Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

Cardiovascular diseases are the major causes of death worldwide. Studying factors leading to initiation and progression of atherosclerosis and its complications leads to a better understanding of underlying mechanisms of this disease and to development of novel treatments. Adhesion of monocytes on the endothelial surface is the initial step of atherosclerosis. The main aim of this study was to establish and test an in vitro model of monocyte adhesion on the endothelial cells and to evaluate the results by means of two methods - measuring the fluorescence signal intensity and counting adhered cells. Because of its well known effects on endothelial cells activation and adhesion molecules expression TNF-α was chosen for endothelial cells stimulation. The lowest concentration of TNF-α affecting the percentage of adhered monocytes in comparision with negative control was 1 ng TNF-α/ml. The optimal concentration of TNF-α increasing the percentage of adhered monocytes was 10 ng TNF-α/ml. The influence of TNF-α on the adhesion was observed already after 5 minutes of coincubation of THP-1 monocytes with HUVEC. Using the optimal concentration of 10 ng/ml led to the highest percentage of adhered monocytes after 30 - 40 minutes of coincubation with HUVEC. Other factor affecting the percentage of adhered...

Despite the availability of effective therapy of hypercholesterolemia and hypertension, cardiovascular mortality continues to be very high in the Western world. Inflammatory changes occurring in the arterial wall as well as in the adipose tissue play a major role in the development of atherosclerosis. Macrophages are involved in the process of atherogenesis as early as atherosclerosis begins to develop, when, still as monocytes, they migrate and adhere to the arterial wall as a result of endothelial activation and stimulation by pro-inflammatory substances. Adipose tissue has long been recognized as an important endocrine organ, with part of adipose tissue made up by a large amount of macrophages capable of producing a large number of pro-inflammatory cytokines, which contribute to the development of low-grade chronic inflammation important in the development of atherosclerosis. In samples of subcutaneous, visceral and perivascular adipose tissue (SAT, VAT, and PVAT, respectively) obtained from healthy subjects (living kidney donors, LKD), we analyzed macrophages and their polarization, gene expression of pro-inflammatory cytokines and the effect of substances released by VAT on the level of monocyte adhesion to the endothelium. In some analyses, we included samples of SAT, VAT and PVAT obtained...

Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...

Introduction: Recent advances in posterior lamellar keratoplasty necessitated the preparation of posterior corneal lamellae even in the Czech Republic. The aim of this work was to introduce and standardize a novel method of manual preparation of corneal lamellae for Descemet Membrane Endothelial Keratoplasty with a Stromal rim (DMEK-S) in an ocular tissue bank. After setting the criteria for endothelial quality, we obtained a licence to provide the tissues for transplantation purposes. The obtained results were analysed after two years. Furthermore, a novel lamellar insertion technique using a cartridge was assessed. The potentials for the long-term storage of posterior corneal lamellae by the vitrification in liquid ethane was studied using human amniotic membrane as a model tissue. Material and Methods: Corneoscleral buttons stored in tissue cultures were used to prepare lamellae consisting of a central zone of endothelium and Descemet membrane supported by a stromal rim at the periphery. The manual preparation was performed on an artificial anterior chamber using the big bubble technique. Endothelial quality was assessed before storage, before and immediately after preparation as well as after 2 days of storage at 31řC. A group of 12 corneas with a live endothelial cell density ≥ 2500 cells/mm2...

Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...

Part I: Endothelial cells form the posterior layer of the cornea and are important for maintaining its transparency. Dysfunctional endothelium can only be restored by transplantation. The global shortage of donor corneas requires the search for alternative treatments. The preparation of the graft by tissue engineering methods is complicated by low proliferative capacity of endothelium. To date, no endothelium-specific marker has been defined and the existence of endothelial stem cells has not been confirmed yet. We have prepared a protocol for culturing endothelial cells from research-grade tissue - corneoscleral rims obtained after transplantation or corneas excluded from the transplant process. We monitored localization of selected proteins, including stem cell markers, in native tissue and in primary cell cultures. We prepared up to 6.4 cm2 of endothelium from one cornea/rim, which had cellular features comparable to the native endothelium. This approach can increase the amount of endothelium for research or transplantation purposes. Using indirect immunohistochemistry, we showed that none of the previously proposed endothelial molecular markers is specific for these cells. We detected the expression of stem cell markers throughout the endothelial layer. In the porcine cornea model, we monitored...

<p>UVOD: Gojaznost je hronično, multifaktorijalno i kompleksno oboljenje povezano sa povećanim rizikom od aterosklerotskih kardiovaskularnih bolesti (KVB). Disfunkcija vaskularnog endotela predstavlja rani događaj u patofiziolo&scaron;kom kontinuumu aterosklerotskog procesa, a produženo izlaganje vaskularnog endotela faktorima rizika za aterosklerozu udruženim sa gojaznosti (insulinska rezistencija, dislipidemija, proinflamatorno/protrombozno stanje), može doprineti procesima aktivacije/disfunkcije endotela i progresiji ateroskleroze u supkliničku, odnosno kliničku formu bolesti. CILJ: Uporediti koncentracije solubilne forme adhezionih molekula &ndash; intracelularnog adhezivnog molekula &ndash;1 (sICAM&ndash;1) i E selektina (sE&ndash;selektin), između ispitanika sa hiperalimentacionim tipom gojaznosti i normalno uhranjenih zdravih ispitanika, kao i utvrditi eventualno postojanje razlika u koncentraciji sICAM&ndash;1 i sE&ndash;selektina između ispitanika kod kojih je merenjem debljine kompleksa intima medija karotidne arterije (IMK) uočen supklinički stadijum ateroskleroze i ispitanika koji imaju normalnu debljinu IMK. Ispitati povezanost parametara telesne kompozicije (ukupne masne mase tela i masne mase abdominalnih depoa), cirkuli&scaron;ućih koncentracija biomarkera disfunkcije vaskularnog endotela (sICAM&ndash;1 i sE&ndash;selektina) i IMK kod ispitanika sa hiperalimentacionim tipom gojaznosti. MATERIJAL I METODE: U istraživanje je uključeno 60 ispitanika sa hiperalimentacionim tipom gojaznosti bez pridruženih komorbiditeta i 30 zdravih normalno uhranjenih učesnika usklađenih sa ispitanicima po godinama života i polu koji su činili kontrolnu grupu. Svim ispitanicima su urađena antropometrijska merenja, analiza komponenata telesne kompozicije (bioelektrična impedansna analiza, Tanita Body Composition Analyzer BC &ndash; 418 MA III), laboratorijska analiza uzoraka krvi na automatizovanim analizatorskim sistemima sa određivanjem parametara metabolizma glukoze (bazalno i 2 h u toku oralnog glukoza tolerans testa), lipida i lipoproteina, inflamacije i homocisteina. Određivanje serumske koncentracije sICAM&ndash;1 i sE&ndash;selektina je vr&scaron;eno ELISA tehnikom (R&amp;D Systems, Inc. Minneapolis, USA). Vrednosti IMK&ndash;a su određivane pomoću karotidnog dupleks ultrazvuka (Aloka SSD&ndash;650 US system, Tokyo), a na osnovu izmerenih (IMK) i normalno očekivanih vrednosti IMK za svakog ispitanika je izračunavan IMK Z&ndash;skor. Supklinički stadijum ateroskleroze je definisan kao vrednost IMK Z&ndash;skora veća od 1 (&scaron;to odgovara vrednosti IMK većoj od 95 percentila normalno očekivane vrednosti u odnosu na pol i godine života ispitanika). REZULTATI: Ispitanici sa hiperalimentacionim tipom gojaznosti su imali statistički značajno vi&scaron;e vrednosti medijane serumske koncentracije sE&ndash;selektina u poređenju sa medijanom serumske koncetracije sE&ndash;selektina učesnika u kontrolnoj grupi (36,2 (33,21&ndash;43.7) vs. 25,14 (23,1&ndash;29,48) ng/mL, P=0,00). Gojazni ispitanici III stepena gojaznosti su imali statistički značajno vi&scaron;u medijanu serumske koncenracije sE&ndash;selektina u odnosu na medijanu sE&ndash;selektina u ispitanika I stepena gojaznosti (41,5 (36,58&ndash;49,48) vs. 34,34 (22,49&ndash;36,62) ng/mL, P=0,00), odnosno medijanu sE&ndash;selektina u ispitanika II stepena gojaznosti (41,5 (36,58&ndash;49,48) vs. 32,1 (26,1&ndash;43,64) ng/mL, P=0,00). Nije uočena statistički značajna razlika u medijani serumske koncentracije sE&ndash;selektina između ispitanika I i II stepena gojaznosti (34,34 (22,49&ndash;36,62) vs. 32,1 (26,1&ndash;43,64) ng/mL, P=0,12). Gojazni ispitanici su imali statistički značajno vi&scaron;e vrednosti medijane serumske koncentracije sICAM&ndash;1 u poređenju sa medijanom serumske koncetracije sICAM&ndash;1 učesnika u kontrolnoj grupi (266,8 (245,8&ndash;326,73) vs.183,32 (167,9&ndash;208,57), P=0,00). U ispitivanoj grupi gojaznih uočena je statistički značajna razlika u medijani koncentracije sICAM&ndash;1 između ispitanika u I, II i III stepena gojaznosti (200,6 (190,26 - 264,4) vs. 278,5 (219,54 - 343,24) vs. 329,6 (259,2 - 350,34) ng/mL, P=0,00). Učestalost IMK Z&ndash;skor &gt; 1 je bila statistički značajno eća u gojaznih ispitanika u odnosu na kontrolnu grupu (36/60 vs. 7/30, P=0,00). Ispitanici sa IMK Z&ndash;skor &gt; 1 su imali statistički značajno vi&scaron;u medijanu koncentracije sICAM&ndash;1 u odnosu na ispitanike kod kojih je IMK Z&ndash;skor &le; 1 (295,4 (238,46&ndash;340,38) vs. 244,2 (227,35&ndash;260,38), P=0.00). Regresionom analizom (R2=0,71, korigovani R2=0,59) je utvrđeno da su parametri hsCRP (&beta;=0,45, P=0,00), HOMA&ndash;IR (&beta;=0,44, P=0,035) i ISI (&beta;=&ndash;0,36, P=0,028) nezavisno i statistički značajno povezani sa serumskom koncentracijom sE&ndash;selektina. Regresionom analizom (R2=0,65, korigovani R2=0,56) je utvrđeno da parametri ITM (&beta;=0,55, P=0,00), trigliceridi (&beta;=0,30, P=0,00), HDL holesterol (&beta;=&ndash;0,31, P=0,00), odnos TG/HDL&ndash;holesterol (&beta;=0,33, P=0,01), hsCRP (&beta;=0,31, P=0,00) i fibrinogen (&beta;=0,34, P=0,00) su nezavisno i statistički značajno povezani sa serumskom koncentracijom sICAM&ndash;1. U faktorskoj analizi je izdvojeno pet faktora &ldquo;gojaznost&rdquo;, &ldquo;insulinska rezistencija&rdquo;, &ldquo;aterogeni faktor&rdquo;, &ldquo;endotelna disfunkcija i vaskularna inflamacija&rdquo; i &ldquo;metabolički faktor&rdquo; koji obja&scaron;njavaju 69.72% ukupne varijanse ispitivanog uzorka. U multivarijabilnom modelu sa svim faktorima zajedno kojim je obja&scaron;njeno ukupno 75% varijanse, jedino je faktor gojaznost imao statički značajan i nezavistan uticaj na vrednost IMK Z&ndash;skor &gt; 1 (OR=2,74 (CI 1,18&ndash;6,33), P=0,019). U faktoru gojaznost su se izdvojili parametri: FAT trunk (%), FAT (%), OS (cm), ITM (kg/m2), LDL &ndash; holesterol (mmol/L), SP (mmHg), HOMA1&ndash;%B, fibrinogen (g/L), ApoB/apoA-I i hsCRP (mg/L). Univarijantom logističkom regresijom je uočeno da porast u koncentraciji LDL&ndash;H (OR=5,33 (CI 1,9&ndash;14,2), P=0,02) i koncentraciji hsCRP&ndash;a (OR=2,53 (CI 1,3&ndash;3,98),P=0,017) povećava rizik za postojanje vrednosti IMK Z&ndash;skor &gt; 1. ZAKLJUČAK: Cirkuli&scaron;uće serumske koncentracije biomarkera disfunkcije vaskularnog endotela, sE&ndash;selektina i sICAM&ndash;1, su značajno vi&scaron;e kod ispitanika sa hiperalimentacionim tipom gojaznosti u odnosu na njihove koncentracije u normalno uhranjenih ispitanika. U gojaznih ispitanika, koncentracija sE&ndash;selektina je povezana sa vrednostima indeksa insulinske rezistencije i biomarkera inflamacije, dok je koncentracija sICAM&ndash;1 značajno povezana sa udelom masne mase u ukupnoj telesnoj masi, vrednostima biomarkera inflamacije i proaterogenih lipidskih parametara. Ispitanici kod kojih postoji uvećanje abdominalnih masnih depoa i ukupnog udela masnog tkiva u telesnoj masi, vrednosti SKP, koncentracije LDL &ndash; holesterola, vrednosti lipoproteinskog indeksa ApoAI/apoB, bazalne insulinemije i biomarkera inflamacije, imaju trostruko povećan rizik od supkliničkog stadijuma ateroskleroze. U gojaznih osoba prilikom procene rizika od aterosklerotskih KVB, potrebno je utvrditi fenotipske osobine vaskularnog endotela i eventualno postojanje supkliničkog stadijuma ateroskleroze, da bi se definisale adekvatne preventivne mere i sagledale potencijalne terapijske mogućnosti.</p> / <p>INTRODUCTION: Obesity is a chronic, multifactorial and complex disease associated with an increased risk of atherosclerotic cardiovascular diseases (CVD). Vascular endothelial dysfunction is an early event in the pathophysiological continuum of atherosclerotic process. The prolonged exposure of vascular endothelium to classical and obesity associated risk factors (insulin resistance, dyslipidemia, proinflammatory state) could further promote deterioration of endothelial function and progression of atherosclerosis to subclinical or clinical form of disease. OBJECTIVE: The aim of the study was to compare the concentration of soluble forms of adhesion molecules, intracellular adhesion molecule-1 (sICAM-1) and E-selectin (sE-selectin), between obese subjects and normal weight healthy subjects, as well as to determine the possible existence of differences in concentration of sICAM-1 and sE-selectin among subjects with subclinical stage of atherosclerosis (assessed by measuring the thickness of the intima media complex of the carotid artery (IMT)), and subjects who have a normal value of IMT. Also, the aim was to determine the association between the parameters of body composition (total body fat mass and fat mass intra-abdominal depots), circulating concentrations of sICAM-1 and sE-selectin, and value of IMT in obese subjects. MATERIALS AND METHODS: The study included 60 obese nondiabetic subjects, without preexisting CVD and other associated comorbidity, and 30 healthy normal weight age and sex matched participants. All subjects underwent anthropometric measurements, analysis of the components of body composition (bioelectrical impedance analysis, Tanita Body Composition Analyzer BC - 418 MA III), laboratory analysis of blood samples (automated analyzer systems) with determining the parameters of glucose metabolism (basal and 2 h during the oral glucose tolerance test), lipids and lipoproteins, inflammation and homocysteine. Serum concentrations of sICAM-1 and sE-selectin were determined by ELISA (R &amp; D Systems, Inc., Minneapolis, USA). The values of IMK were determined by carotid duplex ultrasound (Aloka &ndash; ProSound ALPHA 10). IMK Z-score was calculated using the measured and the normal expected values of IMT for each patient. Subclinical stage of atherosclerosis was defined as the value of IMT Z-score greater than 1 (corresponding to the 95th sex-age-specific percentile of IMT measurements). RESULTS: Obese subjects had significantly higher median sE-selectin serum concentrations compared to median serum concentrations of sE-selectin in the normal weight subjects (36.2 (33.21-43.7) vs 25.14 (23.1-29.48) ng/mL, P=0.00). Morbid obesity subjects had significantly higher sE-selectin median serum concentration compared to the median sE-selectin concentration in moderate obese subjects (41.5 (36.58-49.48) vs 34.34 (22.49-36.62) ng/mL, P=0.00), and compared to the median sE-selectin concentration in severely obese subjects (41.5 (36.58-49.48) vs. 32.1 (26.1-4364) ng / mL, P=0.00). Obese subjects had significantly higher median sICAM-1 serum concentration compared to median sICAM-1 serum concentration in the control group (266.8 (245.8-326.73) vs. 183.32 (167.9-208.57), P=0.00). In the obese group, we observed a statistically significant difference in median sICAM-1 serum concentrations between moderate, severely and morbid obese subjects (200.6 (190.26-264.4) vs. 278.5 (219.54-343.24) vs. 329.6 (259.2-350.34) ng/mL, P=0.00). The frequency of IMT Z-score&gt; 1 was significantly higher in the obese group compared to control group (36/60 vs. 7/30, P=0.00). Subjects with IMT Z-score&gt; 1 had significantly higher median concentrations of sICAM-1 compared to those in which the IMK Z-score &le; 1 (295.4 (238.46-340.38) vs. 244.2 ( 227.35-260.38), P=0.00). In regression analysis (R2=0.71, adjusted R2=0.59), hsCRP (&beta;=0.45, P=0.00), HOMA-IR (&beta;=0.44, P=0.035) and ISI (&beta;=-0.36, P=0.028) were independently and significantly associated with serum sE-selectin concentration. In regression analysis (R2=0.65, adjusted R2=0.56), BMI (&beta;=0.55, P=0.00), triglycerides (&beta;=0.30, P=0.00), HDL cholesterol (&beta;=-0.31, P=0.00), the ratio of TG/HDL-cholesterol ratio (&beta;=0.33, P=0.01), hsCRP (&beta;=0.31, P=0.00 ) and fibrinogen (&beta;=0.34, P=0.00) were independently and significantly associated with serum sICAM-1 concentration. In the Factor analysis, five factors &quot;obesity&quot;, &quot;insulin resistance&quot;, &quot;atherogenic factor,&quot; &quot;endothelial dysfunction and vascular inflammation&quot; and &quot;metabolic factor&quot; explained 69.72% of the total variance of the test sample. In a multivariate model with all the factors together (75% of the total variance), &quot;obesity&quot; factor was significantly and independently associated with IMT Z-score&gt; 1 (OR=2.74 (CI 1.18-6.33), P=0.019). The &quot;obesity&quot; factor consisted of parameters: trunk fat (%), fat (%), waist (cm), BMI (kg/m2), LDL &ndash; cholesterol (mmol/L), systolic blood presure (mmHg), HOMA1-% B, fibrinogen (g/L), Apo B/apoA-I and hsCRP (mg/L). Logistic regression analysis showed that independent predictors of IMT Z-score&gt; 1 were LDL-cholesterol (OR=5.33(CI 1.9-14.2), P=0.02) and hsCRP (OR=2.53 (CI 1.3-3.98), P=0.017). CONCLUSION: Circulating serum concentrations of endothelial dysfunction biomarkers, sE-selectin and sICAM-1, were significantly higher in obese subjects compared to concentration in the normal weight subjects. In obese subjects, the concentration of sE-selectin was associated with insulin resistance and biomarkers of inflammation, whereas sICAM-1 concentration was associated with fat mass, inflammation biomarkers and the proatherogenic lipid parametars. In individuals with increased abdominal fat depots and total proportion of fat mass in the body weight, values of SBP, LDL-C, ApoB/apoA-I, basal insulin levels and biomarkers of inflammation, there is threefold increased risk of subclinical stages of atherosclerosis. In order to define an adequate preventive measures and possible therapeutic options for atherosclerotic CVD in obese subjects, it is necessary to assess the phenotypic characteristics of vascular endothelium and possible presence of subclinical stage of atherosclerosis.</p>

<p>Uvod: Sepsa je jedan od vodećih uzroka smrtnosti u jedinicama intenzivnog lečenja i van njih uprkos implementaciji novih dijagnostičkih i terapijskih protokola &scaron;irom sveta. Multiorganska disfunkcija (MODS), koja predstavlja najtežu formu nepovoljnog toka sepse, je u osnovi svojih patofiziolo&scaron;kih de&scaron;avanja obeležena promenama, koje se de&scaron;avaju na nivou kapilara, pre svega u endotelu. Poremećaji koagulacije koji se javljaju kao posledica ovih promena u endotelu su prepoznati kao jedan od dijagnostičkih kriterijuma prema najnovijim smernicama za dijagnostiku i lečenje sepse, međutim njihov značaj u predviđanju toka i ishoda ovog oboljenja jo&scaron; uvek nije precizno definisan. Cilj istraživanja: Odrediti koncentraciju markera endotelne aktivacije, aktivacije koagulacije, aktivnost prirodnih inhibitora koagulacije i funkcionalnost fibrinolize kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za postavljanje dijagnoze sepse i predikciju pojave komplikacija. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za procenu ishoda kod obolelih od sepse. Materijal i metode: Istraživanje je sprovedeno analitičkom metodom u formi studije preseka, a obuhvatilo je pacijente lečene na Odeljenju anestezije i reanimacije Urgentnog centra Kliničkog centra Vojvodine i na Klinici za infektivne bolesti Kliničkog centra Vojvodine, u Novom Sadu. Istraživanje je sprovođeno tokom 2012. i 2013. godine u trajanju od dve godine. U studiju je bilo uključeno 180 ispitanika od kojih je 150 imalo postavljenu dijagnozu sepse,a 30 ispitanika su činili kontrolnu grupu su klinički i biohemijski zdravih ispitanika, dobrovoljni davaoci krvi. Ispitanici su kategorisani u četiri grupe u odnosu na kliničko stanje i laboratorijske nalaze unutar prvih 24 časa od prijema: bolesnici sa sepsom, te&scaron;kom sepsom, septičkim &scaron;okom i multiorganskom disfunkcijom na prijemu. Nakon kategorizacije ispitanika, izračunati su APACHE II i SOFA numerički pokazatelji procene težine bolesti ispitanika. U roku 24 časa od trenutka postavljanja dijagnoze sepse, iz uzoraka krvi ispitanika, izvr&scaron;ene su predviđene laboratorijske analize u cilju praćenja endotelne aktivacije, aktivacije koagulacije i inhibicije antikoagulantnih mehanizama. U toku 48 časova od prijema, bolesnici koji nisu imali MODS na prijemu su intenzivno praćeni u cilju evidentiranja razvoja multiorganske disfunkcije, dok su bolesnici koji su imali MODS praćeni radi evidentiranja perzistiranja ili eventualne rezolucije MODS-a. Zdravstveno stanje bolesnika je praćeno tokom 28 dana od trenutka uključivanja u studiju i nakon tog perioda je evidentiran ishod lečenja u smislu preživljavanja ili smrtnog ishoda. Statistička analiza je izvr&scaron;ena pomoću statističkog paketa IBM SPSS 20 Statistics. Podaci su predstavljeni tabelarno i grafički, a statistička značajnost određivana je na nivou p&lt; 0,05. Rezultati: Vrednosti biolo&scaron;kih markera endotelne aktivacije i aktivacije koagulacije su statistički značajno povi&scaron;ene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji, dok su vrednosti prirodnih inhibitora koagulacije statistički značajno snižene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Vrednosti APTT-a, PT-a, D-dimera, fibrinogena, prirodnih inhibitora koagulacije i markera endotelne aktivacije (endokan i vWF antigena i aktivnosti) imaju značajan i veoma visok dijagnostički potencijal. Vrednosti biomarkera endotelne disfunkcije i pokazatelja poremećaja hemostaznog mehanizma su značajni prediktori komplikacija kod bolesnika sa sepsom. APTT, PT, D-dimer, broj trombocita, vrednosti priorodnih inhibitora koagulacije, trombomodulina, endokana i ETP-a su jednako validni u inicijalnoj proceni toka kliničke slike sepse kao i prediktivni APACHE II i SOFA skorovi. Koncentracija trombomodulina, D-dimera, ETP-a i PC su dobri prediktori nastanka MODS-a u prvih 48 časova u toku sepse. Endokan, PT, APTT, koncentracija fibrinogena, prirodnih inhibitora koagulacije i vrednosti ETP-a su značajni u predikciji mortaliteta kod bolesnika sa sepsom. Zaključci: Ukoliko bi pokazatelji aktivacije endotela i mehanizma hemostaze bili inkorporirani u određeni sistem skorovanja u cilju procene težine bolesti u smislu ishoda kod bolesnika sa sepsom, to bi moglo doneti doprinos boljoj klasifikaciji bolesnika, te primeni pravovremene i adekvatne terapije u cilju postizanja pozitivnog ishoda kod bolesnika sa sepsom. Prilikom interpretacije pokazatelja inflamacije i koagulacije neophodno je steći uvid u celokupnu sliku pro-i antikoagulantnih de&scaron;avanja koja se odvijaju tokom sepse, odnosno adekvatno proceniti pravac toka disbalansa mehanizma hemostaze da bi se eventualnim terapijskim merama mogao postići pozitivan učinak.</p> / <p>Introduction: Sepsis is one of the main causes of death in intensive care units and other hospital wards in spite of implementation of new sepsis treatment guidelines in everyday hospital practice worldwide. Changes that occur in the microvasculature, affecting primarily endothelial cell, are the basis of the pathophysiology of multiorgan dysfunction (MODS) in sepsis. Coagulation abnormalities which occur as a consequence of endothelial changes are recognized as diagnostic criteria for sepsis, but significance of these changes in the outcome prognosis and prediction of the course of sepsis is still not accurately defined. Aims: Evaluation of hemostasis related parameters and endothelial activation biomarkers values in patients with sepsis and healthy volunteers. Determination whether the levels of hemostasis-related parameters and biomarkers of endothelial activation have diagnostic significance and are they associated with MODS development and persistence in the first 48 hours of hospitalization and 28-day mortality in patients with sepsis. Material and methods: This is cross-sectional study conducted in 2012 and 2013 in the Department of Anesthesia and Reanimation at the Emergency Center of the Clinical Center of Vojvodina and in the Clinic of Infectious Disease at the Clinical Center of Vojvodina. 150 patients who fulfilled criteria for diagnosis of sepsis were included in the study. Patients were divided into 4 groups: sepsis, severe sepsis, septic shock and MODS. 30 healthy volunteers, blood donors were the control group. After the categorization of patients, during the first 24 hours of hospitalization, predictive APACHE II and SOFA scores were calculated. Hemostasis related parameters and endothelial activation biomarkers concentrations were determined within the first 24 hours of the onset of the disease. To assess the development of complication of the disease, patients were monitored for 48 hours for MODS development and persistence or resolution and for 28 days from the onset of sepsis for outcome assessment. Data were analyzed using SPSS 20.0 software and are presented in tables and graphs, statistical significance was set at p&lt; 0,05. Results: Biomarkers of endothelial and coagulation activation are significantly higher in patients with sepsis in comparison to their values in healthy volunteers, while concentrations of natural anticoagulants are significantly lower in patients with sepsis than in healthy volunteers. APTT, PT, D-dimer, fibrinogen, natural anticoagulants and biomarkers od endothelial activation (endocan and vWF antigen and activity) have diagnostic significance in patients with sepsis. Hemostasis related parameters and endothelial activation biomarkers are good prognostic factors for complication development in patients with sepsis. APTT, PT, D-dimer, platelet count, natural anticoagulants, thrombomodulin, endocan and ETP are equally valuable in early prediction of sepsis development as APACHE II and SOFA scores. Thrombomodulin, D-dimer, ETP and PC are good predictors of MODS development during the first 48 hours from sepsis onset. Endocan, PT, APTT, fibrinogen concentration, values of natural anticoagulants and ETP values are significant in 28-day mortality prediction in patients with sepsis. Conclusion: A combination of markers of endothelial dysfunction with widely used ICU scores and organ failure assessment could contribute to an early recognition of complication development and consequent death in patients with sepsis. It is necessary to obtain the full insight in pro-and anticoagulant dynamic evaluation while interpreting coagulation and inflammation processes in sepsis development, in order to accurately lead early resuscitation therapy.</p>

<p>UVOD: Procesi koji obuhvataju endotelnu disfunkciju, oksidativni stres, hroničnu inflamaciju, hiperaktivnost i aktivaciju trombocita te naru&scaron;avanje ravnoteže procesa koagulacije i fibrinolize od najranijih faza razvoja dijabetes melitusa tip 2 (T2DM) promovi&scaron;u aterogenezu i nastanak aterotromboznih komplikacija. Kompleksan terapijski pristup u T2DM ima za cilj ne samo uspostavljanje glikoregulacije, korekciju brojnih metaboličkih poremećaja i modifikaciju pridruženih faktora rizika za nastanak ateroskleroze već i primenu antitrombocitne terapije u cilju primarne ili sekundarne prevencije aterotromboznih komplikacija. Uprkos primenjenoj antiagregacionoj terapiji, deo bolesnika doživi rekurentne aterotrombozne atake. Bolesnici sa T2DM se izdvajaju kao grupa sa posebnim rizikom za recidivantne aterotromboze &scaron;to može biti uslovljeno rezistencijom na primenjenu antitrombocitnu terapiju. Praćenje efekata antitrombocitne terapije i blagovremeno identifikovanje rezistentnih bolesnika ima za cilj optimizaciju primenjene antitrombocitne terapije &scaron;to može biti od izuzetnog kliničkog značaja u smislu sprečavanja progresije aterotromboznog procesa. CILJ: Proceniti i uporediti nivoe biomarkera, pokazatelja endotelne aktivacije, aktivacije i agregabilnosti trombocita u bolesnika sa bole&scaron;ću arterijskih krvnih sudova u tipu 2 dijabetes melitusa u odnosu na njihove vrednosti u zdravoj populaciji. Uporediti efikasnost primenjene antitrombocitne terapije tienopiridinima u bolesnika sa tipom 2 dijabetes melitusa i bole&scaron;ću arterijskih krvnih sudova u odnosu na efikasnost ove terapije u nedijabetičnoj populaciji bolesnika sa bole&scaron;ću arterijskih krvnih sudova. MATERIJAL I METODE: U ispitivanje je uključeno 100 ispitanika oba pola, starosti od 33 do 70 godina života, kod kojih je prethodno utvrđeno postojanje neke od kliničkih manifestacija bolesti arterijskih krvnih sudova (IBS, CVB, PAB) koji kao antitrombocitnu terapiju uzimaju tienopiridinski preparat, klopidogrel. Od toga, 50 uključenih ispitanika imalo je dijagnozu dijabetes melitus tipa 2, a 50 su bili bolesnici bez dijabetesa. Kontrolnu grupu je činilo 30 klinički i biohemijski zdravih ispitanika, nepu&scaron;ača koji su prema polnoj i dobnoj strukturi odgovarali ispitivanim grupama bolesnika. Svim ispitanicima su urađena antropometrijska merenja, laboratorijska analiza uzoraka krvi na automatizovanim analizatorima sa određivanjem parametara metabolizma glukoze, lipida, parametera inflamacije, KKS, parmetara koagulacije i trombocitnih pokazatelja. Određivanje serumske koncentracije sE&ndash;selektina i sP-selektina je vr&scaron;eno ELISA tehnikom (R&amp;D Systems, Inc. Minneapolis, USA). Plazmatska koncentracija vWFAg-a određivana je imunoturbidimetrijskom metodom na koagulacionom analizatoru Siemens Healthcare Diagnostics, Nemačka. Agregabilnost trombocita je određivana impedantnom agregometrijom (Multiple Electrode Aggregometry - MEA) na Multiplate analizatoru, Dynabyte, Minhen, Nemačka. Bazalna agregabilnost trombocita procenjivana je TRAP testom, rezidualna agregabilnost trombocita pod terapijom klopidogrela ADP testom, rezidualna agregabilnost trombocita pod terapijom aspirina, ASPI testom. Individualni odgovor na primenjenu antiagregacionu terapiju je procenjivan i na osnovu procenta sniženja bazalne agregabilnosti trombocita (%SAT) nakon primenjene antiagregacione terapije &scaron;to je izračunato sledećim formulama: procena antiagregacionog efekta klopidogrela:%SATadp =100 x (1-ADP/TRAP) i procena antiagregacionog efekta aspirina:%SATaspi =100 x (1-ASPI/TRAP). REZULTATI: Nivo sE-slektina je bio signifikantno vi&scaron;i u bolesnika sa T2DM u odnosu na bolesnike bez dijabetesa (45,1&plusmn;18,1vs.31,8&plusmn;10,5ng/ml; p&lt;0,001) i kontrolnu grupu zdravih ispitanika (45,1&plusmn;18,1vs.27,2&plusmn;11,2ng/ml; p&lt;0,001). Plazmatski nivo vWF Ag, bio je statistički značajno vi&scaron;i u bolesnika sa T2DM u odnosu na grupu ispitanika bez dijabetesa (172&plusmn;75,2vs. 146&plusmn;40,6%; p=0,045), kao i u odnosu na kontrolnu grupu zdravih (172&plusmn;75,2vs.130&plusmn;33,8%; p=0,007). Nivo sPselektina bio je statistički značajno vi&scaron;i kod bolesnika s T2DM u odnosu na ispitanike u grupi dijabetesa (95,2&plusmn;31,8vs.84,0&plusmn;21,8 ng/ml; p=0,042) i kontrolnoj grupi (95,2&plusmn;31,8vs.76,7&plusmn;16,2ng/ml; p=0,004). Uočeno je da je %rP statistički bio značajno vi&scaron;i u grupi dijabetičara u odnosu na grupu ispitanika bez dijabetesa (3,47&plusmn;1,30vs.2,30&plusmn;1,30%; p&lt;0,001) i kontrolnu grupu zdravih (3,47&plusmn;1,30vs.2,29&plusmn;1,23%; p&lt;0,001). Bolesnici sa T2DM imali su statistički značajno vi&scaron;e vrednosti ADP testa (70,3&plusmn;22,0vs.56,9&plusmn;19,7U; p=0,002) u odnosu na bolesnike bez dijabetesa, a uočen je i značajno niži stepen procenta sniženja bazalne agregabilnosti, %SATadp, u dijabetičara u odnosu na ispitanike bez dijabetesa (31,6&plusmn;12,4vs. 48,6&plusmn;12,6 %; p&lt;0,001). U grupi ispitanika sa T2DM vrednost TRAP testa statistički značajno pozitivno koreli&scaron;e sa brojem neutrofila (r=0,349;p= 0,013) i NLR-om (r=0,472;p=0,001), a multivarijantnom linearnom regresionom analizom dokazana je nezavisna povezanost TRAP testa i fibrinogena (B=9,61;p=0,009). Takođe, u istoj ispitivanoj grupi postoji pozitivna povezanost ADP testa sa HOMAIR (r=0,319;p=0,024), NLR-om (r=0,515;p&lt;0,001), hsCRP-om (r=0,356;p=0,011), kao i sa %rP (r=0,302;p=0,049). Multivarijantnom linearnom regresionom analizom dokazana je nezavisna povezanost ADP testa i ITM (B=1,43;p=0,043). %SATadp u bolesnika sa T2DM negativno je korelisao sa ITM (r= -0,381;p=0,006), OS (r= - 0,387;p=0,006), HOMA-IR (r= -0,349;p=0,013), hsCRP-om (r= -0,288; p=0,043), %rP (r= -0,302;p=0,049), sE-selektinom (r= -0,369; p=0,008) i sP-selektinom (r= - 0,374;p=0,007). U grupi dijabetičara, postoji pozitivna povezanost %rP sa ITM (r=0,365;p= 0,016), OS (r=0,435;p=0,004), HOMA-IR (r=0,409;p=0,006), hsCRP (r=0,374;p=0,014), sP-selektinom (r=0,341;p=0,025) i vWFAg-om (r=0,348;p=0,022). Takođe, sE-selektin pozitivno koreli&scaron;e sa ITM (r=0,380;p =0,006), OS (r=0,380; p=0,007), HOMA-IR (r=0,339;p=0,016), hsCRP-om (r=0,351;p=0,013), a sP-selektin sa ITM (r=0,312;p=0,027), OS (r=0,395;p=0,005), HOMA-IR (r=0,286;p=0,044), hsCRP-om (r=0,369; p=0,008) i nivoom sE &ndash; selektina (r=0,560;p &lt;0,001). Evaluirajući odgovor na terapiju klopidogrelom u podgrupama bolesnika sa dijabetesom, napravljenim prema kvartilnoj distribuciji nivoa ADP-a, tj. stepenu rezidualne agregabilnosti trombocita u toku terapije klopidogrelom, uočeno je da ukupna bazalna agregabilnost trombocita procenjena TRAP testom statistički značajno raste od prvog do četvrtog kvartila (76,50 &plusmn;19,91 vs. 94,54&plusmn;16,67 vs. 112,00&plusmn;10,22 vs. 128,92&plusmn;15,69U;p&lt;0,001), dok se %SATadp od prvog do četvrtog kvartila značajno smanjivao (40,44&plusmn;13,33 vs. 31,20&plusmn;11,82 vs. 33,16&plusmn;7,03 vs. 21,53&plusmn;10,16%). ZAKLJUČAK: Koncentracije cirkuli&scaron;ućih biomarkera endotelne aktivacije, sE &ndash; selektina i vWF Ag-a, solubilnog biomarkera trombocitne aktivacije, sP &ndash; selektina, kao i procenat retikulisanih trombocita, %rP, markera trombocitnog prometa, značajno su povi&scaron;ene kod bolesnika sa bole&scaron;ću arterijskih krvnih sudova u tipu 2 dijabetes melitusa u odnosu na njihove koncentracije kod zdravih ispitanika i bolesnika bez dijabetesa. Bolesnici sa T2DM imali su znatno vi&scaron;i stepen rezistencije na antitrombocitnu terapiju klopidogrelom u odnosu na bolesnike bez dijabetesa, procenjene stepenom rezidualne agregabilnosti trombocita, ADP test, kao i procentom sniženja ukupne bazalne agregabilnosti trombocita, %SATadp, metodom impedantne agregometrije, a &scaron;to je uslovilo i trend učestalijeg ponavljanja ishemijskih ataka u odnosu na bolesnike bez dijabetesa. Međusobna povezanost ispitivanih biomarkera endotelne i trombocitne aktivacije (sE &ndash; selektina, vWF Aga, sP &ndash; selektina), kao i markera prometa trombocita (%rP) sa metaboličko inflamatornim parametrima i sa indikatorima odgovora na antiagregacionu terapiju, može ukazivati na to da nepovoljan metabolički milje dijabetičara može biti jedan od doprinosnih faktora lo&scaron;em odgovoru na antitrombocitnu terapiju klopidogrelom.</p> / <p>INTRODUCTION: Processes involving endothelial dysfunction, oxidative stress, chronic inflammation, platelet activation and the imbalance between coagulation and fibrinolysis promote atherogenesis and atherothrombotic complications at early stage of diabetes mellitus type 2 (T2DM). The complex therapeutic approach in T2DM aims not only to reestablish glycemic control and to correct a number of metabolic disorders, but also to achieve primary or secondary prevention of atherothrombotic complications. Despite the applied antiplatelet therapy, some patients experience recurrent atherothrombotic attacks. Patients with T2DM are the group at particular risk for recurrent atherothrombosis, which can be caused by antiplatelet therapy resistance. Monitoring the effectiveness of antiplatelet therapy and identification of resistant patients aims to optimize the applied antiplatelet therapy, which can be of great clinical significance in terms of preventing progression of atherotrombotic processes. AIM: Evaluate and compare the levels of biomarkers, indicators of endothelial activation, platelet activation and aggregability in patients with arterial vascular disease in type 2 diabetes mellitus compared to their values in a healthy population. Compare the effectiveness of applied antiplatelet therapy with thienopyridines in patients with type 2 diabetes mellitus and arterial vascular disease compared to the efficacy of this therapy in nondiabetic population of patients with arterial vascular disease. MATERIAL AND METHODS: The study included 100 patients, 33 to 70 years of age, with previously established existence of some of the clinical manifestations of arterial vascular disease (CAD, CVD, PAD), taking thienopyridine antiplatelet therapy with clopidogrel. 50 patients was previously diagnosed with diabetes mellitus type 2 and 50 were nondiabetic patients. Control group included 30 age and sex matched healthy participants, non-smokers. All subjects underwent anthropometric measurements and laboratory analysis of blood samples on automated analyzers with determining the parameters of glucose metabolism, lipids, inflammation parameters, complete blood count, coagulation and platelet parameters. Serum concentrations of sEselectin and sP-selectin were determined by ELISA (R&amp;D Systems, Inc., Minneapolis, USA). vWFAg was determined by immunoturbidimetry on coagulometer Siemens Healthcare Diagnostics, Germany. Platelet aggregability was determined by impedance aggregometry (Multiple Electrode Aggregometry - MEA) on Multiplate analyzer, Dynabyte, Munich, Germany. Basal platelet aggregability was estimated by TRAP test, residual platelet aggregability during clopidogrel treatment was estimated by ADP test and during aspirin treatement by ASPI test. Individual response to antiplatelet therapy was estimated by the percentage of decrease in basal platelet aggregability (%DPA) obtained after antiplatelet therapy, calculated bypresented formulas: %DPAadp =100 x (1-ADP/TRAP)and %DPAaspi =100 x (1- ASPI/TRAP). RESULTS: Concentration of sE-selectin was significantly higher in patients with T2DM in order to non-diabetic patients (45,1&plusmn;18,1vs.31,8&plusmn;10,5ng/ml;p&lt;0,001) and healthy control group (45,1&plusmn;18,1vs.27,2&plusmn;11,2ng/ml; p&lt;0,001). vWF Ag was significantly higher in diabetic patients than in non-diabetics (172&plusmn;75,2vs. 146&plusmn;40,6%; p=0,045) and healthy controls (172&plusmn;75,2vs.130&plusmn;33,8%; p=0,007). sP-selectin was also significantly higher in patients with T2DM than in non-diabetics (95,2&plusmn;31,8vs.84,0&plusmn;21,8 ng/ml; p=0,042) and healthy controls (95,2&plusmn;31,8vs.76,7&plusmn;16,2ng/ml; p=0,004). %rP was significantly higher in group of patients with T2DM than in nondiabetic patients (3,47&plusmn;1,30vs.2,30&plusmn;1,30%; p&lt;0,001) and healthy control group (3,47&plusmn;1,30vs.2,29&plusmn;1,23%; p&lt;0,001). T2DM patients had statistically higher values of ADP test (70,3&plusmn;22,0vs.56,9&plusmn;19,7U; p=0,002) compared to patients without diabetes, and significantly lower %DPAadp (31,6&plusmn;12,4vs. 48,6&plusmn;12,6 %; p&lt;0,001). In T2DM group of patients, level of TRAP test correlated positively with number of white blood cells (r=0,349;p= 0,013) and NLR (r=0,472;p=0,001), and multivariant linear regression analisys showed significant independent association of TRAP test with fibrinogen (B=9,61;p=0,009). Statistically significant positive correlation of ADP test with HOMA-IR (r=0,319;p=0,024), NLR (r=0,515;p&lt;0,001), hsCRP (r=0,356;p=0,011) and %rP (r=0,302;p=0,049) was observed in patients with T2DM. Multivariant linear regression analisys showed significant independent association of ADP test with BMI (B=1,43;p=0,043). %DPAadp negatively correlated with BMI (r=-0,381;p=0,006), WC (r= - 0,387;p=0,006), HOMA-IR (r= -0,349;p=0,013), hsCRP (r= -0,288; p=0,043), %rP (r= -0,302;p=0,049), sE-selectin (r= -0,369; p=0,008) and sP-selectin (r= -0,374;p=0,007) in diabetic patients. Significant positive correlation of %rP with BMI (r=0,365;p= 0,016), WC (r=0,435;p=0,004), HOMA-IR (r=0,409;p=0,006), hsCRP (r=0,374;p=0,014), sP-selectin (r=0,341;p=0,025) and vWFAg (r=0,348;p=0,022) was found in diabetics. Also, sE-selectin positively correlated with BMI (r=0,380;p =0,006), WC (r=0,380; p=0,007), HOMA-IR (r=0,339;p=0,016), hsCRP(r=0,351;p=0,013), and sPselectin correlated positively with BMI (r=0,312;p=0,027), WC (r=0,395;p=0,005), HOMA-IR (r=0,286;p=0,044), hsCRP (r=0,369; p=0,008) and sE &ndash; selectin (r=0,560;p &lt;0,001). Evaluating the response to clopidogrel therapy in subgrpoups of diabetic patients accoarding the quartile distribution of ADP test (clopidogrel on-treatment platelet reactivity), it is found that total basal aggregability estimated by TRAP test significantly increased from the first to the fourth quartile (76,50 &plusmn;19,91 vs. 94,54&plusmn;16,67 vs. 112,00&plusmn;10,22 vs. 128,92&plusmn;15,69U;p&lt;0,001) while %DPAadp decreased (40,44&plusmn;13,33 vs. 31,20&plusmn;11,82 vs. 33,16&plusmn;7,03 vs. 21,53&plusmn;10,16%). CONCLUSION: Concentration of circulating biomarkers of endothelial activation, sE-selectin and vWF Ag, soluble marker of platelet activation, sP &ndash; selectin, as well as percentage of reticulated platelets, %rP, marker of platelet turnover, were significantly higher in patients with arterial vascular disease in T2DM compared to healthy controls and non-diabetics. Patients with T2DM had significantly higher degree of resistance to antiplatelet therapy with clopidogrel compared to non diabetics, estimated by ADP test, as well as with %DPAadp, what caused more frequent recurrent ischemic attacks compared to nondiabetic patients. Correlation of biomarkers of endothelial and platelet activation (sE &ndash; selectin, vWF Ag, sP &ndash; selectin) and markers of platelet turnover (%rP) with metabolic profile indicators and poor antiplatelet therapy response suggest that altered metabolic profile can be one of contributing factors of poor antiplatelet response in diabetic patients.</p>