Investigating the interaction of cellulose nanofibers derived from cotton with a sophisticated 3D human lung cell coculture

Cellulose nanofibers are an attractive component of a broad range of nanomaterials. Their intriguing mechanical properties and low cost, as well as the renewable nature of cellulose make them an appealing alternative to carbon nanotubes (CNTs), which may pose a considerable health risk when inhaled. Little is known, however, concerning the potential toxicity of aerosolized cellulose nanofibers. Using a 3D in vitro triple cell coculture model of the human epithelial airway barrier, it was observed that cellulose nanofibers isolated from cotton (CCN) elicited a significantly (p < 0.05) lower cytotoxicity and (pro-)inflammatory response than multiwalled CNTs (MWCNTs) and crocidolite asbestos fibers (CAFs). Electron tomography analysis also revealed that the intracellular localization of CCNs is different from that of both MWCNTs and CAFs, indicating fundamental differences between each different nanofibre type in their interaction with the human lung cell coculture. Thus, the data shown in the present study highlights that not only the length and stiffness determine the potential detrimental (biological) effects of any nanofiber, but that the material used can significantly affect nanofiber-cell interactions.

In vitro effects of thiazolides on Giardia lamblia WB clone C6 cultured axenically and in coculture with Caco2 cells

The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 microM; in comparison, NTZ and tizoxanide had IC50s of 2.4 microM, and MTZ had an IC50 of 7.8 microM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.

New exposure system to evaluate the toxicity of (scooter) exhaust emissions in lung cells in vitro

A constantly growing number of scooters produce an increasing amount of potentially harmful emissions. Due to their engine technology, two-stroke scooters emit huge amounts of adverse substances, which can induce adverse pulmonary and cardiovascular health effects. The aim of this study was to develop a system to expose a characterized triple cell coculture model of the human epithelial airway barrier, to freshly produced and characterized total scooter exhaust emissions. In exposure chambers, cell cultures were exposed for 1 and 2 h to 1:100 diluted exhaust emissions and in the reference chamber to filtered ambient air, both controlled at 5% CO(2), 85% relative humidity, and 37 degrees C. The postexposure time was 0-24 h. Cytotoxicity, used to validate the exposure system, was significantly increased in exposed cell cultures after 8 h postexposure time. (Pro-) inflammatory chemo- and cytokine concentrations in the medium of exposed cells were significantly higher at the 12 h postexposure time point. It was shown that the described exposure system (with 2 h exposure duration, 8 and 24 h postexposure time, dilution of 1:100, flow of 2 L/min as optimal exposure conditions) can be used to evaluate the toxic potential of total exhaust emissions.

Role of hypoxia and growth and differentiation factor-5 on differentiation of human mesenchymal stem cells towards intervertebral nucleus pulposus-like cells

There is evidence that mesenchymal stem cells (MSCs) can differentiate towards an intervertebral disc (IVD)-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß) to the effects of hypoxia, growth and differentiation factor-5 (GDF5), and coculture with bovine nucleus pulposus cells (bNPC). The efficacy of molecules recently discovered as possible nucleus pulposus (NP) markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control) supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 %) or normal (20 %) oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confirmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

Pregnancy in patients with ankylosing spondylitis: do regulatory T cells play a role?

OBJECTIVE: To investigate the numerical and functional changes of CD4+CD25(high) regulatory T (Treg) cells during pregnancy and postpartum in patients with ankylosing spondylitis (AS). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 10 pregnant and 5 nonpregnant patients with AS as well as in 14 pregnant and 4 nonpregnant healthy controls. Pregnant individuals were investigated at the third trimester and 8 weeks postpartum. Treg cells and CD4+CD25- effector T (Teff) cells separated by fluorescence-activated cell sorting were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies, alone or in coculture, to investigate proliferation and cytokine secretion. RESULTS: The frequency of CD4+CD25(high) Treg cells was significantly higher during pregnancy than postpartum in both healthy control subjects and patients with AS. In contrast to Treg cells in healthy pregnant women, Treg cells in pregnant women with AS secreted only small amounts of interleukin-10 and showed lower suppression of tumor necrosis factor alpha and interferon-gamma secretion by CD4+CD25- Teff cells. At the postpartum time point, proinflammatory cytokine levels in the Treg/Teff cell cocultures and Teff cell monocultures were significantly higher in patients with AS than in healthy controls. CONCLUSION: Pregnancy influenced the expansion and cytokine secretion of Treg cells in both patients with AS and control subjects. However, the Treg cells of pregnant patients with AS failed to support an antiinflammatory cytokine milieu, thereby possibly contributing to the persistent disease activity of AS during pregnancy.

Wear particles and surface topographies are modulators of osteoclastogenesis in vitro

Prosthetic and osteosynthetic implants from metal alloys will be indispensable in orthopedic surgery, as long as tissue engineering and biodegradable bone substitutes do not lead to products that will be applied in clinical routine for the repair of bone, cartilage, and joint defects. Therefore, the elucidation of the interactions between the periprosthetic tissues and the implant remains of clinical relevance and several factors are known to affect the longevity of implants. Within this study, the effects of metal particles and surface topography on the recruitment of osteoclasts was investigated in vitro in a coculture of osteoblasts and bone marrow cells. The cells were grown in the presence of particles of different sizes and chemical composition or on metal discs with polished or sandblasted surfaces, respectively. At the end of the culture, newly formed osteoclasts were counted. Osteoclastogenesis was reduced when particles were added directly to the coculture. The effect depended on the size of the particles, small particles exerting stronger effects than larger ones. The chemical composition of the particles, however, did not affect the development of osteoclasts. In cocultures grown on sandblasted surfaces, osteoclasts developed at higher rates than they did in cultures on polished surfaces. The data demonstrate that wear particles and implant surfaces affect osteoclastogenesis and thus may be involved in the induction of local bone resorption and the formation of osteolytic lesions, leading eventually to the loosening of orthopedic implants.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood-brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1(+)) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two (Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y(+)Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma

PURPOSE In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

Use of Genetically Modified Glial Cells Overexpressing Laminin α1-Chain Peptides in Neurite Outgrowth Studies

1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.

Co-culture of Notochordal Cells with Nucleus pulposus and Annulus fibrosus cells under normoxia and hypoxia

Introduction Notochordal cells (NC) are shifted back into focus due to their apparent action of activating other disc cells via indirect release of yet unknown factors into the medium (conditioned medium = CM).1,2 Recent evidence confirms the results from the late 1990s.3,4 Here, we test porcine (p) NC cultured in 3D and the influence of adding serum or using serum-free medium onto the culture on NC cells and its stimulating effects for subsequent coculture with primary bovine (b) nucleus pulposus (bNPC) and annulus fibrous cells (bAFC). Materials and Methods Primary pNC, bNPC, and bAFC were isolated from porcine tails (< 6-12 months age) or bovine tails (∼1 year age), which were obtained from the food chain (N = 4 repeats) within 4 hours postmortem. All cells were seeded into 1.2% alginate, each with a density of 4 × 106/mL. NC were then either cultured for 7 days in serum free medium (SFM = Dulbecco modified eagle medium [DMEM] supplied with ITS+, 50 µg/mL vitamin C and nonessential amino acids) or DMEM + 10% fetal calf serum (FCS). CM was produced from NC collecting 4 mL SFM and keeping approximately 30 beads for 7 days. Then, a coculture was set up in SFM for 14 days using indirect cell-cell contact (culture insert, high density pore, 0.4 µm) using a 50:50% ratio5 of pNC:bNP or bAF, or by addition of CM, respectively. The cell activity, glycosaminoglycan per DNA (GAG/DNA) ratio, and real-time RT-PCR of IVD relevant genes were monitored. Mass spectrometry was performed on the SFM and the cocultured medium as well as the CM of the pNC to identify possible key cytokines to the stimulatory effects. Results The results for cell activity confirmed that pNC are highly responsive on the nutritional condition in the culture (K-W test, p = 0.048) after 7 days of coculture. bNPC and bAFC did not respond significantly different to coculture or addition of CM with respect to cell activity. However, GAG/DNA ratio of pNC was significantly upregulated if they were initially pre-exposed to FCS and in coculture with bNPC after 14 days, for both normoxia and hypoxia (K-W, p = 0.03). The bNPC were stimulated by both, 1:1 coculture with pNC but also by addition of CM only, which resulted in approximately 200% increased GAG/DNA values relative to the day 0 state. However, this doubling of the GAG/DNA ratio was nonsignificant after 14 days. The aggrecan/collagen type 2 ratio as quantified from real-time RT-PCR pointed to a beneficial state of the bNPC if cultured either in indirect coculture with pNC or by the addition of CM (Fig. 1). The mass spectrometric analysis of the CM revealed that there was connecting tissue growth factor present (CTGF) among the cytokine CLC11, a cytokine that has been found to be expressed in skeletal tissues including bone marrow and chondrocytes among other factors that might have immunoregulatory and cell proliferative functions.

Human IgA Fc receptor FcαRI (CD89) triggers different forms of neutrophil death depending on the inflammatory microenvironment

FcαRI (CD89), the human Fc receptor for IgA, is highly expressed on neutrophil granulocytes. In this study, we show that FcαRI induces different forms of neutrophil death, depending on the inflammatory microenvironment. The susceptibility of inflammatory neutrophils from sepsis or rheumatoid arthritis toward death induced by specific mAb, or soluble IgA at high concentrations, was enhanced. Although unstimulated cells experienced apoptosis following anti-FcαRI mAb stimulation, preactivation with cytokines or TLR agonists in vitro enhanced FcαRI-mediated death by additional recruitment of caspase-independent pathways, but this required PI3K class IA and MAPK signaling. Transmission electron microscopy of FcαRI-stimulated cells revealed cytoplasmic changes with vacuolization and mitochondrial swelling, nuclear condensation, and sustained plasma membrane. Coculture experiments with macrophages revealed anti-inflammatory effects of the partially caspase-independent death of primed cells following FcαRI engagement. Our data suggest that FcαRI has the ability to regulate neutrophil viability and to induce different forms of neutrophils depending on the inflammatory microenvironment and specific characteristics of the ligand-receptor interactions. Furthermore, these findings have potential implications for FcαRI-targeted strategies to treat neutrophil-associated inflammatory diseases.

In human basophils, IL-3 selectively induces RANKL expression that is modulated by IgER-dependent and IgER-independent stimuli

BACKGROUND Receptor activator of NF-κB ligand (RANKL) is expressed as either surface (hRANKL1, hRANKL2) or soluble (hRANKL3) form. RANKL is involved in multifaceted processes of immunoregulation and bone resorption such as they occur in rheumatoid arthritis (RA). Interestingly, activated basophils, which are effector cells in allergic inflammation, contribute to the progress of collagen-induced arthritis (CIA), a mouse model for RA. Here, we investigate under which conditions human basophils express RANKL. METHODS Among other stimuli, basophils were cultured with IL-3 alone. Alternatively, as a secondary stimulus, IgER-dependent or IgER-independent agents were added simultaneously either with IL-3 or after prolonged IL-3 culturing. Expression of RANKL protein and mRNA was analyzed by flow cytometry, ELISA, and real-time PCR. A coculture system was applied to investigate biological activity of basophil-derived RANKL. RESULTS We show that in human basophils, IL-3 but no other stimulus induces de novo expression of soluble and surface RANKL, of which the latter enhances survival of MoDC. Upon simultaneous stimulation, IgER cross-linking reduces surface RANKL expression, while IgER-independent stimuli have no effect. This is in contrast to consecutive stimulation, as triggering with both IgER-dependent and IgER-independent stimuli enhances RANKL expression, particularly in its soluble form. Real-time PCR analysis shows that RANKL expression is mainly regulated at the mRNA level. CONCLUSION This study identifies IL-3 as a potent inducer of RANKL expression in human basophils, suggesting them to interact with bone physiology and activation of immune cells. IgER-dependent and IgER-independent stimuli modulate the IL-3-mediated RANKL expression in a time- and stimulus-dependent fashion.