Mohammad Alkhatatbeh

The origin of circulating CD36 in type 2 diabetes

Objective:Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36+MPs in persons with T2DM.Design:An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI)=39.9±6.4 kg m−2; age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI=23.6±1.8 kg m−2 and 33.5±5.9 kg m−2, respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235a, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations.Results:CD36+MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235a+=35.8±14.6%); although this did not correlate with haemoglobin A1c. By contrast, the main source of CD36+MPs in non-T2DM individuals was endothelial cells (CD105+=40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 μg ml−1 and was positively correlated with CD36+MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36+MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively).Conclusions:Both the levels and cellular profile of CD36+MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.

Strategies for enumeration of circulating microvesicles on a conventional flow cytometer: Counting beads and scatter parameters.

Enumeration of circulating microvesicles (MVs) by conventional flow cytometry is accomplished by the addition of a known amount of counting beads and calculated from the formula: MV/μl = (MV count/bead count) × final bead concentration. We sought to optimize each variable in the equation by determining the best parameters for detecting ‘MV count’ and examining the effects of different bead preparations and concentrations on the final calculation. Three commercially available bead preparations (TruCount, Flow-Count and CountBright) were tested, and MV detection on a BD FACSCanto was optimized for gating by either forward scatter (FSC) or side scatter (SSC); the results were compared by calculating different subsets of MV on a series of 74 typical patient plasma samples. The relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations (R 2 ≥ 0.997). However, TruCount beads produced the most consistent (concentration variation = 3.8%) calculated numbers of plasma CD41+/Annexin V+ MV, which were significantly higher from that calculated using either Flow-Count or CountBright (p < 0.001). The FACSCanto was able to resolve 0.5 μm beads by FSC and 0.16 μm beads by SSC, but there were significantly more background events using SSC compared with FSC (3113 vs. 470; p = 0.008). In general, sample analysis by SSC resulted in significantly higher numbers of MV (p < 0.0001) but was well correlated with enumeration by FSC for all MV subtypes (ρ = 0.62–0.89, p < 0.0001). We conclude that all counting beads provided linear results at concentrations ranging from 6 beads/μl to 100 beads/μl, but TruCount was the most consistent. Using SSC to gate MV events produced high background which negatively affected counting bead enumeration and overall MV calculations. Strategies to reduce SSC background should be employed in order to reliably use this technique.

Analysis of circulating adipokines in patients newly diagnosed with solid cancer: Associations with measures of adiposity and tumor characteristics

The development and progression of cancer is a complex and multifactorial process and the global prevalence of obesity is markedly increasing. A number of studies have made an association between obesity and increased rates of epithelial tumors. Obesity is associated with altered adipokine levels, potentially contributing to the process of tumor development and metastasis. In the current study, the associations between circulating adipokines and measures of adiposity and tumor characteristics among patients diagnosed with solid malignancies were examined at the time of presentation, and following the administration of chemotherapy. A total of 30 patients with cancer and matched healthy controls were enrolled in the present study. Plasma adipokine levels of hepatocyte growth factor (HGF), adiponectin and leptin were determined using commercially available ELISA kits. At baseline, plasma HGF, adiponectin and leptin levels were not significantly different between patients with cancer and the healthy controls. Circulating HGF levels were significantly associated with the stage of cancer at diagnosis (P=0.044), but lacked a significant association with lymph node status (P=0.194). Plasma adiponectin and leptin levels were not significantly associated with tumor characteristics at the time of diagnosis. Only leptin was positively correlated with the body mass index of patients with cancer (P<0.001). No significant correlations were detected between the evaluated adipokines and measures of visceral obesity, as determined by waist circumference and the waist-hip ratio at presentation. Following administration of chemotherapy, adiponectin was the only adipokine evaluated in the current study that exhibited a significant difference, when compared with baseline plasma levels (P=0.013), and a significant positive correlation between baseline and follow-up circulating levels (P=0.002) among patients with cancer. In addition, there were no significant inter-correlations between circulating adipokines at baseline level and during follow-up in patients with cancer. Collectively, the findings of the current study suggest a lack of diagnostic roles for the adipokines investigated and no significant association with measures of adiposity. Adiponectin may be a potential adipokine to measure in patients with cancer, in order to further assess its prognostic and predictive potential.

Circulating CD36+ microparticles are not altered by docosahexaenoic or eicosapentaenoic acid supplementation ☆

BACKGROUND AND AIMS Circulating microparticles (MP) are the source of a plasma derived form of the scavenger receptor CD36, termed soluble (s)CD36, the levels of which correlate with markers of atherosclerosis and risk of cardiovascular disease. Long chain n-3 polyunsaturated fatty acids have cardioprotective effects that we have previously reported to be gender specific. The aim of this study was to determine if dietary docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA) supplementation affect circulating CD36 + MP levels, and if this occurs differentially in healthy men and women. METHODS AND RESULTS Participants (43M, 51F) aged 39.6 ± 1.7 years received 4 weeks of daily supplementation with DHA rich (200 mg EPA; 1000 mg DHA), EPA rich (1000 mg EPA; 200 mg DHA), or placebo (sunola) oil in a double-blinded, randomised, placebo controlled trial. Plasma CD36 + MP were enumerated by flow cytometry and differences between genders and treatments were evaluated by Students or paired t-test and one way ANOVA. Males and females had similar levels of CD36 + MP at baseline (mean = 1018 ± 325 vs 980 ± 318; p = 0.577) and these were not significantly changed after DHA (M, p = 0.571; F, p = 0.444) or EPA (M, p = 0.361; F, p = 0.901) supplementation. Likewise, the overall percent change in these levels were not different between supplemented cohorts compared to placebo when all participants were combined (% change in CD36 + MP: DHA = 5.7 ± 37.5, EPA = -3.4 ± 35.4, placebo = -11.5 ± 32.9; p = 0.158) or stratified by gender (M, DHA = -2.6 ± 30.6, EPA = -15.1 ± 20.1, placebo = -21.4 ± 28.7, p = 0.187; F, DHA = 11.7 ± 41.5, EPA = 6.8 ± 42.9, placebo = -2.8 ± 34.7, p = 0.552). CONCLUSION The cardioprotective effects of DHA and EPA do not act through a CD36 + MP mechanism.

Smoking prevalence, knowledge and attitudes among primary healthcare professionals: a study from Jordan

This was a questionnaire-based cross-sectional study of 400 healthcare professionals recruited from primary healthcare centres in northern Jordan between April and October 2015. The questionnaire included questions about smoking behaviour, risks, opinions and providing anti-smoking counselling. More than 80% of participants reported that smoking-free policies were not enforced at primary healthcare centres. Compared to hospitals and the general population, smoking was less prevalent among primary healthcare professionals and more prevalent in men. More than 90% of participants believed that smoking was dangerous and associated with cardiovascular and respiratory diseases. Around 92% believed that they should set a good example to patients by not smoking and advise them about smoking cessation. Only 15.3% of participants felt well prepared when counselling patients about smoking and 92.8% believed that they needed training. This study suggests that primary healthcare professionals should act as anti-smoking role models after receiving professional training.

Non-cardiac Chest Pain and Anxiety: A Possible Link to Vitamin D and Calcium

This study was performed to check the hypothesis that low serum vitamin D and reduced calcium intake may contribute to the comorbidity of psychological symptoms (anxiety and depression) and non-cardiac chest pain (NCCP). The design was a case–control study that involved 40 subjects with NCCP and 40 age and gender-matched healthy controls. Serum vitamin D was assessed using electrochemiluminescence immunoassay; anxiety and depression symptoms were assessed using Hospital Anxiety and Depression Scale, and dietary calcium intake was assessed by self-reporting. Subjects with NCCP had higher anxiety and depression scores and lower vitamin D and dietary calcium intake compared to healthy controls (p < .05). NCCP was associated with anxiety score (odds ratio = 1.40, p < .01). Prevalence of abnormal anxiety score was much higher in subjects with NCCP (47.5%) compared to healthy controls (7.5%). Anxiety score was inversely correlated with vitamin D level and dietary calcium intake (p < .01). Anxiety score was inversely associated with vitamin D level (R2 = .32, p < .05). In conclusion, development of NCCP can be predicted from increased anxiety score which in turn can be predicted from low vitamin D levels. This suggests physicians to consider anxiety and vitamin D deficiency as possible causes for NCCP.

Impaired Awareness of Hypoglycaemia in Insulin-treated Type 2 Diabetes Mellitus

BACKGROUND Patients with Type 2 Diabetes Mellitus (T2DM) may develop hypoglycemia as an adverse effect of insulin therapy. Hypoglycemia has dangerous consequences that result from neuroglycopenia and hypersecretion of counter-regulatory hormones. Patients who recognize early symptoms of hypoglycemia can initiate self-treatment and rectify the situation. Impaired awareness of hypoglycemia (IAH) predisposes patients to severe hypoglycemia and unconsciousness. OBJECTIVE To assess prevalence of IAH, intensity of hypoglycaemic symptoms, frequency of severe hypoglycemia and factors associated with IAH in patients with insulin-treated T2DM. METHOD This is a cross-sectional study that used Clarkes and Golds surveys to assess IAH and Edinburgh survey to assess intensity of hypoglycemic symptoms in patients with insulin-treated T2DM (n= 388). Frequency of hypoglycemia and other data were collected by self-reporting or from medical records. RESULTS The prevalence (95% confidence interval) of IAH was 17.01% (13.27%-20.75%) as determined by Clarkes method and 5.93% (3.58-8.28) by Golds method (Odds= 3.25, p-value<0.00001). Drowsiness, hunger, sweating, tiredness, trembling and weakness, were the most intense hypoglycaemic symptoms, and 6.19% of participants reported at least one episode of severe hypoglycaemia within the past year. Regardless of classification method used, IAH was not dependent on age, gender, duration of T2DM or duration of insulin therapy (p-values>0.05). Instead, IAH was positively associated with frequency of hypoglycaemia during the previous six months (p-value<0.05) and development of severe hypoglycaemia within the past year (p-value <0.05). CONCLUSION This study highlights large variability in IAH depending on the method used for assessment. Increased hypoglycaemia frequency may increase the prevalence of IAH and thus the development of severe hypoglycemia.

Prevalence of musculoskeletal pain in association with serum 25‑hydroxyvitamin D concentrations in patients with type 2 diabetes mellitus

The aim of the present study was to investigate the prevalence of musculoskeletal pain in patients with type 2 diabetes mellitus (T2DM) in association with 25-hydroxyvitamin D levels, anxiety, depression and neuropathy. A cross-sectional study was conducted involving a total of 124 T2DM patients. Musculoskeletal pain was determined by self-reporting of painful body sites. Pain intensity was assessed using a scale of 0-10. Anxiety and depression were assessed using the Hospital Anxiety and Depression Scale. Neuropathy was assessed using the PainDETECT questionnaire. The concentration of 25-hydroxyvitamin D was measured using liquid chromatography-tandem mass spectrometry. Fasting blood sugar (FBS) was determined using the hexokinase method and glycated hemoglobin (HbA1c) level was determined using turbidimetric inhibition immunoassay. The neck, lower back and head were reported as the most common painful sites (affected in 60.5, 60.5 and 56.5% of patients, respectively). Pain in the lower extremities, including the knees, lower legs and feet, was more common compared with pain in the upper extremities. The pain measurements of number of painful sites and pain intensity did not differ significantly among patients with sufficient (>30 ng/ml), insufficient (20-30 ng/ml) and deficient (<20 ng/ml) vitamin D levels (P>0.05). The pain measurements were identified to have no correlation with age, body mass index, FBS, HbA1c level, 25-hydroxyvitamin D concentration, anxiety or depression (P>0.05). However, the pain measurements were correlated with duration of T2DM and neuropathy score (P<0.05). Further regression analysis demonstrated that the pain measurements were significantly associated with the neuropathy score (P<0.05). In conclusion, musculoskeletal pain in patients with T2DM was not associated with 25-hydroxyvitamin D concentration, but was associated with neuropathy score. This may encourage further investigations to assess the etiology of musculoskeletal pain in T2DM, and whether vitamin D supplementation and management of neuropathy would be of value as pain relief treatment.

Effects of antirheumatic drug underutilization on rheumatoid arthritis disease activity

BackgroundFollowing the recommended guidelines is crucial for achieving patient remission in rheumatoid arthritis. The aim of this study was to assess the effect of proper drug utilization of antirheumatic drugs on disease activity and drug safety in Jordan.MethodsIn a retrospective cross-sectional study, patient’s demographics, clinical variables, drug regimens and side effects were recorded and the 28-joint disease activity scores were calculated. Patients were stratified into high, moderate, low disease activity or remission group.ResultsAround 80% of patients were using methotrexate which was under-dosed in 82% of them. Only 25% were using biologic drugs. Surprisingly, only 10% of patients had low disease activity and only 4% were in a remission state. Anaemia (32.3%) and mild renal impairment (27.6%) were the most common side effects.ConclusionsThe low frequency of well-controlled disease activity is interpreted by high occurrence of methotrexate underdosing and biologic agent underprescription. Implementing the role of a clinical pharmacist could have a real impact on tight control of such disease issues in Jordan.

Bio-maleimide-stained plasma microparticles can be purified in a native state and target human proximal tubular HK2 cells

Plasma microparticles (MPs) are heterogeneously sized submicron extracellular vesicles that originate from the cell membrane as a result of cell activation or apoptosis. Circulating MPs express cell-specific molecules that reflect their cell of origin and they are increasingly investigated for their potential role in intercellular communication. The aim of the current study was to determine if size exclusion chromatography could be used to purify fluorescent-labeled MPs in sufficient concentrations to be used experimentally in cell binding assays. Bio-maleimide was used to stain plasma MPs in platelet free plasma before applying to size exclusion chromatography. Collected fractions were analyzed for protein content and MPs were enumerated by flow cytometry. Fractions were ultracentrifuged and MPs further confirmed by western blotting for the putative diabetic marker, cluster of differentiation (CD)36 and platelet-specific CD41 proteins. Fractions that contained MPs were incubated with HK2 cells to determine MP-cell binding. Bio-maleimide-stained MPs were detected across various fractions of size exclusion, and pellets of these fractions confirmed positivity for the MP markers, CD41 and CD36. The addition of the isolated MPs to HK2 renal tubular cells and analysis by epi-fluorescent imaging demonstrated that, in principle, the labeled MPs are able to bind to cells in vitro. Notably, only the first eluted MP fraction bound HK2 cells indicating a possible association between MP size and cell-targeting properties.