| Abstract | Cilj istraživanja: Ciljevi istraživanja bili su utvrditi promjene udjela pojedinih leukocitnih subpopulacija i udio regulacijskih stanica T (T reg) u perifernoj krvi, odrediti koncentracije MCP-1, proupalnih citokina (IL-1, TNF- i IL-6) i protuupalnih citokina ( IL-10, TGF-) u plazmi te odrediti koncentracije VMA i HVA u urinu kod bolesnika s TIA-om i IMU-om. Dobivene podatke postavili smo u korelaciju s tijekom te konačnim ishodom bolesti korištenjem NIHSS-a prvog, trećeg i sedmog dana bolesti u svih ispitanika te NIHSS-om, mRS-om i Barthelovim indeksom nakon 90 dana od nastupa IMU-a.Materijali i metode:U ovu prospektivnu studiju bili su uključeni bolesnici (Tablica 1.) s IMU-om, podijeljeni u tri skupine: bolesnici s tranzitornom ishemijskom atakom (TIA), bolesnici s lakšim IMU-om (NIHSS 12) i bolesnici s težim IMU-om (NIHSS>12). U studiju je uključeno ukupno 126 ispitanika, u svakoj skupini po 42 bolesnika. Kontrolnu skupinu (42 ispitanika) činili su zdravi dobrovoljni davatelji krvi i urina iste zastupljenosti prema dobi i spolu.Kompletna krvna slika učinjena je na hematološkom analizatoru te C-reaktivni protein na biokemijskom analizatoru. Fenotipska obilježja limfocita T (CD3+/CD4+, CD3+/CD8+), limfocita B (CD5+/19+) te stanica NK (CD3-/CD16/56+,) kao T regulacijskih (CD4+CD25+Foxp3+) i CD11b+ stanica, analizirana su metodom imunofenotipizacije na FACS Calibur protočnom citometru. Koncentracija VMA i HVA u urinu ispitivana je metodom tekućinske kromatografije visoke djelotvornosti (prema engl. high performance liquid chomatography - HPLC) s elektrokemijskim detektorom. Koncentracija IL-1, TNF, i MCP-1 te IL-6, IL-10, TGF- u plazmi bolesnika, ispitivana je enzimatsko-imunokemijskom metodom (prema engl. Enzym Linked Immuno Sorbant Assay - ELISA). Rezultati: Zabilježen je postupan lagani pad vrijednosti limfocitne populacije (CD3-/CD16+/CD56+) u skupini bolesnika s lakšim MU-om i sedmog dana vrijednost je izrazito ispod referentne vrijednosti. Statistički značajna razlika izmjerena je sedmog dana u odnosu na treći dan u bolesnika s težim MU-om i TIA-om. Kod bolesnika s NIHSS >12, zabilježeno je statistički značajno povišenje broja monocita sedmoga dana. Koncentracija MCP-1 u plazmi koja je bila inicijalno povišena, snižena je trećeg dana kod pacijenta s NIHSS > 12 te nakon toga povišena i obnovljena sedmoga dana. Negativna korelacija između broja monocita i koncentracije MCP-1, zabilježena je trećeg dana nakon MU-a. Povišene vrijednosti MCP-1 sedmoga dana, povezane su s višim prijednostima mRS-a (što ukazuje na teži ishod) 90-og dana nakon IMU-a u pacijenata s NIHSS >12. Dobiveni rezultati kod bolesnika s lakšim (NIHSS 12) i težim (NIHSS > 12) MU-om govore u prilog neuroprotektivnom djelovanju proupalnog citokina IL-1, sukladno povišenju koncentracije tijekom svih triju dana mjerenja. Rezultati vrijednosti proupalnog citokina TNF- za bolesnike s lakšim MU-om ( NIHSS 12) prvog dana bolesti, u pozitivnoj su korelaciji sa stupnjem invaliditeta što ide u prilog njegovoj neurotoksičnoj ulozi. Trećeg i sedmog dana bolesti dolazi do pojave negativne korelacije, odnosno do povišenje koncentracije TNF- obrnuto proporcionalno stupnju invaliditeta te ga možemo smatrati neuroprotektivnim markerom prognoze MU-a. Kod bolesnika s težim MU-om, koncentracija TNF- u pozitivnoj je korelaciji sa stupnjem invaliditea, što govori u prilog neurotoksičnosti. U skupini bolesnika s lakšim MU-om (NIHSS 12), prisutna je pozitivna korelacija koncentracije proupalnog citokina IL-6 sa stupnjem inavalidnosti, što govori u prilog neurotoksičnosti. Kod bolesnika s težim MU-om (NIHSS > 12), dobivene vrijednosti koncentracije IL-6 prvog, trećeg i sedmog dana bolesti u negativnoj su korelaciji sa stupnjem invaliditeta 90-og dana bolesti te ga možemo smatrati neuroprotektivnim markerom prognoze krajnjeg stupnja invaliditeta.Vrijednosti protuupalnog citokina IL-10 kod bolesnika s lakšim MU-om (NIHSS 12) prvog, trećeg i sedmog dana bolesti, u pozitivnoj su korelaciji sa stupnjem inavliditeta 90-og dana bolesti, što ne potvrđuje njegovu protuupalnu ulugu pri IMU-u, dok bolesnici s težim MU-om pokazuju negativnu korelaciju tijekom svih triju dana mjerenja, potvrđujući protuupalnu ulogu. Koncentracija TGF beta kod bolesnika s lakšim MU-om (NIHSS 12) prvog i sedmog dana bolesti te kod bolesnika s težim MU-om udarom tijekom svih triju dana mjerenja, u pozitivnoj su korelaciji sa stupnjem invaliditeta, što ne ide u prilog njegovu protuupalnom djelovanju. Kod pacijenata s težim MU-om, vrijednosti VMA i HVA povišene su trećeg dana nakon IMU-a, za razliku od bolesnika s blažim IMU-om i TIA-om kod kojih su vrijednosti snižene. Bolesnici kod kojih su vrijednosti HVA i VMA snižene trećeg dana u usporedbi s prvim danom od nastupa IMU-a, imaju niži stupanj invaliditeta 90-og dana, za razliku od bolesnika kod kojih se vrijednosti povisuju.Zaključci: Neuro-imunološke pokazatelje ishemije u bolesnika s ishemijskom cerebrovaskularnom bolesti možemo koristiti kao rane prognostičke biološke indikatore težine bolesti kod bolesnika s IMU-om, kao i potencijalne pretkazatelje razvoja bolesti te indikatore rezultatata liječenja. Buduća istraživanja mogu pripomoći razvoju terapijske molekule u svrhu imunološke manipulacije. |
| Abstract (english) | The aim of this study was to determine changes in various leukocyte subpopulations and regulatory T-cells (T reg) in perihperal blood, and concentrations of MCP-1, proinflammatory cytokines (IL-1, TNF- i IL-6) and anti-inflammatory cytokines (IL-10, TGF-) in plasma, while also measuring the concentrations of VMA and HVA in the urine of patients with TIA and ischaemic stroke. The data was put into correlation with the final outcome of the disease using NIHSS on the 1st, 3rd and 7th day after onset in all patients and with modified Rankin scale and Barthels index 90 days after onset.Materials and methods:In this study to identify prognostic biomarkers for ischemic stroke (IS) outcome we monitored monocyte number and MCP-1 concentration in peripheral blood of 44 patients with IS during the week following IS. According to the severity of IS, patients were allocated to three groups: patients with transient ischemic attack (TIA), patients with National Institute of Health Stroke Scale (NIHSS) score 12 and patients with NIHSS >12(engl. Fluorescence-Activated Cell Sorting)Results:Group of patients with minor stroke showed gradual slight decline in the value of lymphocyte population ( CD3 / CD16 + / CD56 + ) and the seventh-day value is markedly below the reference value.A statistically significant difference was measured on seventh day compared to the third day in patients with severe IS and transient ischemic attack. On 7th day a statistically significant increase in number of monocytes was observed in patients with NIHSS > 12. The concentration of MCP - 1 in plasma initially elevated, decreased on day 3 in patients with NIHSS > 12 , and thereafter raised and restored on day 7. The negative correlation between the number of monocytes and the concentration of MCP - 1 was observed 3 days after stroke.Elevated levels of MCP - 1 on day 7, in patients with NIHSS > 12, are associated with higher mRS values (indicating worse outcome) at 90 days after the IS. The results obtained in patients with minor ( NIHSS 12 ) and severe ( NIHSS > 12 ) IS are in favor of neuroprotective action of proinflammatory cytokines IL - 1, in accordance with increase in the concentration in all three days of measurement. Values of proinflammatory cytokines TNF - in patients with minor stroke ( NIHSS 12 ) the first day of the disease are positively correlated with the degree of disability , which contributed to his neurotoxic role.Negative correlation was observed on third and seventh day of disease as a increase of TNF- which is in inverse proportion to the degree of disability and it can be considered a neuroprotective prognostic marker of IS. In patients with severe IS concentrations of TNF - are in a positive correlation with the degree of disability which speaks in favor of neurotoxicity. In the group of patients with mild stroke ( NIHSS 12 ) there is a positive correlation between concentrations of proinflammatory cytokines IL - 6 with a degree of disability suggesting neurotoxicity.In patients with severe stroke ( NIHSS > 12 ) obtained values of IL - 6 first, third and seventh day of the disease are negatively correlated with the degree of disability on 90th day of illness and it could be considered a neuroprotective prognostic marker of the final degree of disability.The values of anti-inflammatory cytokine IL - 10 in patients with mild stroke ( NIHSS 12 ) first, third and seventh day of the disease are positively correlated with degree of disability 90 days after IS, which does not confirm the anti-inflammatory role in ischemic stroke, while patients with severe IS show a negative correlation for all three days of measurements confirming anti-inflammatory role.Concentration of TGF- beta in patients with minor stroke ( NIHSS 12 ) first and seventh day of the disease and in patients with severe IS through all three days of measurement are positively correlated with the degree of disability , which does not support its anti-inflammatory action .In patients with severe stroke VMA and HVA values are increased on the third day after IS , in contrast to patients with mild IS and transient ischemic attack in which the values were reduced . Patients whose HVA and VMA values were reduced third day compared to first day of IS onset have a lower degree of disability on 90th day, in contrast to patients whose values were increasing .In patients with NIHSS >12 statistically significant increased number of monocytes was observed on day 7. MCP-1 plasma concentration initially increased, decreased at day 3 in patients with NIHSS > 12 and increased and restored on day 7. A negative correlation between the number of monocytes and MCP-1 concentration was observed on day 3 after IS. Higher day-7 MCP-1 level was associated with higher mRS value (indicating worse outcome) at 90 days post-IS in patients with NIHSS >12.Conclusions: Our findings suggest that number of monocytes and plasma MCP-1 level could be clinical prognostic biomarkers as early predictors of disease outcome in patients with IS. |
