Role of Leptin in the Activation of Immune Cells

Adipose tissue is an active endocrine organ that secretes various humoral factors (adipokines), and its shift to production of proinflammatory cytokines in obesity likely contributes to the low-level systemic inflammation that may be present in metabolic syndrome-associated chronic pathologies such as atherosclerosis. Leptin is one of the most important hormones secreted by adipocytes, with a variety of physiological roles related to the control of metabolism and energy homeostasis. One of these functions is the connection between nutritional status and immune competence. The adipocyte-derived hormone leptin has been shown to regulate the immune response, innate and adaptive response, both in normal and pathological conditions. The role of leptin in regulating immune response has been assessed in vitro as well as in clinical studies. It has been shown that conditions of reduced leptin production are associated with increased infection susceptibility. Conversely, immune-mediated disorders such as autoimmune diseases are associated with increased secretion of leptin and production of proinflammatory pathogenic cytokines. Thus, leptin is a mediator of the inflammatory response

Mecanismos de inmunomodulación de la leptina en células T Jurkat

La leptina no es sólo una hormona del tejido adiposo que regula la ingesta y el gasto energético a nivel central, sino que tiene múltiples efectos en tejidos periféricos. Una de las funciones encontrada es su papel en la hermatopoyesis y en la función inmune. Trabajos previos en nuestro grupo habían demostrado la activación de monocitos y linfocitos T de sangre periférica humana. En la presente Tesis, dada la dificultad de trabajar con subpoblaciones linfocitarias decidimos utilizar la línea de células T Jurkat para caracterizar la acción activadora, trófica y antiapoptótica de la leptina sobre las células T. Para la consecución de este cometido nos planteamos los siguientes objetivos: 1. Caracterizar la acción activadora de la leptina en células T Jurkat. 2. Caracterizar el efecto trófico de la leptina en células T Jurkat. 3. Caracterizar el sistema receptor de la leptina y su señalización en células T Jurkat. 4. Determinar la importancia de las diferentes vías de señalización sobre la acción de la leptina utilizando inhibidores farmacológicos de las vías implicadas en la señal del receptor de leptina. Las conclusiones elaboradas acerca de los mecanismos de inmunomodulación de la leptina en células T Jurkat, tras el trabajo expuesto en la presente Tesis son: 1. La leptina potencia la activación de las células T Jurkat coestimuladas con dosis bajas de PHA. 2. La leptina previene la muerte celular programada, apoptosis, producida tanto por estimulación máxima como por falta de suero en las células T Jurkat. 3. Las células T Jurkat presentan las isoformas larga y corta del receptor de leptina en la membrana plasmática y la leptina produce la activación de varias vías de señalización presentes en ellas: quinasa tipo Janus-transductor de señal y activador de la transcripción (JAK-STAT), fosfatidil-inositol 3 quinasa (PI3K) y proteína quinasa activada por mitógeno (MAPK). 4. El efecto activador y antiapoptótico de la leptina en las células T Jurkat está mediado fundamentalmente por la vía MAPK, que es necesaria pero no suficiente para la consecución del efecto máximo trófico y de activación, el cuál requeriría la participación de la vía PI3K

Complete laboratory diagnosis of Insulin Autoimmune Syndrome

The definition of Insulin autoimmune syndrome includes the presence of high levels of blood insulin and insulin autoantibodies. We encountered a 45-years-old white man with a high insulin serum value that do not fit with the C-peptide result. To discard or to confirm an analytical interference and diagnose a possible Insulin Autoimmune Syndrome we performed the following investigations: dilution linearity test, heterophilic antibody blocking, polyethylene glycol precipitation, measurements with alternative assays, and gel filtration chromatography by size exclusion. The latter technique confirmed that most of the insulin was complexed with a 150-kDa protein, corresponding to immunoglobulin G, identified as insulin autoantibodies. These antibodies were responsible for hypoglycemia attacks in the patient, who had a previous autoimmune disease. This case highlights the importance of carefully analyzing the results and ruling out possible interferences, as well as considering all kinds of pathologies, even if they are infrequent

Evaluation of Bio-Rad D-100 HbA1c analyzer against Tosoh G8 and Menarini HA-8180V

AbstractObjectivesTo evaluate the Bio-Rad D-100®, an HPLC analyzer for glycated hemoglobin (HbA1c) determination, and to compare its performance with the Menarini HA-8180V® and Sysmex G8®.MethodsMethod comparison was performed according to Clinical and Laboratory Standards Institute (CLSI) EP9-A2 guidelines. We selected 100 samples from the routine laboratory workload and analyzed them in duplicate with the three analyzers. The imprecision study was performed according to CLSI EP5-A2 guidelines for both inter-assay and intra-assay variability. Bias was assessed with external quality control material. To establish linearity, CLSI EP6-A protocol was followed.ResultsMethod comparison (95% confidence intervals in parentheses): D-100 vs G8: Passing-Bablok regression; y=0.973(0.963–0.983)×−0.07(−0.07−0.069); r=0.9989. Bland-Altman mean difference: −0.229%HbA1c (−0.256: −0.202); Relative bias plot: D-100/G8 vs D100-G8 mean ratio=0.971(0.967−0.975). D-100 vs HA-8180V: Passing-Bablok regression; y=0.944(0.932–0.958)×−0.078(0.024−0.173); r=0.9989. Bland-Altman mean difference: −0.363%HbA1c (−0.401: −0.325); Relative bias plot D-100/HA-8180V vs D100-HA-8180V mean ratio=0.955(0.952−0.958). Inter-assay coefficient of variation (CV): 0.81%. Intra-assay CV: 1.04% (low level), and 0.78% (high level). Bias against target value=2.332%. Linearity: r2=0.998 in the concentration range 4.4−13.9%HbA1c. Carry-over: 0.0024%.ConclusionsThe Bio-Rad D-100 shows good correlation with G8 and HA-8180V. There is a small proportional systematic difference (2.7% and 5.6%, respectively) in both comparisons. Inter and intra-assay CVs are both lower than the lowest CV obtained in studies performed with D-100 and other instruments

Evaluation of two HbA1c point-of-care analyzers.

Journal Article;BACKGROUND Measurement of HbA1c is the most important parameter to assess glycemic control in diabetic patients. Different point-of-care devices for HbA1c are available. The aim of this study was to evaluate two point-of-care testing (POCT) analyzers (DCA Vantage from Siemens and Afinion from Axis-Shield). We studied the bias and precision as well as interference from carbamylated hemoglobin. METHODS Bias of the POCT analyzers was obtained by measuring 53 blood samples from diabetic patients with a wide range of HbA1c, 4%-14% (20-130 mmol/mol), and comparing the results with those obtained by the laboratory method: HPLC HA 8160 Menarini. Precision was performed by 20 successive determinations of two samples with low 4.2% (22 mmol/mol) and high 9.5% (80 mmol/mol) HbA1c values. The possible interference from carbamylated hemoglobin was studied using 25 samples from patients with chronic renal failure. RESULTS The means of the differences between measurements performed by each POCT analyzer and the laboratory method (95% confidence interval) were: 0.28% (p<0.005) (0.10-0.44) for DCA and 0.27% (p<0.001) (0.19-0.35) for Afinion. Correlation coefficients were: r=0.973 for DCA, and r=0.991 for Afinion. The mean bias observed by using samples from chronic renal failure patients were 0.2 (range -0.4, 0.4) for DCA and 0.2 (-0.2, 0.5) for Afinion. Imprecision results were: CV=3.1% (high HbA1c) and 2.97% (low HbA1c) for DCA, CV=1.95% (high HbA1c) and 2.66% (low HbA1c) for Afinion. CONCLUSIONS Both POCT analyzers for HbA1c show good correlation with the laboratory method and acceptable precision.Ye

Predictive value of the kinetics of procalcitonin and C-reactive protein for early clinical stability in patients with bloodstream infections due to Gram-negative bacteria

[Objective] To investigate whether the magnitude of the change in procalcitonin (PCT) and C-reactive protein (CRP) levels between day 1 and day 2 after the blood culture date is associated with early clinical stability (ECS) on day 3 in patients with bacteremia due to Gram-negative bacteria (GNB).[Materials/methods] A prospective cohort study carried out in a 950-bed tertiary hospital in Spain between March 2013 and May 2014. Patients with GNB bacteremia were included. Changes in PCT and CRP kinetics from day 1 to day 2 (∆%PCT, ∆%CRP) were expressed as percentage of decline in blood levels. Logistic regression was used to identify predictors of ECS. Classification and regression tree analysis was performed to identify breakpoints. The discriminatory power of ∆%CRP and ∆%PCT as predictors of ECS was assessed by the area under the ROC (AUROC).[Results] 71 patients were included, and 53 (74.56%) reached ECS. Multivariate analyses showed that SOFA score on day 1, ∆%PCT, and ∆%CRP were associated with ECS after controlling for confounders. ∆%PCT ≥ 30% (decline) and ∆%CRP ≥ 10% (decline) predicted ECS only among patients with SOFA≤3 on day 1 (n = 54; 43 reached ECS). In these patients, the AUROCs for the prediction of ECS were 0.96 (95% CI: 0.90–1) for ∆%CRP and 0.96 (95% CI: 0.90–1) for ∆%PCT, respectively.[Conclusions] In the subgroup of patients with a SOFA score on day 1 ≤3, a ≥30% decline in PCT or a ≥10% decline in CRP between day 1 and day 2 was a very good predictor of ECS (which in turn was associated with a lower 30-day mortality and a greater clinical cure on day 14). Patients who do not achieve this decrease may need more intensive workup. In this subgroup (with a SOFA on day 1 ≤3), CRP may be preferred due to its lower cost.This study was supported by Plan Nacional de I + D + i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (RD16/0016/0008) and co-financed by the European Development Regional Fund “A way to achieve Europe”, Operative Program Intelligent Growth 2014–2020.Peer reviewe

Molecular inflammation and oxidative stress are shared mechanisms involved in both myocardial infarction and periodontitis

Background and Objective. Our aims were to improve the understanding of the pathogenic relationship between cardiovascular diseases and periodontitis and to generate new perspectives in the prevention and treatment of acute myocardial infarction (AMI) and periodontitis. The present study evaluates possible differences in inflammation, oxidative stress and autophagy markers among subject suffering AMI, periodontitis or both, to explore possible common pathogenic mechanisms. Material and Methods. A total of 260 subjects were enrolled in the study, 106 subjects that survived to a first AMI (AMI group) and 154 subjects had no cardiac events in their clinical record (control group). A questionnaire was used to assess age, height, weight, blood pressure and heart rate. The clinical probing depth, clinical attachment loss, number of remaining teeth and average number of sites with bleeding on probing were assessed. Lipid peroxidation and protein levels of phosphorylated AMP-activated protein kinase (p-AMPK) and microtubule-associated proteins 1A/1B-light chain 3-II (LC3-II) were determined in isolated peripheral blood mononuclear cells by thiobarbituric acid reactive substances (TBARS) assay and western-blot, respectively. Plasma levels of interleukin-1β were determined using a commercial ELISA kit. All the obtained variables were compared between subjects suffering an AMI with or without periodontitis and control subject periodontal healthy or with periodontitis. Results. A higher proportion of subjects suffering AMI+periodontitis than only AMI (without periodontitis) was found. Higher levels of TBARS were found in subjects with periodontitis than in subjects without periodontitis in both AMI and control subjects. Positive correlations between IL-1β levels and TBARS and between IL-1β levels and LC3-II were found only in control subjects. Conclusions. Results from the present study are consistent with the suggestion of periodontitis as a potential risk factor for AMI. Periodontitis association with circulating lipid peroxides in both AMI and control subjects were found. The absence of differences in IL-1β levels between AMI subjects (only AMI vs. AMI+periodontitis) suggests that oxidative stress could be the main pathogenic link between AMI and periodontitis