Non-stimulated Cell Culture Research Articles

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.

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.

Cancer Cell Invasion Is Enhanced by Applied Mechanical Stimulation

Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor.

Assessment of humoral and cellular-mediated immune response in chickens treated with tilmicosin, florfenicol, or enrofloxacin at the time of Newcastle disease vaccination

The effect of tilmicosin, florfenicol, or enrofloxacin on humoral and cell-mediated immune response induced by Newcastle disease (ND) vaccination was evaluated in 20-wk-old specific-pathogen-free layer chickens. Humoral immunity was measured by detection of ND virus (NDV) antibody titer and anti-NDV IgG response using the hemagglutination inhibition (HI) test and ELISA, respectively, whereas cell-mediated immunity was evaluated by measurement of chicken interferon γ (ChIFN-γ) produced in splenocytes cell culture stimulated with concanavalin A, inactivated NDV antigen, or live attenuated La Sota strain using ELISA. Florfenicol hampered the ND antibody production measured by both HI and ELISA. Tilmicosin and enrofloxacin reduced the production of ND antibody in the first 3 wk after the last ND vaccination measured by HI test, which suggests that these antibiotics exert their effect mainly on the IgM isotype. The ND-vaccinated, but not treated group, showed an increase in ChIFN-γ production after NDV antigen-specific stimulation above the nonstimulated cell culture, whereas this effect was masked in all the antibiotic-treated groups due to the stronger ChIFN-γ production background value reported in the nonstimulated cell culture. In conclusion, our results showed, for the first time, that tilmicosin, florfenicol, or enrofloxacin reduced the humoral immune response and had beneficial effects on the cell-mediated immune response. In addition, we demonstrated that the combination of both inactivated and attenuated ND vaccine gave a strong immune response at both the humoral and cellular level.

Atopic dermatitis in adults: evaluation of peripheral blood mononuclear cells proliferation response to Staphylococcus aureus enterotoxins A and B and analysis of interleukin‐18 secretion

Atopic dermatitis (AD) is a chronic, inflammatory skin disease with a high prevalence and complex pathogenesis. The skin of AD patients is usually colonized by Staphylococcus aureus (S. aureus); its exotoxins may trigger or enhance the cutaneous inflammation. Several mediators are related to the AD immune imbalance and interleukin-18 (IL-18), an inflammatory cytokine, may play a role in the atopic skin inflammation. To evaluate peripheral blood mononuclear cells (PBMC) proliferation response to staphylococcal enterotoxins A (SEA) and B (SEB) and the levels of IL-18 in adults with AD. Thirty-eight adult patients with AD and 33 healthy controls were analysed. PBMC were stimulated with SEA and SEB, phytohemaglutinin (PHA), pokeweed (PWM), tetanus toxoid (TT) and Candida albicans (CMA). IL-18 secretion from PBMC culture supernatants and sera were measured by ELISA. A significant inhibition of the PBMC proliferation response to SEA, PHA, TT and CMA of AD patients was detected (P < or = 0.05). Furthermore, increased levels of IL-18 were detected both in sera and non-stimulated PBMC culture supernatants from AD patients (P < or = 0.05). A decreased PBMC proliferation response to distinct antigens and mitogens (TT, CMA, SEA and PHA) in adults with AD suggest a compromised immune profile. IL-18 secretion from AD upon stimulation was similar from controls, which may indicate a diverse mechanism of skin inflammation maintained by Staphylococcus aureus. On the other hand, augmented IL-18 secretion from AD sera and non-stimulated cell culture may enhance the immune dysfunction observed in AD, leading to constant skin inflammation.

Cytogenetic abnormalities in mesenchymal stem cells in chronic lymphocytic leukemia (CLL) patients and normal subjects

e22002 Background: Mesenchymal stem cells (MSC) residing in the marrow support hematopoiesis and protect cancer cells from undergoing cell death induced by chemotherapy. Recent reports have described clonal cytogenetic abnormalities in the MSC of acute myeloid leukemia and myelodysplastic syndrome patients. To determine if cytogenetic abnormalities are present in MSC from CLL patients, we analyzed karyotypes of MSC from 13 CLL patients and 5 normal subjects. Methods: Stromal cells from marrow core biopsies of 13 CLL patients and 5 normal control subjects were isolated, cultured and confirmed as MSC based on their immunophenotype and capacity to differentiate into three lineages. After 3–4 non-stimulated cell culture passages, the karyotype was analyzed in 5–40 metaphase cells from each subject Abnormalities were considered clonal using the accepted convention of the same chromosomal gain or rearrangement in 2 or more cells or loss in at least 3 cells. For each CLL patient, interphase FISH analysis to detect the common CLL-associated abnormalities was performed on freshly isolated peripheral blood mononuclear cells (PBMC). Results: Clonal cytogenetic abnormalities of the cultured MSC were observed in 6 of 13 CLL patients (46%) and 3 of 5 control subjects (60%). The 6 CLL MSC had different clonal abnormalities than the PBMC CLL FISH studies. One CLL MSC exhibited trisomy 5, one exhibited trisomy 8, and one had monosomy X clone. One MSC had a balanced t(1;16). One MSC had a balanced t(3;12) and a del(3p), and one had a complex karyotype with 4 unbalanced rearrangements. The clonal aberrations observed in the 3 controls included a del(3p) in two cases, and in one case an inv(5) and a del(10q). There was no correlation of the chromosomal abnormalities in CLL MSC with clinical stage. Conclusions: Marrow MSC derived from CLL patients and normal subjects do show an array of cytogenetic abnormalities including clonal chromosomal abnormalities. However the genetic abnormalities found in both CLL and normal MSC could represent acquired genomic instability associated with advanced age, rather than oncogenesis associated with CLL. [Table: see text]

Evidence of occult HCV genotypes in haemophilic individuals with unapparent HCV mixed infections

Individuals with haemophilia who received non heat-treated factor concentrates were likely to undergo multiple exposures to the hepatitis C virus (HCV). Therefore, HCV mixed-genotype infections might be more frequent in these patients than in the general population. Their prevalence is extremely variable in similar groups of patients tested by different assays due to the fact that currently available genotyping techniques are not suitable to detect multiple HCV genotypes in a viral population. As an HCV viral reservoir, the peripheral blood mononuclear cell (PBMC) might harbor viral variants distinct from the genotypes detected in plasma. We investigated the presence of HCV genotypes in a group of chronically infected haemophilic patients in the PBMC compartment using a non-stimulated cell culture system that allows the detection of the HCV genome in culture supernatants. We compared them to the HCV genotypes found in plasma samples. Cell culture experiments performed with PBMC demonstrated the presence of additional HCV genotypes that were undetected in the corresponding plasma samples with the same genotyping technique. Although mixed infections at HCV genotype level became evident in 5.6% of the patients (16/288), the culture methodology increased the number of HCV infections with multiple genotypes to 62.5% (10/16) (P < 0.0001). Once more, the role of mononuclear cells as HCV viral reservoirs is emphasized. Considering minor strains could influence the outcome of treatment, detection of covert HCV mixed-genotype infections might be essential for choosing the adequate therapeutic regimen.

The adaptive potential of human immunity during strength training

Quantitative and functional characteristics of human T-and B-cell-related immunity and natural cytotoxicity were studied during nine-week strength training. Long-term classic strength training and low-intensity strength training without relaxation did not change the peripheral blood contents of the main subpopulations of immunocompetent cells, the phytohemagglutinin-induced proliferative activity of T lymphocytes, serum levels of immunoglobulins A, M, and G (IgA, IgM, and IgG, respectively) and the cytotoxic activity of natural killer cells. At the same time, training was accompanied by activation of the immune system, which was evident from increased counts of CD25+ lymphocytes observed in the peripheral blood and in the mitogen-stimulated and nonstimulated cell cultures, as well as from the higher spontaneous and mitogen-induced productions of IgA, IgM, and IgG by B lymphocytes.

Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries

Bighorn sheep are more susceptible to respiratory infection by Mannheimia haemolytica than are domestic sheep. In response to bacterial challenge, macrophages produce a number of molecules that play key roles in the inflammatory response, including highly reactive nitrogen intermediates such as nitric oxide (NO). Supernatants from monocyte-derived macrophages cultured with M. haemolytica LPS were assayed for nitric oxide activity via measurement of the NO metabolite, nitrite. In response to LPS stimulation, bighorn sheep macrophages secreted significantly higher levels of NO compared to levels for non-stimulated macrophages. In contrast, levels of NO produced by domestic sheep macrophages in response to M. haemolytica LPS did not differ from levels detected in non-stimulated cell cultures. Nitrite levels detected in supernatants of LPS-stimulated bighorn macrophage cultures treated with an inducible nitric oxide synthase (INOS) inhibitor, N(G)-monomethyl-L-arginine, were similar to that observed in non-stimulated cultures indicating a role for the iNOS pathway.

Adjuvant Effect of Synthetic Oligodeoxyribonucleotides (CpG‐ODN) From the Paracoccidioides brasiliensis gp43 gene on the Th2–Th1 Immunomodulation of Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis. Immunostimulatory effects of P. brasiliensis DNA and CpG-oligodeoxyribonucleotides (CpG-ODN) have shown a Th2-Th1 immunomodulation of the isogenic murine model of susceptibility, which develops a progressive and disseminating disease. In this study, we investigated the optimum time interval and doses of CpG-ODN which are able to induce Th2-Th1 immunomodulation. The optimum concentrations for the induction of a decrease in antibody production were 0.5 and 1 microg. Mice immunized twice with CpG-ODN and gp43 (5 and 7 days before the challenge) showed a 60% higher chance of survival compared with the control group (nonimmunized), and an increase in Th1 isotype (IgG2a) was also observed. In vitro assays of naive and preimmunized mice showed discrete cellular proliferation when stimulated by suitable concentrations of CpG-ODN. Type 1 cytokines interleukin-12 (IL-12) and interferon-gamma were increased in cell culture supernatants, but no significant difference was found in Th2 IL-4 cytokines in stimulated or nonstimulated cell cultures. Concerning the Th2-Th1 kinetics in experimental PCM models by adjuvant effect of CpG-ODN, there are still many questions to be answered and clarified. However, the gathering of data obtained in this investigation has led us to suggest that the modulation of Th2-Th1 in experimental PCM depends on time and CpG-ODN concentration.

Induction of Tumor Necrosis Factor and Interleukin‐6 mRNA in Human Cytotrophoblast Cells Exposed to Lipopolysaccharide

Objective: The cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) have previously been identified in placental tissue and are known to be mediators of infection-associated induction of the host immune system. This study was undertaken to better characterize the in vitro regulation of these cytokines in cytotrophoblast cells when challenged with the bacterial product lipopolysaccharide (LPS). Methods: Term placentas were freshly collected, digested with trypsin/DNase, and subjected to Percoll gradient centrifugation to isolate cytotrophoblasts. Either immediately or after overnight incubation, LPS (1 μg/ml) or media alone was added to the cell cultures for 0, 1, 2, 4, 8, 24, 48, and 72 h. Total cellular RNA was isolated by the guanidinium thiocyanate/cesium chloride methodology. RNA samples were run on 1% agarose-formaldehyde gels and subsequently transferred to nylon filters. Blots were hybridized with the appropriate P32-radiolabeled probe. Results: In non-LPS-treated cells, minimal amounts of TNF mRNA could be detected at zero time, or throughout the incubation periods. Conversely, LPS exposure resulted in detectable signal starting at 1 h and peaking at 2 h after the addition of LPS. Overnight incubation gave stronger TNF signals in the LPS stimulated cells, although the kinetics of this response remained similar to zero time exposure. IL-6 was likewise minimally expressed at zero time, although non-stimulated cell cultures demonstrated progressive increases in mRNA expression which was maximal at 16 h after plating. LPS further augmented the transcription of IL-6 mRNA, with peak signals seen at 4 h after LPS stimulation. Again, overnight incubation of the cytotrophoblasts increased baseline and LPS-induced IL-6 mRNA responses. Long-term constant exposure of cells to LPS did not demonstrate any evidence of prolonged signaling. LPS did not alter mRNA expression of the placental gene H19 or the oncogene FOS. Conclusions: These data demonstrate the induction of TNF and IL-6 mRNA in cytotrophoblast cells with LPS. These transcriptional events are kinetically distinct and short term in nature. Overnight incubation accentuates the TNF and IL-6 mRNA signal and allows for an augmented response to LPS.

Placental isoferritin associated p43 antigen correlates with features of high differentiation in breast cancer.

Placental isoferritin (PLF), an acidic isoform of ferritin, and its unique superheavy chain p43 have been recently described to be synthesized by breast cancer cell lines but not by normal breast epithelial cells. Since previous reports have demonstrated a correlation between the content of 'normal' ferritin in breast cancer tissue, degree of differentiation, and prognosis, we have tried to evaluate the correlation of p43 in the cytosol of 122 breast cancer samples with commonly applied prognostic factors and features of proliferation and differentiation. In parallel, we investigated the correlation of p43 expression in MCF-7 and T47-D breast cancer cell lines during proliferation induced by estradiol plus fetal calf serum (assessed by 3H-thymidine incorporation), compared to p43 expression in stationary non-stimulated cell cultures. The levels of p43 in breast cancer cytosols correlated significantly negatively with tumor size (p = 0.0001), histologic grading (p = 0.0038), nuclear pleomorphism (p = 0.0019), rate of mitosis (p = 0.0002), and lymphocytic reaction (p = 0.0001), and significantly directly with the estrogen receptor status (p = 0.0009). Although patients with a higher p43 content showed a trend for a better outcome (median follow-up: 61.4 months), an independent influence of the cytosolic p43 content on survival could not be confirmed by a multivariate Cox model. In accordance with the observed negative correlation of features of differentiation vs. p43 expression, induction of proliferation by estradiol plus FCS added to serum-free tissue culture medium correlated with a decrease of p43 synthesis in both cell lines. Expression of p43 in estrogen and FCS-absent media revealed also a decrease in relation to a low spontaneous proliferation. However, the drop of p43 synthesis was significantly stronger in cell lines with estrogen-stimulated proliferation. Our in vitro and cytosol results confirm recent clinical observations describing an inverse correlation of p43 synthesis with the degree of proliferation and differentiation in breast cancer. However, the pathologic mechanisms leading to this phenomenon as well as the negative correlation with lymphocytic infiltration are still unclear and need to be further elucidated.

Thyrotrophin‐Induced Aggregation and Reorganization into Follicles of Isolated Porcine‐Thyroid Cells in Culture

Porcine thyroid cells isolated by trypsinization grow in culture as a monolayer. At high cell concentration and in the presence of thyrotrophin, cells aggregate and rearrange in structures resembling cross‐sections of intact thyroid tissue. Longitudinal and transverse sections of thyrotrophin‐stimulated and non‐stimulated cell cultures have been examined by electron microscopy after fixation of the cell layers in the culture flasks. Cells cultured in the presence of thyrotrophin undergo an organizational rearrangement into typical three‐dimensional follicles as shown by the presence of junctional complexes, differentiated apical poles with microvillae, follicular lumen sometimes containing material dense to electrons, and apical vesicles. In contrast, cells cultured in the absence of thyrotrophin develop as a two‐dimensional layer. No differentiation of the plasma membrane is visible. Cells are flat and partially overlap each other. All cells are linked by junctional complexes. Except for this zone of association, the undifferentiated plasma membranes of the contiguous cells are not adherent and limit more or less distended intercellular spaces. Cells associated in follicles show the ultrastructural aspects of polarized and stimulated follicular cells. The apical pole is rich in organelles such as lysosomes, vesicles, Golgi complexes and rough endoplasmic reticulum. To the contrary in monolayer cells, the organelles are perinuclear; Golgi complexes are scarce and a very dense network of microfilaments and microtobules is visible in the peripheral part of the cytoplasm. Therefore, thyroid cells cultured with or without thyrotrophin, each form a homogeneous population of cells in a given organizational arrangement and at different level of differentiation.