Non-stimulated Cultures Research Articles

Cucurbituril-based Supramolecular complexes with platinum compounds influence on expression of CTLA-4 on Regulatory T cells

Tumors are a leading pathology in the population. Chemotherapy cannot provide adequately and effectively to cure patients. Some medicine, such as cytostatic, are characterized by a wide range of side effects and resistance of solid tumors to chemotherapy by these medicines. In recent research, the mechanisms of action of cytotoxic agents have been described, and the most appropriate causes of resistance have been suggested. Drug delivery system based on Cucurbit[7]uril (CB[7]) was used to minimize side effects and overcome resistance. CB[7] has ability to form host-guest supramolecular complexes with oxaliplatin and carboplatin.It is important to consider the immune system maintain to a great role, and platinum compounds are able to have an immunomodulatory effect on immunocompetent cells. There is convincing evidence about the cytotoxic response against tumor cells is also associated with immunomodulating properties. A specific immune microenvironment with high frequency of suppressor cells is made by tumors. FoxP3+ regulatory T cells are recruited by the tumor, an increased number of these cells and expression levels of CTLA-4 and PD-1 on them contribute to the progression of the tumor process. These markers correlate with recurrence and poor survival of the patients. Therefore, it is necessary that antitumor therapy agents have an effect on a subpopulation of regulatory T cells and their functional activity. This study evaluated the effects of cucurbit[7] uril, platinum compounds, and supramolecular complexes on FoxP3+ regulatory T cells and the expression of immune checkpoint molecules.In this study peripheral blood cells from volunteers (n = 8, average 29.0±2.4) were used. Mononuclear cells obtained in the standard protocol were incubated for 72 h at concentrations of 0.3 and 0.1 mM for carboplatin and oxaliplatin, respectively, as well as complexes and CB[7] in equivalent dosages. Next, the samples were labeled with monoclonal antibodies to determine the phenotype and expression of immune checkpoint molecules by flow cytometry.We obtained the following results: The CB[7]-carboplatin complex in stimulated and non-stimulated cultures significantly reduced the number of FoxP3+ regulatory T cells compared to the control. At the same time, carboplatin and the CB[7]-carboplatin complex reduced the expression of CTLA-4 in an non-stimulated culture compared to CB[7].Complexes of Cucurbit[7]urils with platinum compounds are a perspective antitumor agent with immunomodulatory properties.

Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model

Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease, caused by Dirofilaria immitis, causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction, and hypoxia. The aim of this study was to analyze whether the excretory/secretory and surface-associated antigens of adult worms interact and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEFG-A (Vascular Endothelial Growth Factor A), as well as DiES+VEFG-A and Cut+VEFG-A were used. The production of VEFG-A and other proangiogenic [soluble VEFGR-2 (sVEFGR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEFGR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non‑stimulated cultures were used as controls. DiES+VEFG-A and Cut+VEFG-A significantly increased the production of VEFG-A and sVEFGR-2, and only Cut+VEFG-A significantly increased the production of VEFGR-1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEFG-A produced a significant increase in viable cell number and significant decrease cell migration, as well as in the organization and number of cell connections. Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.

DC electrical stimulation enhances proliferation and differentiation on N2a and MC3T3 cell lines

BackgroundElectrical stimulation is a novel tool to promote the differentiation and proliferation of precursor cells. In this work we have studied the effects of direct current (DC) electrical stimulation on neuroblastoma (N2a) and osteoblast (MC3T3) cell lines as a model for nervous and bone tissue regeneration, respectively. We have developed the electronics and encapsulation of a proposed stimulation system and designed a setup and protocol to stimulate cell cultures.MethodsCell cultures were subjected to several assays to assess the effects of electrical stimulation on them. N2a cells were analyzed using microscope images and an inmunofluorescence assay, differentiated cells were counted and neurites were measured. MC3T3 cells were subjected to an AlamarBlue assay for viability, ALP activity was measured, and a real time PCR was carried out.ResultsOur results show that electrically stimulated cells had more tendency to differentiate in both cell lines when compared to non-stimulated cultures, paired with a promotion of neurite growth and polarization in N2a cells and an increase in proliferation in MC3T3 cell line.ConclusionsThese results prove the effectiveness of electrical stimulation as a tool for tissue engineering and regenerative medicine, both for neural and bone injuries. Bone progenitor cells submitted to electrical stimulation have a higher tendency to differentiate and proliferate, filling the gaps present in injuries. On the other hand, neuronal progenitor cells differentiate, and their neurites can be polarized to follow the electric field applied.

Effect of cucurbiturils on cytokine production by peripheral blood mononuclear cells of healthy donors

Many drug delivery systems are currently under study, e.g., nanosized cavitands cucurbiturils, which, due to the presence of a cavity, can incorporate drug molecules. Since the immune system is quite sensitive to influence of nanomaterials and other cell-damaging factors, it is necessary to study immunosafety of the new delivery systems, i.e., immunotoxicity and immunomodulatory properties. The aim of this study was to investigate the effect of nanosized cucurbituril cavitands on the cytokine-producing ability of peripheral blood mononuclear cells in apparently healthy donors.
 Blood mononuclear cells (106/mL) were cultured in the presence of cucurbiturils at the following concentrations: 0.3 mM cucurbit[6]uril, 0.3 mM cucurbit[7]uril, and 0.01 mM cucurbit[8]uril for 72 h, under additional stimulation with aCD3 antibodies (1 g/mL), or without it. The level of cytokines in the supernatants was determined using enzyme immunoassay.
 It was shown that cucurbit[6]uril increased the level of spontaneous IL-4 production by 1.5 times (p 0.01) compared with the control. In the case of stimulated cytokine production, we found that cucurbit[6]uril reduced the level of IL-6, and also shows a tendency (p = 0.09) towards an increase in the IL-4 level. When cells were cultured with cucurbit[7]uril, we gave revealed a trend for increased production of pro-inflammatory TNF. It was also found that cucurbit[7]uril is able to suppress the production of IL-10 in aCD3-stimulated cell culture by 1.5 times. Cucurbit[8]uril was shown to inhibit production of cytokines in non-stimulated cell cultures. A significant decrease in the level of IFN and IL-10 was revealed as compared with the production of these cytokines in control cultures. When assessing the effect of cucurbit[8]uril on the IFN production upon stimulation with aCD3 antibodies, no significant differences were found, but there is also a trend for a decreased concentration of this cytokine agains control levels.
 Cucurbiturils can influence both spontaneous and stimulated production of cytokines by the blood mononuclear cells. The effect on cytokine-producing ability of the cells depends on the tested homologue compound.

Metabolic and immune/inflammatory alterations induced by a triathlon under extreme conditions.

PurposeTo investigate the effects of triathlon racing under extreme conditions on metabolic and immune/inflammatory responses.MethodsThirteen amateur athletes participated in an extreme triathlon competition (swim – 3.8 km; cycling – 180 km; running – 4 2 km; with a 3,700 m accumulated altitude). Blood samples were collected on three different occasions: pre-competition (baseline), immediately post-competition (IM), and 12 h post-competition (12 h) to evaluate glycemic and lipid profiles, leukocytes count, and cytokines levels in plasma and in whole-blood cell culture supernatant stimulated or not with LPS.ResultsDecreased glucose and triglycerides levels, increased LDL, and a significant leukocytosis were observed at IM and 12 h compared to baseline. In addition, higher serum levels of IL-6, IL-8, and IL-10 were found at IM than in baseline and post-12 h. Whereas increased IL-12p40 levels were observed for 12 h compared to baseline. At baseline, in LPS-stimulated cell culture, IL-6, IL-8, and IL-12p70 were higher, while IL-12p40 levels were lower than non-stimulated cell culture. At IM, IL-12p40 levels were unchanged, while higher levels of other cytokines were found in LPS-stimulated cell culture compared to non-stimulated cell culture. The 12 h results showed higher levels of IL-6, IL-8, and IL-10 in LPS-stimulated cell culture than in non-stimulated cell culture. Additionally, a significant negative correlation between circulating glucose levels and IL-6 was found.ConclusionThe triathlon competition's performance under extreme conditions has remarkable impacts on the lipid profile and systemic immune/inflammatory responses. For the first time, significant alterations in the cytokine responses of whole blood cell culture to LPS-stimulation in baseline, IM, and 12h were demonstrated.

Is there an Inflammation Role for MYD88 in Rheumatoid Arthritis?

Rheumatoid arthritis (RA) is an autoimmune and inflammatory disease with strong genetic influence, especially upon immune response components. Several cytokines from the toll-like receptors activation pathway display recognized role for RA establishment. However, few studies have verified the role of key mediators such as MYD88 gene and its genetic variants. In the present study, we aim to evaluate the rs6853 functional single-nucleotide variation (SNV) role in RA etiopathogenesis, clinical severity status, and its impact in MYD88 mRNA levels and IL-lβ protein levels. For the association study, a total of 423 RA patients and 346 health individuals, enrolled as control, from Northeast and Southeast Brazil were genotyped using specific Taqman probe. For the gene expression assays, we performed a MYD88 rs6853 genotype-guided monocyte cell culture divided into non-stimulated and lypopolysaccharides (LPS)-stimulated cells from healthy individuals. MYD88 gene expression was measured using primer specifics while IL-1β levels were evaluated by ELISA. We observed that A allele and AA genotype were associated to an increased risk to RA development (OR = 1.60; 95% CI 1.24-2.08; p = 0.0004/OR = 2.83; 95% CI 1.25-6.41; p = 0.0152). The AA genotype exhibited lower MYD88 mRNA levels than GG genotype in non-stimulated monocyte cell culture (FC-3.83; p = 0.003). Additionally, we verified an increase of IL-1β levels when AA genotype non-stimulated monocytes were compared to AA genotype LPS-stimulates (p = 0.021). In summary, MYD88 rs6853 polymorphism associated to RA development in our Brazilian cohort and showed influence upon MYD88 mRNA levels' expression and IL-lβ production.

Effect of electrical stimulation on chondrogenic differentiation of mesenchymal stem cells cultured in hyaluronic acid – Gelatin injectable hydrogels

Electrical stimulation (ES) has provided enhanced chondrogenesis of mesenchymal stem cells (MSCs) cultured in micro-mass without the addition of exogenous growth factors. In this study, we demonstrate for the first time that ES of MSCs encapsulated in an injectable hyaluronic acid (HA) – gelatin (GEL) mixture enhances the chondrogenic potential of the hydrogel. Samples were stimulated for 21 days with 10 mV/cm at 60 kHz, applied for 30 min every 6 h a day. Mechanical properties of hydrogels were higher if the precursors were dissolved in Calcium-Free Krebs Ringer Buffer (G′ = 1141 ± 23 Pa) compared to those diluted in culture media (G′ = 213 ± 19 Pa). Cells within stimulated hydrogels were rounder (55%) than non-stimulated cultures (32%) (p = 0.005). Chondrogenic markers such as SOX-9 and aggrecan were higher in stimulated hydrogels compared to controls. The ES demonstrated that normalized content of glycosaminoglycans and collagen to DNA was slightly higher in stimulated samples. Additionally, collagen type II normalized to total collagen was 2.43 times higher in stimulated hydrogels. These findings make ES a promising tool for enhancing articular cartilage tissue engineering outcomes by combining hydrogels and MSCs.

Equine Fetal, Adult, and Embryonic Stem Cell-Derived Tenocytes Are All Immune Privileged but Exhibit Different Immune Suppressive Properties In Vitro.

In horses and humans, tendon injuries are a significant problem. Not only can they occur in both athletes and nonathletes, they require lengthy periods of recuperation and undergo poor natural regeneration, which leads to high reinjury rates. Embryonic stem cells (ESCs) may provide a renewable source of allogeneic cells to use in clinical applications to aid tissue regeneration. Equine ESCs can undergo tenocyte differentiation in vivo and in vitro, but the immune properties of tenocytes isolated from either ESCs or tissues have not previously been characterized. Here, we demonstrate that equine tenocytes derived from fetal and adult tendon tissue and ESCs express robust levels of major histocompatibility complex (MHC) I but no MHC II in response to inflammatory cytokine interferon gamma (IFNγ). However, MHC expression does not affect their allorecognition by peripheral blood mononuclear cells in vitro. Adult and fetal tenocytes remain immune privileged and strongly immune suppressive in both the presence and absence of exogenously applied IFNγ. In contrast, ESC-derived tenocytes are immune privileged even in the presence of IFNγ, but they are only weakly immune suppressive in the presence but not in the absence of exogenously applied IFNγ. This is despite ESC-tenocytes expressing a number of genes involved in immune modulation at significantly higher levels than those expressed by adult and fetal tenocytes when in standard, nonstimulated monolayer culture. Together, this work suggests that, similar to other fibroblasts, tenocytes have immune modulatory properties, and that culture-expanded tenocytes derived from primary tissues or ESCs may be safe to use in clinical transplantations to injured tendons of unrelated animals.

Angiogenic response in an in vitro model of dog microvascular endothelial cells stimulated with antigenic extracts from Dirofilaria immitis adult worms

BackgroundAngiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells.MethodsThe expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls.ResultsFirst, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections.ConclusionsThe ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

Schistosoma mansoni rSm29 Antigen Induces a Regulatory Phenotype on Dendritic Cells and Lymphocytes From Patients With Cutaneous Leishmaniasis.

The immune response induced by Schistosma mansoni antigens is able to prevent immune-mediated diseases. Conversely, the inflammatory response in cutaneous leishmaniasis (CL), although responsible for controlling the infection, is also associated with the pathogenesis of disease. The aim of this study was to evaluate the potential of the S. mansoni Sm29 antigen to change certain aspects of the profiles of monocyte derived dendritic cells (MoDCs) and lymphocytes from subjects with CL in vitro. Expression of surface molecules and intracellular cytokines in the MoDCs and lymphocytes as well as the proliferation of Leishmania braziliensis were evaluated by flow cytometry. Levels of cytokines were evaluated in culture supernatants by ELISA. It was observed that stimulation by rSm29 increased the frequency of expression of CD83, CD80, CD86, and IL-10R in MoDCs compared to non-stimulated cultures. Additionally rSm29 decreased the frequency CD4+ and CD8+ T cells expressing CD28 and increased the frequency of CD4+CD25hi and CD4+CTLA-4+ T lymphocytes. Addition of rSm29 to cultures increased IL-10 levels and decreased levels of IL-12p40 and IFN-γ, while not altering TNF levels compared to non-stimulated cultures. This study showed that rSm29 induced a regulatory profile in MoDCs and lymphocytes and thereby regulated the exaggerated inflammation observed in CL. Considering that there are few therapeutic options for leishmaniasis, the use of rSm29 may be an alternative to current treatment and may be an important strategy to reduce the healing time of lesions in patients with CL.

A Chip for Estrogen Receptor Action: Detection of Biomarkers Released by MCF-7 Cells through Estrogenic and Anti-Estrogenic Effects.

The fluorescence-based multi-analyte chip platform for the analysis of estrogenic and anti-estrogenic substances is a new in vitro tool for the high throughput screening of environmental samples. In contrast to existing tools, the chip investigates the complex action of xenoestrogens in a human cell model by characterizing protein expression. It allows for the quantification of 10 proteins secreted by MCF-7 cells, representing various biological and pathological endpoints of endocrine action and distinguishing between estrogen- and anti-estrogen-dependent secretion of proteins. Distinct protein secretion patterns of the cancer cell line after exposure to known estrogen receptor agonists ß-estradiol, bisphenol A, genistein, and nonylphenol as well as antagonists fulvestrant and tamoxifen demonstrate the potential of the chip. Stimulation of cells with Interleukin-1ß shifts concentrations of low abundant biomarkers towards the working range of the chip. In the non-stimulated cell culture, Matrix Metalloproteinase 9 (MMP-9) and Vascular Endothelial Growth Factor (VEGF) show differences upon treatment with antagonists and agonists of the estrogen receptor. In stimulated MCF-7 cells challenged with receptor agonists secretion of Monocyte Chemoattractant Protein (MCP-1), Interleukin-6 (IL-6), Rantes, and Interleukin-8 (IL-8) significantly decreases. In parallel, the proliferating effect of endocrine-disrupting substances in MCF-7 cells is assessed in a proliferation assay based on resazurin. Using ethanol as a solvent for test substances increases the background of proliferation and secretion experiments, while using dimethyl sulfoxide (DMSO) does not show any adverse effects. The role of the selected biomarkers in different physiological processes such as cell development, reproduction, cancer, and metabolic syndrome makes the chip an excellent tool for either indicating endocrine-disrupting effects in food and environmental samples, or for screening the effect of xenoestrogens on a cellular and molecular level.

Hyaluronan oligosaccharides stimulate matrix metalloproteinase and anabolic gene expression in vitro by intervertebral disc cells and annular repair in vivo.

The role of hyaluronan (HA) oligosaccharides in disc cell-mediated matrix metalloproteinase (MMP) and anabolic gene expression in vitro and annular repair in vivo were examined. Monolayer and alginate bead cultures of ovine intervertebral disc cells were stimulated with 10-12 mer hyaluronan oligosaccharides (HA-oligos). Annulus fibrosus (AF) monolayers were poorly responsive to the HA-oligos, proMMP-2 levels were marginally elevated and levels were MMP-9 unaffected. ProMMP-2 displayed a strong dose-dependent increase in the nucleus pulposus (NP) monolayers. In AF alginate bead cultures, proMMP-2 and active MMP-9 increased up to day 10, in NP cultures proMMP-2 was progressively converted to active MMP-2 over days 7-10 and active MMP-9 levels were elevated on day 10. A steady decline in MMP-2 and MMP-9 activity was evident over days 2-10 in the non-stimulated NP cultures. Disc cell viabilities were ≥92±5% in all cultures indicating that the HA-oligo was not cytotoxic. Reverse-transcription polymerase chain reaction demonstrated an upregulation in MMP1, MMP113 and ADAMTS1 and the anabolic matrix repair genes ACAN, COL1A1 and COL2A1 in the NP by HA-oligos, whereas AF MMP13, ADAMTS1, ADAMTS4 and ADAMTS5, ACAN and COL2A1 were down-regulated; this differential regulation is expected to promote clearance of granulation/scar tissue from AF defects and matrix replenishment. The AF defect sites contained enlarged annular lamellae in vivo in response to the HA oligos, which is consistent with an active repair response. Masson trichrome and PicroSirius red histology and immunolocalization of type I collagen supported active remodelling in the outer lesion zone by the HA-oligo treatment but not the inner lesion. Copyright © 2016 John Wiley & Sons, Ltd.

Modulation of Immune Responses by Extracellular Vesicles From Retinal Pigment Epithelium.

PurposeExtracellular vesicles (EV), such as exosomes, are important mediators of intercellular communication and have been implicated in modulation of the immune system. We investigated if EV released from retinal pigment epithelium (RPE) modulate immune responses in vitro.MethodsExtracellular vesicles were isolated from ARPE-19 cultures stimulated or not with the inflammatory cytokines IL-1β, IFN-γ, and TNF-α. Isolated EV were characterized by nanoparticle flow and Western blot analyses. Retinal pigment epithelium–derived EV were cultured with human peripheral blood mononuclear cells, which were assayed for T-cell proliferation by 3H-thymidine incorporation. Retinal pigment epithelium–derived EV were also independently cultured with enriched lymphocytes or monocytes. Cell phenotype and cell death were evaluated by flow cytometric analysis. Cytokine levels were assayed in culture supernatants by multiplex bead analysis.ResultsThe concentration of ARPE-derived EV from cytokine-stimulated cultures was slightly higher than from nonstimulated cultures. The size of EV was approximately 100 nm in both groups. Extracellular vesicles from both nonstimulated and cytokine-stimulated ARPE-19 significantly inhibited T-cell proliferation without affecting T-cell viability. Culture of EV from nonstimulated ARPE-19 with undifferentiated human monocytes induced an immunoregulatory phenotype with a significantly higher percentage of CD14++CD16+ monocytes and upregulation of TGF-β1. Culture of EV from cytokine-stimulated ARPE-19 cells with human monocytes induced upregulation of several proinflammatory cytokines and monocyte death.ConclusionsRetinal pigment epithelium cells constitutively secrete EV in the size range of exosomes, with increased release from RPE cells stimulated with inflammatory cytokines. Extracellular vesicles from both nonstimulated and cytokine-stimulated RPE inhibited T-cell stimulation. Extracellular vesicles from nonstimulated ARPE-19 cells promoted an immunoregulatory CD14++CD16+ phenotype in human monocytes, while EV from cytokine-stimulated ARPE-19 cells caused human monocyte death. These findings suggest that RPE cells use EV to induce a downregulatory immune environment under homeostatic conditions. In an inflammatory milieu, RPE-derived EV may mitigate a potentially harmful inflammatory response through killing of monocytes.

PRODUCTION OF PROINFLAMMATORY CYTOKINES AND ALPHA-2-MACROGLOBULIN BY PERIPHERAL BLOOD CELLS IN THE PATIENTS WITH COLORECTAL CANCER

Colorectal cancer (CRC) is the third most common cancer worldwide, being quite complicated, with respect to diagnostics and postoperative prognosis. Proinflammatory cytokines are shown to be involved into CRC pathogenesis. However, the changes in alpha-2-macroglobulin (α2-MG), a known regulator of cytokine production, still remain unclear. The aim of this work was to compare contents and production of a2-MG and several pro-inflammatory cytokines in blood serum and supernates from short-term blood cell cultures. The samples were taken from the patients with CRC at initial terms and after surgical removal of the tumor. Studies of cytokines and a2-MG concentrations in serum and supernates of 24-h blood cell cultures from the patients with verified CRC (stages T2-3N0-1M0 and T4N0-1M0) have shown some sufficient differences from healthy volunteers (control group). Pre-surgery IL-6 and TNFα contents in blood of CRC patients was significantly increased agains healthy controls (respectively, 29.9±5.4 and 3.4±1.5 pg/mL) versus control group (1.0±0.3 and 0 pg/mL, respectively). Following surgical treatment, the cytokine levels were decreased by 40- 60% after the operation, however, without significant differences from initial values. The supernates of blood cultures stimulated with polyclonal mitogens exhibited significant reduction of IFNγ levels prior to surgery (273±123 pg/ml versus 804±154 pg/mL), and elevated IL-6 levels (14412±2570 pg/mL versus 1970±457 pg/mL). The mean α2-MG concentrations before CRC surgery comprised 1.96±0.11 g/L for blood serum, 0.0304±0.0047 g/L, for non-stimulated blood cell cultures, and 0.0300±0.0052 g/L in mitogen-induced cultures. These parameters did not significantly differ from control values (2.21±0.17 g/L, 0.0328±0.0018 g/L, and 0.0314±0.0019 g/L, respectively). Similar results have been yielded with the samples obtained after surgical treatment of the CRC patients. The obtained data indicate that surgical treatment of CRC does not lead to normalization of the serum levels and secretion of pro-inflammatory IL-6 and TNFα cytokines by the patients’ blood cells. Marginal changes in a2-MG levels in colorectal cancer seem to be not of such importance, as functional state of the molecules which may abnormally respond to changing cytokine contents. The revealed post-surgical suppression of mitogen-induced IFNγ secretion makes it necessary to suggest administration of IFNγ and IL-2 after surgical treatment of CRC.

ASSESSMENT OF GOLD NANOPARTICLE EFFECT ON PROSTATE CANCER LNCaP CELLS

In recent years gold nanoparticles (AuNPs) have received considerable attention for various biomedical applications including diagnostics and targeted drug delivery. However, more research is still needed to characterize such aspects of their use in clinical oncology as permeability, retention and functional effect on tumor cells. This study was designed to describe the effect of non-functionalized AuNPs on LNCaP prostate cancer cells growth. LNCaP cells were cultured in RPMI-1640 medium containing AuNPs covered by polyvinylpyrrolidone of average size 26.4 nm (10.0 μg/ml). Counts of cells were calculated and their morphology was examined. AuNPs conglomerates have been visualized in cultured cells. After 4-day incubation in presence of AuNPs significant retardation of LNCaP cells growth was observed both in 5α-dihydrotestosterone stimulated and non-stimulated cultures. No morphological changes of live LNCaP cells were seen in any experiment. Given absence of morphological changes in live cells and dribble and relatively constant numbers of dead cells, it was concluded that inhibitory effect of AuNPs on LNCaP cells growth was caused by alterations of proliferation.

Immunomodulatory effect of non-viable components of probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus on holoxenic mice.

BackgroundCompetition of probiotic bacteria with other species from the intestinal microbiota involves different mechanisms that occur regardless of probiotics’ viability. The objective of this paper was to assess the cytokine serum levels in holoxenic mice after oral administration of non-viable components (NVC) of Enterococcus faecium probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus in comparison to NVC of unstimulated E. faecium probiotic culture.MethodsProbiotic E. faecium CMGb 16 culture, grown in the presence of heat-inactivated cultures of E. coli and B. cereus CMGB 102, was subsequently separated into supernatant (SN) and heat-inactivated cellular sediment (CS) fractions by centrifugation. Each NVC was orally administered to holoxenic mice (balb C mouse strain), in three doses, given at 24 hours. Blood samples were collected from the retinal artery, at 7, 14, and 21 days after the first administration of the NVC. The serum concentrations of IL-12 and tumor necrosis factor-alpha (TNF-α) interleukins were assessed by ELISA method.ResultsAfter the oral administration of SN component obtained from the probiotic culture stimulated with heat-inactivated cultures of B. cereus CMGB 102 and E. coli O28, the serum concentrations of IL-12 were maintained higher in the samples collected at 7 and 14 days post-administration. No specific TNF-α profile could be established, depending on stimulated or non-stimulated probiotic culture, NVC fraction, or harvesting time.ConclusionThe obtained results demonstrate that non-viable fractions of probiotic bacteria, stimulated by other bacterial species, could induce immunostimulatory effects mediated by cytokines and act, therefore, as immunological adjuvants.

AB0060 Cross-Talk between the Adipogenic and the Chondrogenic Programs Elicited by A Glycosaminoglycan Mixture

BackgroundMesenchymal stem cells (MSCs) are multipotent cells able to differentiate into chondrocytes, osteoblasts and adipocytes in a competitively balanced manner. MSCs play a role in the homeostasis of adipose tissue...

Regulation of activity of P2X7 receptor by its splice variants in cultured mouse astrocytes

Of purinergic receptors, P2X7 receptor (P2X7R, defined as a full-length receptor) has unique characteristics, and its activation leads to ion channel activity and pore formation, causing cell death. Previously, we demonstrated that P2X7R expressed by nonstimulated astrocyte cultures obtained from SJL-strain mice exhibits constitutive activation, implying its role in maintenance of cellular homeostasis. To obtain novel insights into its physiological roles, we examined whether constitutive activation of P2X7R is regulated by expression of its splice variants in such resting astrocytes, and whether their distinct expression profiles in different mouse strains affect activation levels of astrocytic P2X7Rs. In SJL- and ddY-mouse astrocytes, spontaneous YO-PRO-1 uptake, an indicator of pore activity of P2X7R, was detected, but the uptake by the formers was significantly greater than that by the latter. Between the two mouse strains, there was a difference in their sensitivity of YO-PRO-1 uptake to antagonists, but not in the expression levels and sequences of P2X7R and pannexin-1. Regarding expression of splice variants of P2X7R, expression of P2X7R variant-3 (P2X7R-v3) and -4 (P2X7R-v4), but not variant-2 and -k, was lower in SJL-mouse astrocytes than in ddY-mouse ones. On transfection of P2X7R-v3 and -v4 into SJL-mouse astrocytes, the pore activity was attenuated as in the case of the HEK293T cell-expression system. These findings demonstrate that basal activity of P2X7R expressed by resting astrocytes is negatively regulated by P2X7R-v3 and -v4, and that their distinct expression profiles result in the different activation levels of astrocytic P2X7Rs in different mouse strains.

Cellular immunophenotypic profile in the splenic compartment during canine visceral leishmaniasis

To determine the role of the spleen in the pathogenesis of canine visceral leishmaniasis (CVL), we analyzed cellular immunophenotypic profiles of 52 dogs naturally infected with Leishmania infantum, clinically classified as follows: asymptomatic dogs-I (AD-I), seronegative/PCR+; asymptomatic dogs-II (AD-II), seropositive/PCR+; oligosymptomatic dogs (OD) and symptomatic dogs (SD). Seven non-infected dogs (CD) were included as a control group. AD-II presented higher levels of CD8+ T splenocytes and lower TCD4+/TCD8+ ratio in comparison with CD. OD and SD showed lower percentages of CD21+ as compared with AD-II. All seropositive dogs presented lower levels of CD45RA+ than CD. Regardless of the stimuli used, the proliferation index from splenocytes in vitro was inversely correlated with clinical status. After LSA stimulation, there was a higher percentage of specific CD8+ T in AD-II than CD and non-stimulated culture. In contrast, splenocytes from SD under in vitro LSA stimulation induced decreased MHC-II+ expression in comparison with all groups, and non-stimulated culture. In conclusion, the role of CD8+ T splenocytes seems to be important for an effective immunological response, a hallmark of asymptomatic CVL, whereas the pronounced loss of MHC-II expression upon LSA stimulation is a biomarker of symptomatic CVL.

Human platelet‐rich plasma induces chondrogenic differentiation of subchondral progenitor cells in polyglycolic acid–hyaluronan scaffolds

Cartilage repair approaches may be improved by addition of human platelet-rich plasma (PRP) that increases chondrogenic differentiation of mesenchymal stem and progenitor cells. The aim of our study was to evaluate the effect of human PRP on the differentiation of multipotent human subchondral progenitor cells in resorbable polyglycolic acid-hyaluronan (PGA-HA) scaffolds. PGA-HA scaffolds were loaded with subchondral progenitor cells and stimulated with transforming growth factor-beta3 (TGFB3) or 5% PRP, whereas nonstimulated cultures served as controls. Chondrogenic differentiation was evaluated by real-time gene expression analysis of typical chondrogenic marker genes and by immunohistochemical staining of extracellular cartilage matrix molecules such as proteoglycans and collagen type II. TGFB3 and PRP induced the expression of chondrogenic marker genes collagen type II and IX, aggrecan, and cartilage oligomeric matrix protein in subchondral progenitor cells cultured in PGA-HA scaffolds compared with nonstimulated controls. Progenitor cells in PGA-HA scaffolds formed an extracellular matrix rich in proteoglycans and collagen type II on treatment with PRP, but to a lesser extent, than in cultures stimulated with TGFB3. The results suggest that PRP induces chondrogenic differentiation of progenitor cells in PGA-HA scaffolds and may be therefore beneficial in scaffold-assisted cartilage repair approaches involving stem and progenitor cells.