Effects of immunomodulatory drugs on galectin-3

Galektin-3, lektin koji specifično prepoznaje -galaktozidne strukture, djeluje kao jak proinflamatorni signal koji modulira staničnu proliferaciju i adheziju, kemotaksiju, fagocitozu te sintezu upalnih medijatora. Mnoge tvari s imunomodulatornim djelovanjem djeluju na signalne puteve u kojima sudjeluju i transkripcijski faktori čija su vezna mjesta prisutna u promotorskoj regiji gena za galektin-3 (LGALS3). Zbog česte uporabe ovih tvari u terapijske svrhe te važnosti galektina-3 u fiziologiji stanica monocitno-makrofagne loze, u ovom je radu ispitan utjecaj nesteroidnih (aspirina i indometacina) i steroidnih (hidrokortizona i deksametazona) tvari s imunomodulatornim djelovanjem primijenjenih u različitim terapijskim rasponima na ekspresiju LGALS3 i galektina-3 u nediferenciranim i diferenciranim stanicama monocitne stanične linije THP-1 tijekom 72-satnog kultiviranja. Niti jedna od ispitanih tvari u primijenjenim koncentracijama tijekom 72 sata kultiviranja nije citotoksično djelovala na stanice. Količina mRNA za galektin-3 određena je primjenom metode RT-PCR praćene analizom na uređaju AbiPrism 310 Genetic Analyser, a količina galektina-3 u staničnim homogenatima Western-imunoblot metodom. Rezultati su pokazali da ispitivane tvari mijenjaju ekspresiju LGALS3 i galektina-3 te da njihovi učinci ovise o diferenciranosti stanica, koncentraciji primijenjene tvari kao i vremenu izlaganja. U nediferenciranim stanicama sve ispitane tvari svih primijenjenih koncentracija inhibiraju ekspresiju i LGALS3 i galektina-3, a inhibitorni je učinak u korelaciji s vremenom izlaganja. Tijekom 48-satne preobrazbe nediferenciranih stanica u diferencirane, dolazi do snažne indukcije ekspresije i LGALS3 (3 puta) i galektina-3 (3,5 puta). U diferenciranim stanicama sve ispitivane tvari u početku inhibiraju ekspresiju LGALS3 nakon čega dolazi do uspostave konstitutivne razine mRNA, dok na proteinskoj razini dugotrajnije izlaganje ispitivanim tvarima potiče ekspresiju galektina-3. U oba slučaja, intenzitet promjena kao i njihov vremenski slijed ovise o vrsti i koncentraciji primijenjene tvari. Na temelju dobivenih rezultata moguće je zaključiti da primijenjene tvari s imunomodulatornim djelovanjem utječu na različite signalne puteve kao i mehanizme regulacije ekspresije galektina-3 na genskoj i proteinskoj razini, a da promjene ovise o vrsti i koncentraciji tvari te vremenu izloženosti, kao i o diferencijacijskom stupnju stanica. Dobiveni rezultati predstavljaju važan korak u razumijevanju djelovanja tvari s imunomodulatornim djelovanjem na galektin-3 u stanicama monocitno-makrofagne loze, a time i na njihove brojne fiziološke funkcije.Galectin-3, a β-galactoside binding lectin, acts as a strong pro-inflammatory signal that modulates cell proliferation and adhesion, chemotaxis, phagocytosis and synthesis of inflammatory mediators. Many immunomodulatory drugs affect signaling pathways that comprise transcriptional factors which binding sites are present in the promoter region of galectin-3 gene (LGALS3). Because of the frequent therapeutic application of immunomodulatory drugs and the importance of galectin-3 in the physiology of monocytes and macrophages, we investigated effects of non-steroidal (aspirin and indomethacin) and steroidal (hydrocortisone and dexamethasone) immunomodulatory drugs, applied in different therapeutic ranges, on the expression of LGALS3 and galectin-3 in nondifferentiated and differentiated cells of monocytic THP-1 cell-line during 72 hours of cultivation. None of the studied drugs in applied concentrations during 72 hours of cultivation had cytotoxic effect on the cells. The targeted mRNA level was evaluated using relative RT-PCR technique and analysis on the AbiPrism 310 Genetic Analyser. Galectin-3 expression in cell homogenates was determined by western-immunoblot analysis. The results showed that the chosen immunomodulatory drugs affect LGALS3 and galectin-3 expression and that their effects depend on cell differentiation level, concentration of the applied drug, as well as time of exposure. In undifferentiated cells all of the studied drugs (in all applied concentrations) inhibit expression of LGALS3 and galectin-3, and inhibitory effect correlates with time of exposure. Differentiation of monocytic THP-1 cells into macrophages during 48-hours strongly induces expression of LGALS3 (3 times) and galectin-3 (3,5 times). In differentiated cells, all studied drugs inhibited LGALS3 expression at the beginning, but during further incubation constitutive level of galectin-3 mRNA was reestablished. On the protein level, prolonged exposure to all applied drugs induce galectin-3 expression. Intensity and timecourse of both, mRNA and protein level changes depend on drug type and on applied concentration. These findings indicate that the applied immunomodulatory drugs affect different signaling pathways and mechanisms of regulation of galectin-3, on both mRNA and protein level. The changes depend on drug type and concentration, time of exposure as well as cell differentiation level. The results obtained in this study represent important step in the understanding of the effects of immunomodulatory drugs on galectin-3 in monocytes/macrophages, hence on their numerous physiological roles

Utjecaj aspirina i indometacina na galektin-3

Galectin-3, a β-galactoside binding lectin, acts as a strong pro inflammatory signal. Many immunomodulatory drugs affect signaling pathways that comprise transcription factors involved in regulation of galectin-3 gene (LGALS3) expression. The aim of this study was to investigate the effects of non-steroidal anti-inflammatory drugs aspirin and indomethacin on the expression of galectin-3, both on mRNA and protein levels. The human monocytic cell line THP-1 was exposed to various therapeutic concentrations of aspirin and indomethacin for 72 hours. Relative RT-PCR method and the GeneScan analysis software were used for assessing the galectin-3 mRNA level and chemiluminescent-western blot analysis was used for measuring the galectin-3 level. The results showed that galectin-3 is a new target molecule of NSAIDs, which cause reduction of both gene and protein expression of galectin-3, but the intensity and time-course of the changes strongly depend on the kind and concentration of the drugs.Galektin-3, lektin koji veže β-galaktozidne jedinice, djeluje kao jak proinflamatorni signal. Mnoge imunomodulatorne tvari utječu na signalne putove i transkripcijske faktore uključene i u regulaciju ekspresije gena za galektin-3 (LGALS3). Cilj je ovoga rada bio ispitati učinke nesteroidnih tvari s imunomodulatornim djelovanjem – aspirina i indometacina – na ekspresiju galektina-3, kako na razini mRNA tako i na razini proteina. Stanice humane stanične linije THP-1 izložene su različitim terapijskim koncentracijama aspirina i indometacina tijekom 72 sata. Količina mRNA za galektin-3 odre|ena je primjenom metode RT-PCR i analize programom GeneScan, a razina galektina-3 kemiluminescentnom western-blot analizom. Rezultati su pokazali da galektin-3 predstavlja novu ciljnu molekulu djelovanja nesteroidnih imunomodulatornih tvari, koje uzrokuju smanjenje ekspresije gena za galektin-3 i samoga proteina, premda jakost promjena i vrijeme njihove pojave jako ovise o vrsti i koncentraciji primijenjene tvari

Kurkumin – snažan inhibitor ekspresije galektina-3

The expression of galectin-3, a b-galactoside binding lectin, was found to be affected by different kinds of stressors, and is strongly modified in numerous physiological and pathophysiological conditions. Although no precise regulatory mechanisms of galectin-3 expression are unraveled, transcription factors AP-1 (activator protein 1) and NF-kB (nuclear factor kappa B) play an important role in these processes. Activities of both transcription factors are affected by curcumin, a biologically active compound extracted from rhizomes of Curcuma species. We have analyzed the impact of curcumin on the expression of galectin-3 in glioblastoma cells under basal conditions and under stress invoked by the cell exposure to alkylating agent N-methyl-N\u27-nitro-N-nitrosoguanidine (MNNG) and ultraviolet C (UV-C) light. Galectin-3 level was measured by western-blot technique using M3/38 monoclonal antibody. Curcumin has decreased the basal level of galectin-3, while the pretreatment of cells with curcumin has considerably reduced the inducible effect of UV-C radiation and abolished the inducible effect of alkylating agent. Thus, curcumin has been identified as a potent inhibitor of galectin-3 expression.Galektin-3, lektin koji specifično prepoznaje b-galaktozidne strukture glikoproteina, sudjeluje u različitim biološkim procesima. Patofiziološka stanja i različiti uzročnici stresa bitno utječu na ekspresiju galektina-3. Premda nisu poznati svi čimbenici u regulacijskom mehanizmu ekspresije galektina-3, pokazano je da u tom procesu sudjeluju transkripcijski faktori AP-1 i NF-kB. Kurkumin, biološki aktivan spoj izoliran iz biljnih podanaka vrste Curcuma, interferira s djelovanjem obaju transkripcijskih faktora. Istražili smo učinak kurkumina na ekspresiju galektina-3 u stanicama glioblastoma u bazalnim uvjetima te u stresu koji je bio izazvan izlaganjem stanica alkilirajućem agensu N-metil-N\u27-nitro-N-nitrozogvanidinu (MNNG) i ultraljubičastom C (UV-C) svjetlu. Razina galektina-3 određena je tehnikom western-blot uz uporabu monoklonskih protutijela M3/38. Utvrđeno je da kurkumin smanjuje bazalnu razinu galektina-3 te da predinkubacija stanica kurkuminom jako smanjuje inducibilni učinak UV-C zračenja, a potpuno uklanja induktivni učinak alkilirajućeg agensa MNNG. Rezultati nedvojbeno pokazuju da je kurkumin snažan inhibitor ekspresije galektina-3

Oleuropein in olive leaf, branch, and stem extracts: stability and biological activity in human cervical carcinoma and melanoma cells

Olive leaves as a main byproduct of olive oil and fruit industry are a valuable source of phytochemicals such as polyphenols, with multiple biomedical effects. Apart from leaves, olive branches and stems make up a significant amount of olive waste. It is well known that the drying process and long-term storage affect the stability and concentration of polyphenols present in raw materials. For that matter, two different means of storing olive waste, at room temperature and +4 °C, were compared by determining the content of the polyphenol oleuropein (OLE) in olive leaf, branch, and stem extracts (LE, BE, and SE) by HPLC-DAD method. Total phenols (TPC), o-diphenols (o-DPC), and total flavonoids (TFC) content in extracts were assessed by UV-Vis measurements. LE prepared from leaves stored at +4 °C had the highest OLE content, 30.7 mg g–1 of dry extract (DE). SE from stems stored at +4 °C was the richest in TPC and TFC (193 mg GAE/g DE and 82.9 mg CE/g DE, respectively), due to the higher purity of the extract. The biological activity of extracts was determined on cervical cancer (HeLa), melanoma (A375), metastatic melanoma (A375M) tumor cell lines, and on spontaneously immortalized cell line of keratinocytes (HaCaT), using the MTT assay. The data show that all extracts had a similar dose-dependent effect on cell viability in HeLa cells, while the effect of LE on melanoma A375 and A375M, and HaCaT cells was cell-line dependent

Increased HSP70 and TLR2 mRNA expression and association of HSP70 polymorphism rs6457452 with the risk of chronic obstructive pulmonary disease in the Croatian population

Heat shock protein 70 (Hsp70) engages Toll-like receptors (TLR) 2 and 4 when found in the extracellular compartment and contributes to inflammation in chronic obstructive pulmonary disease (COPD). Since there is growing evidence for the genetic risk factors for COPD, the gene expression of HSP70, TLR2 and TLR4 was determined, as well as the association between HSP70, TLR2 and TLR4 single nucleotide polymorphisms, (SNPs) and COPD. The gene expression was assessed in peripheral blood cells of 137 COPD patients and 95 controls by a quantitative polymerase chain reaction (qPCR), while a total of nine SNPs were genotyped by TaqMan allelic discrimination real-time PCR. HSP70 and TLR2 gene expression was increased in COPD patients compared to the controls, regardless of the disease severity and smoking status of participants. The rs6457452 SNP of HSP70 was associated with COPD, indicating the protective role of the T allele (OR = 0.46, 95% CI = 0.24-0.89, p = 0.022). Furthermore, COPD C/T heterozygotes showed a decreased HSP70 mRNA level compared to COPD C/C homozygotes. In conclusion, HSP70 and TLR2 may have a role in the pathogenesis of COPD, and the HSP70 rs6457452 variant might influence the genetic susceptibility to COPD in the Croatian population

Visoke vrijednosti omjera vjerojatnosti ne moraju biti dostatne u procesu identifikacije žrtava rata s pomo}u analize DNA

With the advance in typing tools and extraction procedures in recent years, DNA analysis has developed into an amazingly powerful method for forensic analysis. For a number of years, autosomal STR (Short Tandem Repeat) typing has been used as a tool in the process of identification of war victims in Croatia. Although DNA typing is very effective in detecting possible identities of exhumed skeletal remains, this approach bears some risk of false identification. The paper presents the case of a match between skeletal remains and the son and wife of a missing person in 13 STR loci. Even though these skeletal remains also matched in 13 loci the mother of the same missing person, additional genetic testing (Y-chromosome and mitochondrial DNA) unequivocally excluded the proposed identity. Although likelihood ratio is the best measure of the significance of a genetic match between exhumed skeletal remains and relatives of the missing person, the meaning of likelihood ratio is not as clear in database matching as in simple paternity cases and great care is needed to avoid wrong interpretation. To reduce the risk of possible false identifications, in addition to DNA evidence, other types of evidence (such as information about the time, place and other conditions of disappearance), as well as on anthropological and other »classical« forensic data are being used as a »control mechanism« in the DNA-lead process.Zahvaljujući stalnom napretku u metodama izolacije i karakterizacije, analiza DNA razvila se u vrlo moćnu metodu forenzičke analize. Već niz godina analizu autosomnih STR regija primjenjuje se kao temeljnu metodu u procesu identifikacije žrtava rata u Hrvatskoj. Iako je analiza DNA vrlo učinkovita u pronalaženju mogućega identiteta ekshumiranih posmrtnih ostataka, ovakav pristup nosi i određeni rizik pogrešne identifikacije. Ovdje je opisan slučaj poklapanja posmrtnih ostataka sa sinom i suprugom nestale osobe u 13 STR lokusa. Iako su se isti posmrtni ostaci također poklapali i s majkom iste nestale osobe u 13 lokusa, dodatnim genetičkim ispitivanjima (Y kromosom i mitohondrijska DNA) nedvojbeno je isključena mogućnost da se radi o toj osobi. Omjer vjerojatnosti najbolji je pokazatelj značajnosti genetičkoga podudaranja skeletnih ostataka i rodbine nestalih osoba, no kad je to podudaranje pronađeno nasumičnim pretraživanjem baze podataka, značaj omjera vjerojatnosti nije tako jasan kao u jednostavnome utvrđivanju očinstva i potreban je velik oprez kako ne bi došlo do njegove pogrešne interpretacije. Radi smanjivanja rizika pogrešne identifikacije, osim na rezultate analize DNA, pri utvrđivanju identiteta velik značaj potrebno je dati i drugim tipovima dokaza (poput vremena, lokacije i uvjeta nestanka) te antropološkim i ostalim »klasičnim« forenzičkim dokazima, kao kontrolnome mehanizmu u procesu identifikacije predvođenom analizom DNA

Synthesis, antibacterial and cytotoxic activity evaluation of hydroxyurea derivatives

Synthesis and biological evaluation for a series (N = 16) of cyclic and acyclic hydroxyurea derivatives including benzotriazole-, isocyanuirc acid- and biuret-containing compounds, are disclosed. 1-N-(benzyloxycarbamoyl)benzotriazole was used as benzyloxyisocyanate donor, a useful intermediate in the preparation of substituted hydroxyurea. Antibacterial activities of synthesized hydroxyurea derivatives were tested on three E. coli strains, i.e., strain susceptible to antibiotics, strain resistant to macrolide antibiotics and strain resistant to aminoglycoside antibiotics. Six compounds (three acyclic and three cyclic hydroxyureas) showed the growth inhibition of tested E. coli strains, with different specificity toward each strain. Results of the cytotoxic activity evaluation revealed that twelve out of sixteen test compounds were cytotoxic to human acute monocytic leukemia THP-1 and/or on human acute T cell leukemia Jurkat cell line. 1-(N-hydroxycarbamoyl)benzotriazole (5) increased metabolic activity of both cell lines. Two compounds, 1-(N-hydroxycarbamoyl)benzotriazole (5) and N,N’,N’’-trihydroxybiuret (15) were identified as potential NO donors

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one- and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopreserved PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples

Extra Virgin Olive Oil Secoiridoids Modulate the Metabolic Activity of Dacarbazine Pre-Treated and Treatment-Naive Melanoma Cells

Nowadays, many individuals, whether healthy or diagnosed with disease, tend to expose themselves to various easily accessible natural products in hopes of benefiting their health and well-being. Mediterranean populations have traditionally used olive oil not only in nutrition but also in cosmetics, including skincare. In this study, the phenolic profile&mdash;composed of twelve compounds altogether, including the secoiridoids oleocanthal (OCAL) and oleacein (OCEIN)&mdash;of extra virgin olive oil (EVOO) from autochthonous cultivars from Croatia was determined using 1H qNMR spectroscopy and HPLC-DAD analysis, and its biological activity was investigated in melanoma cell lines. The EVOO with the highest OCEIN content had the strongest anti-cancer activity in A375 melanoma cells and the least toxic effect on the non-cancerous keratocyte cell line (HaCaT). On the other hand, pure OCAL was shown to be more effective and safer than pure OCEIN. Post-treatment with any of the EVOO phenolic extracts (EVOO-PEs) enhanced the anti-cancer effect of the anti-cancerous drug dacarbazine (DTIC) applied in pre-treatment, while they did not compromise the viability of non-cancerous cells. The metastatic melanoma A375M cell line was almost unresponsive to the EVOO-PEs themselves, as well as to pure OCEIN and OCAL. Our results demonstrate that olive oils and/or their compounds may have a potentially beneficial effect on melanoma treatment. However, their usage can be detrimental or futile, especially in healthy cells, due to inadequately applied concentrations/combinations or the presence of resistant cells