Early lipid and inflammatory markers of atherosclerosis in children with type 1 diabetes mellitus: effects of metabolic control and comorbidities

Cilj ove disertacije je bio određivanje lipidnih i inflamatornih markera ateroskleroze kod dece sa tipom 1 dijabetes melitusa (T1DM) (N=201) i zdrave dece (N=141), ispitivanje njihove međusobne povezanosti, kao i uticaja metaboličke kontrole i komorbiditeta na analizirane parametre. To je uključilo određivanje standardnog lipidnog profila, veličine i raspodele subfrakcija lipoproteina niske (LDL) i visoke (HDL) gustine, koncentracije proprotein konvertaze subtilisin keksin tip 9 (PCSK9), koncentracije i genske ekspresije receptora za krajnje produkte uznapredovale glikacije (RAGE) i transformišućeg faktora rasta β1 (TGF-β1) u mononuklearnim ćelijama periferne krvi. U grupi sa T1DM bilo je 30 pacijenata sa pridruženim autoimunskim tireoiditisom i 15 sa pridruženom celijačnom bolešću. Koncentracije HDL-holesterola su bile više kod dece sa T1DM u odnosu na zdravu decu, dok se drugi parametri lipidnog profila nisu značajno razlikovali. Pacijenti sa neadekvatnom metaboličkom kontrolom su imali više koncentracije PCSK9 i veće udele malih HDL subfrakcija. U grupi sa dobrom metaboličkom kontrolom je utvrđena negativna korelacija između koncentracija PCSK9 i udela malih LDL čestica. Ekspresija gena za RAGE i TGF-β1 je bila značajno niža, a koncentracije značajno više kod dece sa T1DM. Koncentracije TGF-β1 u plazmi pacijenata sa T1DM su pozitivno korelirale sa udelom malih HDL subfrakcija, a genska ekspresija sa povećanom ekskrecijom albumina. Pacijenti sa pridruženom celijačnom bolešću su imali najniže koncentracije HDL-holesterola, najmanje dijametre HDL čestica i najveći indeks ateroskleroze. Rezultati su ukazali na značaj određivanja lipidnih i inflamatornih markera i ranog povećanja ekskrecije albumina u prevenciji dijabetesnih komplikacija.The aim of this thesis was to determine lipid and inflammatory markers of atherosclerosis in children with type 1 diabetes mellitus (T1DM) (N=201) and healthy children (N=141), to investigate their mutual relationships and the effects of metabolic control and comorbidities on examined markers. Standard lipid profile, low-density (LDL) and high-density (HDL) size and subclasses distribution, concentration of proprotein convertase subtilisin kexin type 9 (PCSK9), and concentrations and gene expression of receptor for advanced glycation end-products (RAGE) and transforming growth factor β1 (TGF-β1) in peripheral blood mononuclear cells were determined. In T1DM group 30 patients had associated autoimmune thyroiditis and 15 patients had celiac disease. HDL-cholesterol (HDL-C) levels were higher in children with T1DM, while other lipid status parameters did not differ between the groups. Patients with inadequate metabolic control had increased PCSK9 levels and higher proportion of small HDL subfractions. In the group with good metabolic control an inverse association between PCSK9 level and proportion of small LDL particles was found. Gene expression of RAGE and TGF-β1 was significantly lower and plasma concentrations were significantly higher in children with T1DM. TGF-β1 levels in plasma of T1DM patients were positively associated with the proportion of small HDL subfractions, and higher gene expression was associated with increased albumin excretion. Patients with associated celiac disease had the lowest concentrations of HDL-C, the smallest diameter of HDL particles, and the highest index of atherosclerosis. The results indicated importance of lipid and inflammatory markers and early increase of albumin excretion determination for prevention of diabetic complications

Nishodna regulacija MAPK/MAK/MRK preklapajuće kinaze u mononuklearnim ćelijama periferne krvi pedijatrijskih pacijenata sa tip1 diiabetes mellitus-om

Background: Type 1 diabetes mellitus (T1DM) is one of the most common endocrine diseases in children. T-cell autore- activity toward b-cells is controlled by significant changes in metabolism of T cells. Mammalian target of rapamycin (mTOR) is an important intracellular regulator of metabolism and cell growth. MAPK/MAK/MRK overlapping kinase 1 (MOK1) is one of the less known regulators of mTOR. We sought to investigate if MOK1 and mTOR mRNA levels in peripheral blood mononuclear cells (PBMCs) of T1DM pedi- atric patients are different compared to healthy subjects. Methods: This study included 172 adolescents with T1DM and 36 healthy adolescent volunteers designated for control group (CG). MOK1 and mTOR mRNA levels were deter- mined in PBMCs by qPCR. Results: T1DM patients have significant downregulation of MOK1 mRNA levels in PBMCs compared CG (P=0.018), while there was no significant difference in mTOR mRNA levels (P=0.891). Furthermore, in T1DM patients, MOK1 significantly correlated with age, triglycerides and mTOR, while mTOR correlated significantly with BMI and systolic blood pressure. Overweight T1DM subjects had significantly lower MOK1 (P=0.034) and mTOR (P=0.017) mRNA levels, together with significantly higher levels of systolic blood pressure (P<0.001), total cholesterol (P=0.001), LDL-cholesterol (P=0.001) and CRP (P<0.001). Multi- variate analysis showed that MOK1 was independently negatively associated with T1DM when adjusted for sex, age, HDL-C and CRP (OR=0.417 (95%CI: 0.175–0.997), p=0.049). Conclusions: Our study demonstrated for the first time that T1DM is associated with MOK1 downregulation. In addition, downregulation of both mTOR and MOK1 gene expressions was associated with cardiovascular risk factors in overweight T1DM patients.Uvod: Dijabetes melitus tip 1 (T1DM) jedno je od najčešćih endokrinih oboljenja kod dece. Autoreaktivnost T-ćelija prema b-ćelijama kontroliše se značajnim promenama u metabolizmu T ćelija. Cilj delovanja rapamicina kod sisara je važan unutarćelijski regulator metabolizma i rasta ćelija. MAPK/MAK/MRK preklapajuće kinaze koja se preklapa (MOK1) jedan je od manje poznatih regulatora mTOR-a. Cilj istraživanja je bio da se ispita da li su nivoi iRNK MOK1 i mTOR u mononuklearnim ćelijama periferne krvi različiti kod pedijatrijskih pacijenata sa T1DM u odnosu na zdrave ispitanike. Metode: Ovo istraživanje je obuhvatilo 172 adolescenta sa T1DM i 36 zdravih adolescenata dobrovoljaca koji su činili kontrolnu grupu (CG). Nivoi MOK1 i mTOR mRNA određeni su u PBMC-ima pomoću qPCR-a. Rezultati: Pacijenti sa T1DM imali su značajno niže nivoe iRNK MOK1 u PBMC u odnosu na CG, dok razlike u nivoima iRNK mTOR nisu bile značajne (P = 0,891). Štaviše, kod pacijenata sa T1DM, MOK1 je značajno korelirao sa godinama, trigliceri dima i mTOR, dok je mTOR značajno korelirao sa BMI i sistolnim krvnim pritiskom. Ispitanici sa prekomernom težinom T1DM imali su značajno niže nivoe iRNK MOK1 (P = 0,034) i mTOR (P = 0,017), zajedno sa značajno većim nivoima sistolnog krvnog pritiska (P <0,001), ukupnog holesterola (P = 0,001), LDL-holesterola (P = 0,001) i CRP (P <0,001). Multivarijantna analiza je pokazala da je MOK1 nezavisno negativno povezan sa T1DM kada je prilagođen polu, starosti, HDL-C i CRP (OR = 0,417 (95%CI: 0,175-0,997), p = 0,049). Zaključak: Naša studija je prva koja je pokazala da je T1DM udružen sa nishodnom regulacijom MOK1. Pored toga, snižena regulacija ekspresije gena mTOR i MOK1 bila je povezana sa kardiovaskularnim faktorima rizika kod pacije nata sa T1DM sa prekomernom težinom

Association between proprotein convertase subtilisin/kexin 9 (PCSK9) and lipoprotein subclasses in children with type 1 diabetes mellitus: Effects of glycemic control

Background and aims: Dyslipidemia in type 1 diabetes mellitus (T1DM) is characterised by altered distributions of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) subclasses. Recent studies suggested that proprotein convertase subtilisin/kexin 9 (PCSK9) may contribute to the development of dyslipidemia in T1DM. In this cross-sectional study, we investigated the association between PCSK9 and lipoprotein subclasses in young T1DM patients, with respect to glycemic control. Methods: Plasma PCSK9 and lipoprotein subclasses were determined in 207 patients with T1DM (106 boys and 101 girls), aged 13.9 +/- 3.0 years and treated by intensive insulin therapy. Results: Plasma PCSK9 levels significantly increased with worsening of glycemic control (p lt 0.001). T1DM patients with poor glucoregulation had the highest proportion of small, dense LDL (sdLDL) and smaller HDL particles, as well. PCSK9 was positively associated with markers of glucose homeostasis and serum lipid parameters only in patients with suboptimal/poor glucoregulation. In well-controlled T1DM, plasma PCSK9 level was inversely associated with a relative proportion of sdLDL particles (p lt 0.01) and this association remained significant in multivariate analysis. In T1DM patients with suboptimal/poor glycemic control, PCSK9 was positively associated with the proportion of the smallest HDL3c particles (p lt 0.001), but negatively with HDL size (p lt 0.05). Conclusions: The extent of achieved metabolic control modifies the association between PCSK9 and lipoprotein subclasses in T1DM. Further investigations are needed to reveal whether the observed effects of glycemic control on PCSK9 and sdLDL levels have causal consequences on CVD risk in young patients with T1DM

Uloga laboratorijskih biomarkera u dijagnostici i praćenju pacijenata sa COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a highly transmittable and heterogenic infection of the respiratory tract, characterized by a broad spectrum of clinical manifestations with a different degree of severity. Medical laboratories play an important role in early diagnosis and management of Coronavirus Disease 2019 (COVID-19) patients. Indeed, the results of several laboratory tests are essential for assessing the severity of the disease, selecting appropriate therapeutic procedures and monitoring treatment response. Routine laboratory testing in COVID-19 patients includes biomarkers of acute phase reaction, hematological and biochemical parameters that indicate tissue injury. The aim of this review paper is to describe the role of these biomarkers in the diagnostics and management of adult and pediatric COVID-19 patients.Novi koronavirus SARS-CoV-2 je uzročnik lako prenosive i veoma heterogene infekcije respiratornog trakta, koju karakteriše širok spektar kliničkih manifestacija, različitog stepena težine. Medicinske laboratorije igraju važnu ulogu u ranoj dijagnostici i praćenju pacijenata sa COVID-19. Rezultati nekoliko laboratorijskih testova su od suštinskog značaja za procenu težine bolesti, odabir odgovarajućih terapijskih procedura i praćenje odgovora na terapiju. Rutinska laboratorijska ispitivanja kod pacijenata sa COVID-19 uključuju biomarkere akutne faze, hematološke i biohemijske parametre koji ukazuju na oštećenje tkiva. Cilj ovog rada je da se prikaže uloga tih biomarkera u dijagnostici i praćenju toka bolesti kod odraslih i pedijatrijskih pacijenata sa COVID-19

Transforming growth factor-β1 and receptor for advanced glycation end products gene expression and protein levels in adolescents with type 1 diabetes mellitus

Objective: Type 1 diabetes (T1D) mellitus is one of the most frequent autoimmune diseases in childhood. Chronic complications are the main causes of cardiovascular morbidity and mortality in T1D. Although interactions between advanced glycation end products (AGE) and their receptors (RAGE) and transforming growth factor-β1 (TGF-β1) are implicated in development and progression of diabetic micro-and macro-vascular complications, they also have important roles in immune system regulation. Methods: Blood samples were obtained from 156 adolescents with T1D and 80 apparently healthy controls. T1D patients diagnosed with any other autoimmune disease and receiving any kind of drugs except insulin therapy were excluded from this study. Exclusion criteria for controls were positive family history of T1D and drugs/supplements application. TGF-β1 and transmembrane full-length RAGE (flRAGE) messenger ribonucleic acid (mRNA) levels in peripheral blood mononuclear cells (PBMC) were obtained by quantitative polymerase chain reaction (qPCR) method. Circulating levels of biochemical markers, TGF-β1 and soluble RAGE (sRAGE) levels were also determined. Results: TGF-β1 and flRAGE mRNA levels were significantly higher in controls compared to patients (p<0.001, for both). However, TGF-β1 and sRAGE levels were higher in patients than controls (p<0.001, for both). There were significant independent associations of all mRNA and protein levels with T1D. TGF-β1 mRNA was the only marker independently negatively associated with urinary albumin excretion rate in T1D adolescents (p=0.005). Conclusion: Our results indicated gene expression downregulation of TGF-β1 and flRAGE in PBMC of T1D adolescents. TGF-β1 mRNA downregulation may be useful for predicting early elevation of urinary albumin excretion rate

Effects of co-existing autoimmune diseases on serum lipids and lipoprotein subclasses profile in paediatric patients with type 1 diabetes mellitus

Objective: Paediatric patients with type 1 diabetes mellitus (T1DM) frequently develop other autoimmune disorders; most commonly autoimmune thyroiditis (ATD) and celiac disease (CD). In this study we evaluated whether co-existing autoimmune diseases had significant impact on lipid and lipoprotein subclasses, as known cardiovascular risk factors in T1DM. Design and methods: Study included 201 subjects with T1DM (14.1 +/- 2.9 years) and 141 age-and gender-matched controls. ATD was presented in 30 and CD in 15 T1DM patients. Serum lipid parameters were determined by routine laboratory methods and plasma low-density (LDL) and high-density lipoprotein (HDL) subclasses by gradient-gel electrophoresis method. Results: Both groups of T1DM patients with concomitant autoimmune disease had significantly lower HDL-C levels (P lt 0.05) than the patients with T1DM only, but comparable to control group (P = 0.436). T1DM patients had significantly higher (P lt 0.001) proportion of small HDL subclasses than controls. Mean value of atherosclerosis index in patients with T1DM + CD was the highest (1.75 +/- 0.86) and it was significantly higher than the index in patients with T1DM only (1.33 +/- 0.51; P lt 0.05). LDL size did not differ between the groups of T1DM patients and control group (P= 0.619). The size of HDL particles was significantly reduced (P lt 0.05) in the groups with associated autoimmune diseases. The patients with co-existing autoimmune diseases had higher risk of low HDL-C level (OR: 2.96; P lt 0.05). Conclusions: The results have shown significant impact of co-existing autoimmune diseases on lipid profile in patients with T1DM. The most prominent changes were found in HDL lipoprotein characteristics in T1DM + CD group

The Role of Advanced Glycation End Products on Dyslipidemia

Disorders of lipoprotein metabolism and glucose homeostasis are common consequences of insulin resistance and usually co-segregate in patients with metabolic syndrome and type 2 diabetes mellitus (DM). Insulin-resistant subjects are characterized by atherogenic dyslipidemia, a specific lipid pattern which includes hypertriglyceridemia, reduced high-density lipoprotein cholesterol level, and increased proportion of small, dense low-density lipoprotein (LDL). Chronic hyperglycemia favors the processes of non-enzymatic glycation, leading to the increased production of advanced glycation end products (AGEs). Apart from direct harmful effects, AGEs are also potent inducers of oxidative stress and inflammation. In addition, increased AGEs’ production may induce further qualitative modifications of small, dense LDL particles, converting them to glycated LDLs. These particles are even more atherogenic and may confer an increased cardiovascular risk. In this narrative review, we summarize the available evidence of the pathophysiological role and clinical importance of circulating AGEs and glycated LDLs in patients with dyslipidemia, particularly those with DM and related complications. In addition, we discuss recent advances and the issues that should be improved regarding laboratory assessment of AGEs and glycated LDLs, as well as the possibilities for their therapeutic modulation

Nishodna regulacija MAPK/MAK/MRK preklapajuče kinaze u mononuklearnim ćelijama periferne krvi pedijatrijskih pacijenata sa tip1 Diiabetes mellitus-om

Background Type 1 diabetes mellitus (T1DM) is one of the most common endocrine diseases in children. T-cell autoreactivity toward b-cells is controlled by significant changes in metabolism of T cells. Mammalian target of rapamycin (mTOR) is an important intracellular regulator of metabolism and cell growth. MAPK/MAK/MRK overlapping kinase 1 (MOK1) is one of the less known regulators of mTOR. We sought to investigate if MOK1 and mTOR mRNA levels in peripheral blood mononuclear cells (PBMCs) of T1DM pediatric patients are different compared to healthy subjects. Methods This study included 172 adolescents with T1DM and 36 healthy adolescent volunteers designated for control group (CG). MOK1 and mTOR mRNA levels were determined in PBMCs by qPCR. Results T1DM patients have significant downregulation of MOK1 mRNA levels in PBMCs compared CG (P=0.018), while there was no significant difference in mTOR mRNA levels (P=0.891). Furthermore, in T1DM patients, MOK1 significantly correlated with age, triglycerides and mTOR, while mTOR correlated significantly with BMI and systolic blood pressure. Overweight T1DM subjects had significantly lower MOK1 (P=0.034) and mTOR (P=0.017) mRNA levels, together with significantly higher levels of systolic blood pressure (P<0.001), total cholesterol (P=0.001), LDL-cholesterol (P=0.001) and CRP (P<0.001). Multi - variate analysis showed that MOK1 was independently negatively associated with T1DM when adjusted for sex, age, HDL-C and CRP (OR=0.417 (95%CI: 0.175-0.997), p=0.049). Conclusions Our study demonstrated for the first time that T1DM is associated with MOK1 downregulation. In addition, downregulation of both mTOR and MOK1 gene expressions was associated with cardiovascular risk factors in overweight T1DM patients.Uvod: Dijabetes melitus tip 1 (T1DM) jedno je od največih endokrinih oboljenja kod dece. Autoreaktivnost T-čelija prema beta-čelijama kontroliče se značajnim promenama u metabolizmu T }elija. Cilj delovanja rapamicina kod sisara je važan unutarćelijski regulator metabolizma i rasta ćelija. MAPK/MAK/MRK preklapaju}e kinaze koja se preklapa (MOK1) jedan je od manje poznatih regulatora mTOR-a. Cilj istraživanja je bio da se ispita da li su nivoi iRNK MOK1 i mTOR u mononuklearnim }elijama periferne krvi različiti kod pedijatrijskih pacijenata sa T1DM u odnosu na zdrave ispitanike. Metode: Ovo istraživanje je obuhvatilo 172 adolescenta sa T1DM i 36 zdravih adolescenata dobrovoljaca koji su činili kontrolnu grupu (CG). Nivoi MOK1 i mTOR mRNA određeni su u PBMC-ima pomoću qPCR-a. Rezultati: Pacijenti sa T1DM imali su značajno niže nivoe iRNK MOK1 u PBMC u odnosu na CG, dok razlike u nivoima iRNK mTOR nisu bile zna~ajne (P = 0,891). Štaviše, kod pacijenata sa T1DM, MOK1 je značajno korelirao sa godinama, trigliceri dima i mTOR, dok je mTOR značajno korelirao sa BMI i sistolnim krvnim pritiskom. Ispitanici sa prekomernom težinom T1DM imali su znaćajno niže nivoe iRNK MOK1 (P = 0,034) i mTOR (P = 0,017), zajedno sa značajno večim nivoima sistolnog krvnog pritiska (P <0,001), ukupnog holesterola (P = 0,001), LDL-holesterola (P = 0,001) i CRP (P <0,001). Multivarijantna analiza je pokazala da je MOK1 nezavisno negativno povezan sa HDL-C and CRP (OR=0.417 (95%CI: 0.175–0.997), p=0.049). Zaključak: Naša studija je prva koja je pokazala da je T1DM udružen sa nishodnom regulacijom MOK1. Pored toga, snižena regulacija ekspresije gena mTOR i MOK1 bila je povezana sa kardiovaskularnim faktorima rizika kod pacije nata sa T1DM sa prekomernom težinom

The usefulness of advanced lipid and oxidative stress testing for diagnosis and management of low HDL-cholesterol phenotype: A case report

Objective: Plasma high-density lipoprotein cholesterol (HDL-C) level is a strong inverse predictor of cardiovascular disease (CVD) development. Tangier disease, a consequence of mutations in the ATP binding cassette transporter 1 (ABCA1) gene, is associated with very low HDL-C levels. Still, the relationship between Tangier disease and CVD is not always evident. The study investigates usefulness of lipoprotein subfractions, oxidative stress and paraoxonase 1 (PON1) status assessment for evaluation and management of patient with low HDL-C phenotype. Patient and methods: A 12-year-old boy was hospitalised due to hypertension. Laboratory evaluation revealed low HDL-C level, and subsequent molecular diagnostic confirmed Tangier disease. Lipoprotein subfractions were assessed by gradient-gel electrophoresis. Oxidative stress status was estimated by measuring total antioxidative status, total oxidative status, prooxidative-antioxidative balance, malondialdehyde and advanced oxidation protein products levels. Activity of paraoxonase 1 in serum and its distribution within HDL subclasses was also determined (ten healthy boys aged 13.1 +/- 3.4 years served as the reference group). Results: Analysis of oxidative stress status biomarkers revealed a state of prolonged prooxidants activity. In turn, serum PON1 activity was substantially reduced. The majority of PON1 activity was present on HDL 2 particles. Conclusion: Impaired antioxidative potential of HDL may point toward hidden cardiovascular risk in isolated low HDL-phenotype

Probiotic reduced the impact of phthalates and bisphenol A mixture on type 2 diabetes mellitus development: Merging bioinformatics with in vivo analysis

Linkage between bis(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and bisphenol A (BPA) coexposure and type 2 diabetes mellitus (T2DM), as well as ability of multi-strained probiotic to reduce DEHP, DBP and BPA mixture-induced oxidative damage in rat pancreas were investigated. The Comparative Toxicogenomics Database, Cytoscape software and ToppGene Suite were used for data-mining. Animals were sorted into seven groups (n = 6): (1) Control group: corn oil, (2) P: probiotic: Saccharomyces boulardii + Lactobacillus rhamnosus + Lactobacillus plantarum LP 6595 + Lactobacillus plantarum HEAL9; (3) DEHP: 50 mg/kg b.w./day, (4) DBP: 50 mg/kg b.w./day, (5) BPA: 25 mg/kg b.w./day, and (6) MIX: 50 mg/kg b.w./day DEHP + 50 mg/kg b.w/ day DBP + 25 mg/kg b.w./day BPA; (7) MIX + P. Rats were sacrificed after 28 days of oral exposure. In silico investigation highlighted 44 DEHP, DBP and BPA mutual genes linked to the T2DM, while apoptosis and oxidative stress were highlighted as the main mechanisms of DEHP, DBP and BPA mixture-linked T2DM. In vivo experiment confirmed the presence of significant changes in redox status parameters (TOS, SOD and SH groups) only in the MIX group, indicating possible additive effects, while probiotic ameliorated mixture-induced redox status changes in rat pancreatic tissue