particular sports betting web site, or by way of the managers of unethical web sites themselves. In such a case you need to safeguard your finances and a strong e-check facility is one particular method which can ensure that your funds continues to be safe and sound. You can now wager safely within on line pro sports betting with e-check service and revel in your own profits without any issues.
Just about all online sports wagering web sites accept credit cards as means of performing inbound and outgoing transactions. However, there are hackers which victimize sites that have inadequate security measures in addition to the ones that might permeate into your own laptop or computer too, in an effort to grab essential information like PIN numbers or even other important codes. This may severely endanger your safety and result in losing crucial finances since your money could easily be re-directed towards the hacker's accounts without your own knowledge.
It is exactly because of these kinds of conditions that many reputed internet sites have used the particular e-check facility to accept deposits from bettors. An e-check is just like writing away any paper check in favor of your sports book every time you might want to deposit money from your personal banking account in the sports book account. This would be necessary before you place your own wagers at that site. You will also have to present extra verification to the sports book internet site in the form of any cancelled check of your bank account before employing this e-wallet feature. This facility simply leaves no room for any mischief and is consequently preferred by many esteemed online gambling sports books.
Irrespective of whether you love to bet on golf, baseball, football, football, basketball, horse racing or even even if you prefer to bet on car races on a professional or perhaps college level, you will certainly come across on line sportsbooks that are prepared to carry out dealings using the e check service. Whether you are into betting merely as a hobby to inject some fun into your daily life or have made it a paying enterprise, the e-check facility will certainly benefit you by means of holding your own hard-earned money safe and sound within your account. Since on-line bookies could be based outside of the borders of USA, it would be very difficult to kick-off any legitimate action if your money ended up being siphoned-off in a illegal manner into some other account.
You might really like making straight bets or even parimutuel bets just before each game as well as your attention too would be focused on winning your own bets instead of pondering on ways to keep your finances safe. An e-check service will simply help you to securely understand each betting line as well as compute the odds of winning rather than worrying about the security of your funds. Now you can get pleasure from viewing and wagering on your preferred sport and collecting your winnings without any problems.
An e-check service simply enhances the security element whilst dealing with online sports betting sites. You need to thus seek out online pro sports betting with e-check facilities in order to securely place your own gambling bets within their virtual fortified walls and gather your winnings without any worries.


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Home » News » Car News » 5 SUVs worth waiting for in 2017 under Rs. 30 lakh
5 SUVs worth waiting for in 2017 under Rs. 30 lakh
By Rahul Kapoor | on December 31, 2016
A number of SUVs and 4X4s are expected to arrive in India with some making their global debut already. Here is a list of SUVs under the Rs. 30 lakh mark that we feel are worth waiting for in 2017.
Audi will launch their smallest SUV they have ever made, the Audi Q2 in India in 2017. The baby Audi SUV measures in at 4191mm in length, 1794mm wide and 1508mm tall with a wheelbase of 2601mm. The Q2 will sit below the Q3 in the price bracket of Rs. 25-30 lakh. Powering Audi's smallest SUV will get a host of engines internationally, but a turbo petrol 1.4-litre four-cylinder engine and 1.6-litre four-cylinder turbo diesel will be on offer with 7-speed DSG automatic gearbox.
Expected launch date: Mid 2017
In January, Tata will finally launch the much awaited Hexa in India which the have been carefully developing for quite some time. The Tata Hexa will be in competition to Toyota's Innova Crysta and the Mahindra XUV500. The Hexa will come with two engine options, the Varicor 320 2.2-litre, 4-cylinder, diesel engine mated to a 5-speed manual gearbox which develops 148bhp and 320Nm of torque. In addition, the higher spec car will be offered with the same engine tuned to Varicor 400 spec which develops 154bhp and 400Nm of torque. The more powerful engine will come with a 6-speed manual with a 4X4 option and a 6-speed automatic with only rear-wheel drive. Tata will launch this 7-seater all-wheel drive MPV in mid January and will price it competitively and with a vehicle fully loaded with features and gadgets, it might be very good value for money. We expect the Hexa to be priced from Rs. 14 lakh onwards.
Expected launch date: Mid January 2017
After arriving in India with a bang, Jeep will commence its operations at their facility in Ranjangaon when they will start to produce the Jeep Compass SUV which will also be exported to global markets. The Compass will be arriving with a host of engines on offer globally, however it is still unconfirmed which of the 17 powertrain combinations would be available for Indian buyers. The Compass will get A 1.4-litre multi air-turbo, a 1.6-litre and a 2.0-litre turbo diesel engines and a 2.4-litre Tigershark petrol engine. As for gearboxes go, a a 6-speed manual and automatic are available globally in addition to a 7-speed and 9-speed automatics. The car will also get 4WD system called "Jeep Active Drive – SelecTerrain 4×4 system. Jeep will price the Compass around the Rs. 20-25 lakh price bracket and will compete against the Ford Endeavour and the Toyota Fortuner.
Last year Volkswagen had announced that they will renter an SUV in the Indian market. After the Touareg failed to gain popularity in India, VW has decided to aim a little bit lower. Targeting Fortuner and the Endeavour as its main rivals, Volkswagen will launch the Tiguan in India. The Tiguan will be powered in India by a 2.0-litre, 4-cylinder TDI diesel or TSI petrol engines, both mated to a 7-speed automatic DSG gearbox. Pricing will be key for the Tiguan for it to be a success, and some amount of localisation and smaller engines expected to aide its competitiveness in the market in terms of pricing
Expected launch date: First half of 2017
Of similar size as the sister Tiguan, Skoda will also enter India with brand new Kodiaq. Engines will be the same 2.0-litre TSI petrol and TDI diesel which will come with a 7-speed DSG transmissions and all-wheel drive with driving modes. The turbocharged petrol engine produces 177bhp and 320Nm of torque. The TDI diesel engine in the Kodiaq will churn out 147bhp and 340Nm of torque. Skoda is likely to cram the Kodiaq with creature comforts and features with an all new infotainment screen with Apple CarPlay and Android Auto in addition to navigation. The Kodiaq in India, will also get three-row seating and will compete with the Fortuner, Endeavour, SantaFe and its sister Tiguan when both arrive on Indian shores.
Expected launch date: Second half of 2017
Tags: volkswagen tiguan Skoda Kodiaq Tata Hexa Jeep Compass Audi Q2
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MG Hector vs Tata Harrier vs Jeep Compass: Spec Comparison
Jeep Compass Trailhawk bookings officially open
Skoda Kodiaq RS Review: First Drive
Jeep Compass Trailhawk - Photo Gallery
2019 Jeep Compass Trailhawk Review: First Drive


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By Amy Wobbema
Independence Day Fun
Amy Wobbema
B & B Racers, LLC of Minot brought excitement downtown Saturday with their portable go-kart track. "We went nonstop," said Chris Brown, co-owner of B & B Racers, LLC. "This was the busiest small town celebration we've ever done." Residents and visitors were all smiles, even as they waited in line. Scattered showers that passed quickly overhead gave attendees a reprieve from the heat. The go-karts were sponsored by the New Rockford Area Chamber of Commerce, New Rockford Eagles Club and New Rockford Lions Club, which made it possible for everyone to ride free. "This is the coolest thing that's ever come to New Rockford," one resident said as he watched.
New Rockford and Sheyenne had a lot of fun on tap for Independence Day weekend. Scroll through this photo gallery to get the rundown.


We need a very simple splash page and landing page for a community (free) WiFi mesh network we are building. This s...very simple splash page and landing page for a community (free) WiFi mesh network we are building. This site will be linked to our organization's main website. I work in Photoshop, and will prepare all images myself, including logos.
Hi, i have a logo which i need modifying and developing.
We have an amazing opportunity for you we are looking for a PHP developer who is dedicated to the craft, writes code that they are proud of and c...ways. You will be a part of a full-stack creative team that is responsible for all aspects of the ongoing software development from the initial specification, through to developing, testing and launching.
Hi All, I am looking for a beautiful and meaningful logo design for one of our Call Center Software ...than 3 colors) and design everything in those one or two or three shades. Focus on combination of Intelligence and Call Center concept. Once awarded, we will need photoshop PSD, JPG/PNG and necessary files for the same. Best of Luck !!!


text:
Power Hour: The Ultimate Test of Endurance Drinking Game - Disclaimer / Photo by cottonbro studio
Are you looking for a new and exciting drinking game to play with friends? Look no further than the Power Hour, the ultimate test of endurance. This game is a combination of drinking and music, and it's a guaranteed good time.
Equipment needed to play Power Hour:
Shot glasses (1 per person)
A timer or clock
A music device with a playlist of 60 songs (1 song per minute)
Optional: additional rules, tasks or challenges to make the game more challenging
Optional: snacks and food to enjoy while playing
Optional: a designated driver or other transportation options
Optional: a camera to capture the moments
Optional: a whiteboard or chalkboard to keep track of the rules and score
Optional: prizes for the winner(s)
The objective of the Power Hour is to drink one shot of beer every minute for an hour. The game is best played with a group of friends and a playlist of 60 songs, with each song lasting one minute. When the first song starts, everyone takes their first shot of beer. As each song plays, everyone takes another shot. The game continues until the end of the hour, and whoever makes it to the end without stopping is the winner.
To make the game more challenging and fun, you can add different rules, such as taking two shots during a certain song or finishing your beer before the next song starts. You can also assign different tasks or challenges to be completed at certain points during the game.
It's important to remember to drink responsibly and to pace yourself throughout the game. It's also a good idea to have a designated driver or other transportation options available.
The Power Hour is a great way to spend an evening with friends, and it's sure to provide plenty of laughs and memories. Just remember to drink responsibly and have a designated driver or other transportation options available. Have fun and enjoy the game!
Previous article Never Have I Ever: The Truth-Telling Drinking Game
Next article Cheers to the Governor: A Guide to the Ultimate Counting Game


text:
Q: implementing google auth code in Xamarin forms project (not the authentication through google) I want google authentication for my Xamarin froms app
I have tried the soultions uploaded by Hossem dellai and I have tried the get and post method after creating client id and secret through google apis and got the access token but where do we implement the provided code in visual studio because there is no more portable class library and you cant create a webview as well.I have also tried with the xamarin.Auth nuget package and yet my issue isnt still resolved.All these last solutions have been updated atleast 3 years ago so need some fresh expert advise.Im using visual studio 2019.I want to know where exactly should I place the code in my project.

A: You can do most of the operations in PCL , and then you just need to do a callback in the Android or ios project(Close authorized page and Load redirectUrl page).
For example,Android CallBack like (you also could integrate on MainActivity and then get the data in the OnNewIntent method):
[Activity(Label = "ActivityCustomUrlSchemeInterceptor", NoHistory = true, LaunchMode = LaunchMode.SingleTop)]
[IntentFilter(
    new[] { Intent.ActionView },
    Categories = new[] { Intent.CategoryDefault, Intent.CategoryBrowsable },
    DataSchemes = new[] { "<your data scheme>" },
    DataPath = "/oauth2redirect")]
public class ActivityCustomUrlSchemeInterceptor : Activity
{
    protected override void OnCreate(Bundle savedInstanceState)
    {
        base.OnCreate(savedInstanceState);
        global::Android.Net.Uri uri_android = Intent.Data;
        // Convert Android.Net.Url to Uri
        var uri = new Uri(uri_android.ToString());

        // Close browser
        var intent = new Intent(this, typeof(MainActivity));
        //intent.SetFlags(ActivityFlags.ClearTop | ActivityFlags.SingleTop);
        StartActivity(intent);

        // Load redirectUrl page
        OAuthAuthenticatorHelper.AuthenticationState.OnPageLoading(uri);

        this.Finish();
    }
}

Ios is similar to this,you can refer to other more
AuthExample

redpajama_set_name: RedPajamaStackExchange