Ocena zależności pomiędzy leptyną, rezystyną i adiponektyną a naturalnymi limfocytami regulatorowymi T w postaci nawracająco-zwalniającej stwardnienia rozsianego

Background and purpose Data suggest that adipocytokines and natural regulatory T (nTreg) cells play a pivotal role in the immunopathogenesis of multiple sclerosis and the associated inflammation. The purpose of this study was to evaluate selected adipocytokines and nTreg cells and to assess their relationship with relapsing-remitting multiple sclerosis (RRMS). Material and methods The study was conducted among 25 patients with RRMS and 25 healthy individuals. Blood samples were collected within two weeks after the beginning of acute relapse of RRMS. The body mass index (BMI) of each patient was calculated. Serum adipocytokine concentrations were determined by ELISA and nTreg cells were evaluated using multicolour flow cytometry. Results Patients and controls had similar BMI, regardless of gender. Significantly higher leptin and resistin levels and significantly lower adiponectin levels were found in patients with RRMS in comparison to the control group (p < 0.0001). The percentage of nTreg cells (p < 0.01) and the mean fluorescence channel (MFC) of FoxP3 were significantly reduced in patients with RRMS (p < 0.001). There was an inverse correlation between leptin concentration and MFC of the transcription factor Foxp3 nTreg in patients with RRMS (r = −0.7, p < 0.05). Conclusions Proinflammatory adipocytokine profile and decreased percentage of nTreg cells suggest their implication in the inflammatory response in RRMS regardless of corticosteroid therapy. The correlation between leptin and the MFC of the transcription factor Foxp3 in nTreg cells in patients with RRMS suggests its inhibitory effect on FoxP3 expression.Wstęp i cel pracy Dane z piśmiennictwa sugerują, że w immunopatogenezie i w przebiegu procesu zapalnego w stwardnieniu rozsianym szczególną rolę mogą odgrywać adipocytokiny oraz naturalne limfocyty regulatorowe T. Dlatego celem pracy była ocena wybranych adipocytokin i limfocytów nTreg oraz analiza zależności pomiędzy nimi u chorych na stwardnienie rozsiane w postaci nawracająco-zwalniającej (RRMS). Materiał i metody Badania przeprowadzono u 25 chorych na RRMS. Krew pobrano w ciągu 2 tygodni od początku ostrego rzutu choroby. Grupę kontrolną stanowiło 25 zdrowych osób. U każdego pacjenta wyliczono wskaźnik masy ciała (BMI). Stężenie adipocytokin oznaczono w surowicach techniką ELISA. Ocenę immunofenotypu limfocytów nTreg wykonano przy użyciu wielokolorowej cytometrii przepływowej. Wyniki Nie stwierdzono statystycznie istotnej różnicy we wskaźniku BMI pomiędzy pacjentami z RRMS a grupą kontrolną niezależnie od płci. Wykazano znamiennie większe stężenie leptyny i rezystyny oraz znamiennie mniejsze stężenie adiponektyny u chorych na RRMS w porównaniu z grupą kontrolną (p < 0,0001). Odsetek komórek nTreg (p < 0,01) i wskaźnik średniej intensywności fluorescencji (MFC) dla FoxP3 (p < 0,001) były znamiennie mniejsze u chorych na RRMS. Stwierdzono ujemną korelację pomiędzy stężeniem leptyny a MFC dla czynnika transkrypcyjnego FoxP3 w limfocytach nTreg u chorych na RRMS (r = −0,7; p < 0,05). Wnioski Prozapalny profil adipocytokin i zmniejszenie odsetka limfocytów nTreg sugeruje ich udział w przebiegu reakcji zapalnej u chorych na RRMS niezależnie od terapii kortykosteroidami. Korelacja pomiędzy stężeniem leptyny i wskaźnikiem MFC dla czynnika transkrypcyjnego FoxP3 w limfocytach nTreg u chorych na RRMS wskazuje na hamujący wpływ leptyny na jego ekspresję

The use of multi-color flow cytometry for identification of functional markers of nTregs in patients with severe asthma

Wstęp: Ciężka postać astmy oskrzelowej stanowi obecnie szczególny problem kliniczny. W patogenezie astmy istotną rolę przypisuje się dysfunkcji subpopulacji limfocytów nTreg. Dlatego celem pracy była identyfikacja markerów czynnościowych limfocytów nTreg u chorych na astmę ciężką i umiarkowaną&#8211;łagodną. Materiał i metody: Do badania zakwalifikowano 60 pacjentów z rozpoznaną astmą oskrzelową (w tym 30 z ciężką i 30 z umiarkowaną&#8211;łagodną postacią choroby). Grupę kontrolną stanowiło 30 zdrowych ochotników. Diagnoza astmy oskrzelowej została postawiona zgodnie z powszechnie akceptowanymi zaleceniami &#8212; GINA 2008. Materiałem badanym była krew żylna pobrana na K2EDTA. Ocenę immmunofenotypu CD4/CD25/CD127/FoxP3/GITR/CD152/CCR5/CCR7, limfocytów nTreg wykonano techniką wielokolorowej cytometrii przepływowej. Wyniki: Wykazano, znamienne obniżenie odsetka limfocytów nTreg (76%) i ekspresji CD152 (46,2%) u chorych na astmę ciężką w porównaniu z astmą umiarkowaną&#8211;łagodną (85,5% i 86,7%; p < 0,05). Zaobserwowano, że ekspresja czynnika transkrypcyjnego FoxP3 w limfocytach nTreg dodatnio korelowała z FEV1 u chorych na astmę ciężką (r = 0,53; p < 0,05). Stwierdzono także, że obliczony współczynnik nTregCCR5+/TeffCCR5+ był znamiennie obniżony u chorych na astmę ciężką (0,91) w porównaniu do astmy umiarkowanej-łagodnej (1,58) i grupy kontrolnej (1,55; p < 0,001). Wnioski: Istnieją różnice fenotypowe w subpopulacji limfocytów nTreg między chorymi na astmę ciężką i umiarkowaną&#8211; &#8211;łagodną. Fakt ten może potwierdzić dysfunkcję limfocytów nTreg i wskazać na potencjalne markery (FoxP3, CD152, CCR5), które mogą być użyte do monitorowania efektywności leczenia astmy oskrzelowej, a zwłaszcza ciężkich postaci choroby.Introduction: At present, severe asthma is a particular clinical problem. An important role is attributed to dysfunction of nTreg subpopulations of lymphocytes in the pathogenesis of asthma. Therefore, the purpose of this study was to identify markers of nTreg cell function in patients with severe and mild to moderate asthma. Material and methods: The study included sixty patients with asthma (30 with severe and 30 with mild to moderate asthma). The control group comprised 30 healthy volunteers. The diagnosis of asthma was confirmed accordance with generally accepted recommendations (GINA 2008). nTreg immunophenotype CD4/CD25/CD127/FoxP3/GITR/CD152/CCR5/ /CCR7 was evaluated by multicolor flow cytometry. Results: We showed a significant reduction in the percentage of nTreg (76%) cells and the expression of CD152 (46.2%) in patients with severe asthma compared with mild-moderate asthma (85.5% and 86.7%; p < 0.05). It was observed that the transcription factor FoxP3 expression in nTreg cells positively correlated with FEV1 in patients with severe asthma (r = 0.53; p < 0.05). It was also found that the ratio nTregCCR5&lowast;/TeffCCR5&lowast; was significantly reduced in patients with severe asthma (0.91) compared with mild-moderate (1.58) asthma and control groups (1.55; p < 0.001). Conclusions: There are phenotypic differences in nTreg lymphocytes between patients with severe and mild-moderate asthma. This fact may confirm nTreg cell dysfunction and indicate that the potential markers (FoxP3, CD152, CCR5), can be used to monitor the effectiveness of treatment of bronchial asthma, especially severe disease

Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant

To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir

Tissue Factor in Dermatitis Herpetiformis and Bullous Pemphigoid: Link between Immune and Coagulation System in Subepidermal Autoimmune Bullous Diseases

Dermatitis herpetiformis (DH) and bullous pemphigoid (BP) are skin diseases associated with eosinophilic and neutrophilic infiltrations. Although chemokines are critical for the selective accumulation and activation of various leukocyte subsets in the inflammatory process, there are few findings concerning inflammatory cells and production of coagulation factors in blistering diseases. Skin biopsies were taken from 14 patients with DH, 27 with BP, and 20 control subjects. The localization and expression of tissue factor (TF) in skin lesions and perilesional skin were studied by immunohistochemistry and confirmed by Western Blot. Moreover the plasma concentrations of TF were measured by immunoassays. D dimers, fibrinogen, and selected coagulation parameters were measured by routine methods. Expression of TF in the epidermis and in inflammatory influxed cells in dermis was detected in skin biopsies from BP patients. Examined TF expression was detected in perilesional skin of all BP patients too. The expression of TF was not observed in biopsies from healthy people and DH patients. The findings of the study show an increased expression of tissue factor in the lesional and perilesional skin of patients with bullous pemphigoid. The difference in chemokine pattern expression and variations in the cellular infiltration in BP and DH cause variable expression of TF

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field