Transforming growth factor-beta1 and myeloid-derived suppressor cells: A cancerous partnership

Transforming growth factor-beta1 (TGF-beta 1) plays a crucial role in tumor progression. It can inhibit early cancer stages but promotes tumor growth and development at the late stages of tumorigenesis. TGF-beta 1 has a potent immunosuppressive function within the tumor microenvironment that largely contributes to tumor cells' immune escape and reduction in cancer immunotherapy responses. Likewise, myeloid-derived suppressor cells (MDSCs) have been postulated as leading tumor promoters and a hallmark of cancer immune evasion mechanisms. This review attempts to analyze the prominent roles of both TGF-beta 1 and MDSCs and their interplay in cancer immunity. Furthermore, therapies against either TGF-beta 1 or MDSCs, and their potential synergistic combination with immunotherapies are discussed. Simultaneous TGF-beta 1 and MDSCs inhibition suggest a potential improvement in immunotherapy or subverted tumor immune resistance

Estramustine Phosphate Inhibits TGF-beta-Induced Mouse Macrophage Migration and Urokinase-Type Plasminogen Activator Production

Transforming growth factor-beta (TGF-beta) has been demonstrated as a key regulator of immune responses including monocyte/macrophage functions. TGF-beta regulates macrophage cell migration and polarization, as well as it is shown to modulate macrophage urokinase-type plasminogen activator (uPA) production, which also contributes to macrophage chemotaxis and migration toward damaged or inflamed tissues. Microtubule (MT) cytoskeleton dynamic plays a key role during the cell motility, and any interference on the MT network profoundly affects cell migration. In this study, by using estramustine phosphate (EP), which modifies MT stability, we analysed whether tubulin cytoskeleton contributes to TGF-beta-induced macrophage cell migration and uPA expression. We found out that, in the murine macrophage cell line RAW 264.7, EP at noncytotoxic concentrations inhibited cell migration and uPA expression induced by TGF-beta. Moreover, EP greatly reduced the capacity of TGF-beta to trigger the phosphorylation and activation of its downstream Smad3 effector. Furthermore, Smad3 activation seems to be critical for the increased cell motility. Thus, our data suggest that EP, by interfering with MT dynamics, inhibits TGF-beta-induced RAW 264.7 cell migration paralleled with reduction of uPA induction, in part by disabling Smad3 activation by TGF-beta

Regulation of the mesenchymal stem cell fate by interleukin-17: Implications in osteogenic differentiation

Bone regeneration is a tightly regulated process that ensures proper repair and functionality after injury. The delicate balance between bone formation and resorption is governed by cytokines and signaling molecules released during the inflammatory response. Interleukin (IL)-17A, produced in the early phase of inflammation, influences the fate of osteoprogenitors. Due to their inherent capacity to differentiate into osteoblasts, mesenchymal stem/stromal cells (MSCs) contribute to bone healing and regeneration. This review presents an overview of IL-17A signaling and the leading cellular and molecular mechanisms by which it regulates the osteogenic differentiation of MSCs. The main findings demonstrating IL-17A’s influence on osteoblastogenesis are described. To this end, divergent information exists about the capacity of IL-17A to regulate MSCs’ osteogenic fate, depending on the tissue context and target cell type, along with contradictory findings in the same cell types. Therefore, we summarize the data showing both the pro-osteogenic and anti-osteogenic roles of IL-17, which may help in the understanding of IL-17A function in bone repair and regeneration

The Metabolic Features of Tumor-Associated Macrophages: Opportunities for Immunotherapy?

Besides transformed cells, the tumors are composed of various cell types that contribute to undesirable tumor progression. Tumor-associated macrophages (TAMs) are the most abundant innate immune cells in the tumor microenvironment (TME). Within the TME, TAMs exhibit high plasticity and undergo specific functional metabolic alterations according to the availability of tumor tissue oxygen and nutrients, thus further contributing to tumorigenesis and cancer progression. Here, we review the main functional TAM metabolic patterns influenced by TME, including glycolysis, amino acid, and fatty acid metabolism. Moreover, this review discusses antitumor immunotherapies that affect TAM functionality by inducing cell repolarizing and metabolic profiles towards an antitumoral phenotype. Also, new macrophage-based cell therapeutic technologies recently developed using chimeric antigen receptor bioengineering are exposed, which may overcome all solid tumor physical barriers impeding the current adoptive cell therapies and contribute to developing novel cancer immunotherapies

Uticaj pro- i anti-inflamacijskih faktora na produkciju proteaza vanćelijskog matriksa uPA i MMP9 mišijih makrofaga

Macrophages are one of the major effector cells in inflammation. They are responsible for the recognition and processing of foreign materials, activation of the adaptive immune response, regulation of tissue repair/regeneration, and overall homeostasis maintenance. Multiple functions of macrophages are facilitated by their high plasticity in response to environmental or intrinsic signals. Two polarized macrophage modalities are critical in the regulation of different stages of tissue repair after injury. Classical M1 macrophages, exposed to pro-inflammatory cytokines, IFNs and microbial products, such as lipopolysaccharide (LPS), have major role in inducing inflammation and clearing of pathogens, whereas alternative M2 macrophages, in response to IL-4 and IL- 13, IL-10, TGF-β or glucocorticoids, resolve inflammation and are crucial in tissue remodeling and wound healing. In addition to cellular changes, reorganization/remodeling of critical extracellular matrix (ECM) proteins also occurs in injured tissues. During this ECM reorganization, several important ECM proteinases are triggered in a space-temporal fashion through tightly regulated mechanisms. Proinflammatory M1 macrophages are able to secrete matrix metalloproteinases, such as MMP9, which help in degradation of the ECM in affected tissue, thus facilitating the recruitment of inflammatory cells to the site of tissue injury. Moreover, an antifibrinolytic-coagulation cascade, that activates clotting and development of a provisional ECM, is also triggered, and later replaced by normal ECM during the resolution of inflammation. The urokinase-type plasminogen activator (uPA) is a serine protease that plays a major role in fibrinolytic processes, where it converts plasminogen to plasmin which further degrades coagulation components. Moreover, uPA is a key regulator of tissue inflammation and wound-healing processes and tissue regeneration, and is also secreted by macrophages. Considering macrophage’s importance in tissue repair, it is crucial to elucidate how MMP9 and uPA are involved in M1-M2 transition, as well as what are the underlying molecular mechanisms...Makrofazi su centralne ćelije imunskog sistema jer poseduju jedinstvene osobine: učestvuju u održavanju fiziološke homeostaze tkiva i organizma, proceni vrste moguće pretnje po organizam i pokretanju adekvatnog imunskog odgovora angažovanjem odgovarajućih komponenti imunskog sistema, ali i njegovom završetku kada više nije neophodan, aktivno učestvuju u obnavljanju oštećenih tkiva i tako omogućavaju povratak homeostaze. Ostvarivanje ovako raznovrsnih uloga omogućuje visoka prilagodljivost ovih ćelija spoljašnjim signalima. Različiti podtipovi makrofaga imaju odlučujuću ulogu u različitim fazama inflamacije. Klasični M1 makrofazi, izloženi dejstvu pro-inflamacijskih citokina, kao što je interferon γ, i/ili produktima mikroorganizama, kao što je lipopolisaharid (LPS), imaju glavnu ulogu u indukciji inflamacije i eliminaciji patogena, dok alternativni M2 makrofazi, u odgovoru na stimulaciju anti-inflamacisjkim/imunoreglatornim citokinima IL-4, IL-13, IL-10, TGF-β ili glukokortikoidima, obustavljaju inflamaciju i ključni su za obnavljanje tkiva i zarastanje rana. Tokom inflamacije odvijaju se značajne promene na nivou proteina vanćelijskog matriksa (ESM, extracellular matrix), koje su strogo regulisane proteazama ESM, čija se produkcija ostvaruje u definisanom prostorno-vremenskom kontekstu. Sekrecijom matriksnih proteaza (MMR) kao što je MMR9, M1 makrofazi učestvuju u degradaciji ESM i razgradnji tkiva, što olakšava ulazak drugih inflamacijskih ćelija u oštećeno tkivo. Istovremeno se pokreće kaskada koagulacije tokom koje se formira privremena struktura ESM od fibrina, koja se kasnije u fazi rezolucije razgrađuje, a ESM se obnavlja. Urokinaza (uPA) je serinska proteaza koja razgradnjom plazminogena u plazmin ostvaruje glavnu ulogu u procesu razgradnje fibrinske mreže. Osim toga, uPA je važan regulator tkivne inflamacije, procesa zarastanja rana i tkivne regeneracije. Otuda je važno rasvetliti molekulski mehanizam regulacije MMR9 i uPA tokom promene polarizacije makrofaga iz M1 u M2. Na osnovu ovih podataka, pretpostavljeno je da se u inflamacijskim makrofazima MMR9 i uPA regulišu na različit način..

Influence of pro- and anti -inflammatory factors on production of extracellular matrix proteases uPA and MMP9 in mouse macrophages

Макрофази су централне ћелије имунског система јер поседују јединствене особине: учествују у одржавању физиолошке хомеостазе ткива и организма, процени врсте могуће претње по организам и покретању адекватног имунског одговора ангажовањем одговарајућих компоненти имунског система, али и његовом завршетку када више није неопходан, активно учествују у обнављању оштећених ткива и тако омогућавају повратак хомеостазе. Остваривање овако разноврсних улога омогућује висока прилагодљивост ових ћелија спољашњим сигналима. Различити подтипови макрофага имају одлучујућу улогу у различитим фазама инфламације. Класични М1 макрофази, изложени дејству про-инфламацијских цитокина, као што је интерферон γ, и/или продуктима микроорганизама, као што је липополисахарид (LPS), имају главну улогу у индукцији инфламације и елиминацији патогена, док алтернативни М2 макрофази, у одговору на стимулацију анти-инфламацисјким/имунореглаторним цитокинима IL-4, IL-13, IL-10, TGF-β или глукокортикоидима, обустављају инфламацију и кључни су за обнављање ткива и зарастање рана. Током инфламације одвијају се значајне промене на нивоу протеина ванћелијског матрикса (ЕСМ, extracellular matrix), које су строго регулисане протеазама ЕСМ, чија се продукција остварује у дефинисаном просторно-временском контексту. Секрецијом матриксних протеаза (ММР) као што је ММР9, М1 макрофази учествују у деградацији ЕСМ и разградњи ткива, што олакшава улазак других инфламацијских ћелија у оштећено ткиво. Истовремено се покреће каскада коагулације током које се формира привремена структура ЕСМ од фибрина, која се касније у фази резолуције разграђује, а ЕСМ се обнавља. Урокиназа (uPA) је серинска протеаза која разградњом плазминогена у плазмин остварује главну улогу у процесу разградње фибринске мреже. Осим тога, uPA је важан регулатор ткивне инфламације, процеса зарастања рана и ткивне регенерације. Отуда је важно расветлити молекулски механизам регулације ММР9 и uPA током промене поларизације макрофага из М1 у М2. На основу ових података, претпостављено је да се у инфламацијским макрофазима ММР9 и uPA регулишу на различит начин...Macrophages are one of the major effector cells in inflammation. They are responsible for the recognition and processing of foreign materials, activation of the adaptive immune response, regulation of tissue repair/regeneration, and overall homeostasis maintenance. Multiple functions of macrophages are facilitated by their high plasticity in response to environmental or intrinsic signals. Two polarized macrophage modalities are critical in the regulation of different stages of tissue repair after injury. Classical M1 macrophages, exposed to pro-inflammatory cytokines, IFNs and microbial products, such as lipopolysaccharide (LPS), have major role in inducing inflammation and clearing of pathogens, whereas alternative M2 macrophages, in response to IL-4 and IL- 13, IL-10, TGF-β or glucocorticoids, resolve inflammation and are crucial in tissue remodeling and wound healing. In addition to cellular changes, reorganization/remodeling of critical extracellular matrix (ECM) proteins also occurs in injured tissues. During this ECM reorganization, several important ECM proteinases are triggered in a space-temporal fashion through tightly regulated mechanisms. Proinflammatory M1 macrophages are able to secrete matrix metalloproteinases, such as MMP9, which help in degradation of the ECM in affected tissue, thus facilitating the recruitment of inflammatory cells to the site of tissue injury. Moreover, an antifibrinolytic-coagulation cascade, that activates clotting and development of a provisional ECM, is also triggered, and later replaced by normal ECM during the resolution of inflammation. The urokinase-type plasminogen activator (uPA) is a serine protease that plays a major role in fibrinolytic processes, where it converts plasminogen to plasmin which further degrades coagulation components. Moreover, uPA is a key regulator of tissue inflammation and wound-healing processes and tissue regeneration, and is also secreted by macrophages. Considering macrophage’s importance in tissue repair, it is crucial to elucidate how MMP9 and uPA are involved in M1-M2 transition, as well as what are the underlying molecular mechanisms..

Podklasa teških lanaca i nivo ekspresije sijalinske kiseline na njima određuju hromatografsku raspodelu humanih monoklonskih imunoglobulina G

Anion exchange chromatography is a widely accepted method for purification of immunoglobulins. In this work, we used human monoclonal immunoglobulin G (IgG) with structure and solubility of normal human IgG as a model for studying chromatographic behavior of particular molecular forms of IgG. Human sera with monoclonal IgG were fractionated on a strong anion exchanger, Q Sepharose Fast Flow. With 20 mM Tris pH 7.5 as a start buffer, 42% of human monoclonal IgG passed through column, and 58% of them remained adsorbed. Bound monoclonal IgG were eluted from the exchanger by linear increasing of concentration of NaCl from 0 to 0.5 M. The chromatographic distribution of human monoclonal IgG correlated with their electrophoretic mobilities in agarose gels, and it was dependent on ã heavy chain isotype. Light chain type, as well as serum concentration of monoclonal IgG did not influence their chromatographic behavior. The level of heavy chain sialic acid expression, but not of galactose and N-acetylglucosamine, significantly determined chromatographic distribution of serum monoclonal IgG. In addition to the information on the chromatographic behavior of human monoclonal IgG, we believe that the presented data could provide useful information about the possible use of Q Sepharose Fast Flow matrix for the isolation of specific molecular forms of human IgG.Zbog velike molekulske heterogenosti humanih imunoglobulina G (IgG) (4 izotipa, 20 alotipova i preko 600 mogućih glikoformi) ne postoji jedinstven protokol za izolovanje svih molekulskih formi IgG. Jonoizmenjivačka hromatografija je široko prihvaćen metod za izolovanje i prečišćavanje humanih IgG, pre svega zbog činjenice da se separacija odvija u blagim uslovima, što pored efikasnog prečišćavanja omogućava da struktura i funkcija IgG kao antitela ostane očuvana. Za izolovanje humanih IgG mogu se koristiti i slabi i jaki anjonski izmenjivači. Međutim, koji će od brojnih matriksa koji danas postoje na tržištu biti odgovarajući za izolovanje specifičnih molekulskih formi humanih IgG, može se znati samo nakon testiranja. U ovom radu, humani monoklonski IgG iz seruma bolesnika sa monoklonskim gamapatijama su izolovani preparativnom hromatografijom na Q Sepharose Fast Flow anjonskom izmenjivačkom matriksu. Kada je kao startni pufer korišćen 20 mM Tris-HCl, pH 7,5, 42% analiziranih monoklonskih IgG nije se vezivalo za matriks i bilo je moguće izolovati ih u čistoj formi. Preostalih 58% monoklonskih IgG, različitim intenzitetom vezivali su se za matriks, i sa njega su eluirani 0,09-0,43 M Tris, pH 7,5/NaCl. Hromatografska raspodela monoklonskih IgG je bila u korelaciji sa njihovom elektroforetskom pokretljivosti u gelu agaroze i podklasom γ teškog lanca, a nije bila određena tipom lakog lanca i serumskom koncen- tracijom monoklonskog IgG. Nivo ekspresije sijalinske kiseline na teškim lancima je značajno uticao na hromatografsku raspodelu humanih monoklonskih IgG, dok je nivo ekspresije druga dva terminalna šećera, galaktoze i N-acetilglukozamina, bio bez uticaja. Smatramo da prikazani rezultati mogu biti od koristiti u kreiranju protokola za izolovanje humanih monoklonskih IgG jer pored toga što daju informacije o njihovom hromatografskom ponašanju daju i informaciju o mogućnosti korišćenja Q Sepharose Fast Flow anjonskog matriksa za izolovanje pojedinih molekulskih formi humanih IgG

Interaction between fibronectin fragments and immunoglobulin G in gingival crevicular fluid of patients with periodontal disease

Introduction Fibronectin (FN) can interact with immunoglobulin G (IgG) molecules affecting the process of physiological elimination and causing abnormal deposition of immune complexes. The aim of the study was to analyze interaction between FN fragments and IgG molecules with different glycosylation profiles in gingival crevicular fluid (GCF) of patients with periodontal disease and healthy controls. Material and Methods The study included 30 patients with moderate and advanced periodontitis and 22 healthy subjects. IgG and FN content in GCF were measured as well as the presence of FN and galactose expression on IgG molecules. Results IgG content in GCF was five times higher in patients with moderate (p<0.01) and eight time higher in patients with advanced periodontitis (p<0.001) compared to healthy subjects. Also, hypogalactosylated forms of IgG were found in higher concentration in GCF of patients with advanced periodontitis compared to moderate periodontitis and healthy subjects (p<0.05). FN fragments of molecular mass 48 - 53 kDa were the most commonly found fragments in all three groups. Furthermore, in patients with advanced periodontitis, fibronectin fragments were attached to IgG molecules. Conclusion IgG and FN fragments form complexes in GCF in patients with periodontal disease and healthy subjects

Gene expression profile of circulating CD34(+) cells and granulocytes in chronic myeloid leukemia

Purpose: We compared the gene expression profile of peripheral blood CD34(+) cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR-ABL oncogene. Methods: The microarray analyses have been performed in circulating CD34(+) cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR-ABL positive CML patients were in chronic phase, with a mean value of 2012 +/- SD of CD34(+) cells/mu l in peripheral blood. Results: The gene expression profile was more prominent in CML CD34(+) cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34(+) cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR-ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34(+) cells. Among genes linked to the inhibition of cellular proliferation by BCR-ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. Conclusion: The presence of BCR-ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34(+) cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects

Kompleksi fibronektinskih fragmenata i imunoglobulina G u gingivalnoj tečnosti osoba obolelih od parodontopatije

Introduction Fibronectin (FN) can interact with immunoglobulin G (IgG) molecules affecting the process of physiological elimination and causing abnormal deposition of immune complexes. The aim of the study was to analyze interaction between FN fragments and IgG molecules with different glycosylation profiles in gingival crevicular fluid (GCF) of patients with periodontal disease and healthy controls. Material and Methods The study included 30 patients with moderate and advanced periodontitis and 22 healthy subjects. IgG and FN content in GCF were measured as well as the presence of FN and galactose expression on IgG molecules. Results IgG content in GCF was five times higher in patients with moderate (p lt 0.01) and eight time higher in patients with advanced periodontitis (p lt 0.001) compared to healthy subjects. Also, hypogalactosylated forms of IgG were found in higher concentration in GCF of patients with advanced periodontitis compared to moderate periodontitis and healthy subjects (p lt 0.05). FN fragments of molecular mass 48 - 53 kDa were the most commonly found fragments in all three groups. Furthermore, in patients with advanced periodontitis, fibronectin fragments were attached to IgG molecules. Conclusion IgG and FN fragments form complexes in GCF in patients with periodontal disease and healthy subjects.Uvod Fibronektin može da interreaguje s molekulima imunoglobulina G (IgG) i utiče na normalan klirens ili poremećeno deponovanje imunskih kompleksa. Cilj ovog rada je bio da se ispita veza između fibronektina i IgG različitih glikoformi u gingivalnoj tečnosti osoba obolelih od parodontopatije i parodontalno zdravih ispitanika. Materijal i metode rada U studiju je uključeno 30 pacijenata s umerenom i uznapredovalom parodontopatijom i 22 parodontalno zdrave osobe. U gingivalnoj tečnosti određivan je sadržaj IgG i fibronektina dot blot i imunoblot tehnikama. IgG iz gingivalnih tečnosti su afinitetno izolovani i analizirani na prisustvo fibronektina i ekspresiju galaktoze. Rezultati Sadržaj IgG u gingivalnoj tečnosti osoba s umerenom parodontopatijom bio je oko pet puta veći u odnosu na sadržaj IgG kod zdravih osoba (p lt 0,01), dok je kod uznapredovalih oblika bio oko osam puta veći (p lt 0,001). Takođe, hipogalaktozilovane forme IgG su većoj meri postojale kod osoba sa uznapredovalom parodontopatijom u odnosu na zdrave i osobe s umerenom parodontopatijom (p lt 0,05). U sve tri analizirane grupe dominirali su fibronektinski fragmenti od 48 do 53 kDa. Uočeno je da su IgG izolovani iz gingivalne tečnosti vezani za fragmente fibronektina, pri čemu su IgG osoba sa uznapredovalom parodontopatijom, imali najveću količinu ovih vezanih fragmenata. Zaključak Dobijeni rezultati pokazuju da IgG gingivalne tečnosti zdravih i osoba s parodontopatijom formiraju komplekse sa fibronektinom