Carbon nanotubes have a deleterious effect on the nose: the first in vitro data.

BACKGROUND: The information currently available concerning carbon nanotubes toxicity is disturbing and conflicting. Moreover, little is known about their effect on the nasal cavities, which are the first target for nanoparticles. MATERIAL AND METHOD: We investigated the cytotoxicity (50 to 0.5 microg/mL) of double-walled carbon nanotube with two independent tests (MTT, Wst-1) on normal human nasal epithelial cells after 12-day exposure (control untreated nasal cells and A549). Nasal cell differentiation function, oxidative stress, the morphological features of cells in contact with DWCNTs and the localizations of the latter were also investigated. RESULTS: Exposure revealed a dose-dependent decrease in cell metabolic activity and cell growth. In nearly all conditions, normal human nasal epithelial cells were more sensitive than malignant ones. Even with both tests, the cytotoxic threshold dose could not be accurately determined because of dye adsorption by DWCNTs. Nasal cells showed stronger cytokeratin 7 and persistent UEA-I immunostaining. Cytokeratin 19 production was increased at 25 microg/mL and mucus production was stimulated from 0.5 microg/mL. A significant increase in Reactive Oxygen Species was observed from 25 microg/mL. The cell plasma membrane showed several holes and DWCNTs were present in the cytoplasm. CONCLUSION: DWCNTs seem to have a deleterious effect on nasal cells after 12-day exposure.

In vitro endothelialized ePTFE prostheses: clinical update 20 years after the first realization.

The replacement of arteries with synthetic vascular prostheses often leads to failure when small-diameter or low-flow locations are concerned, due in part to the thrombogenicity of the graft surface. In order to improve long-term patency of these grafts, the concept of endothelial cell seeding has been suggested, the composite structure resulting from the combination of biologically active cells to prosthetic materials thus creating more biocompatible vascular substitutes. To achieve endothelialization of synthetic grafts, previous efforts aimed at "one-stage" procedure in the 1980's seemed clinically feasible but results of reported clinical trials were controversial and mostly disappointing. An alternative method is an in vitro complete and preformed endothelial lining at the time of implantation: the "two-stage" procedure which implies harvest and culture of autologous endothelial cells. Up to date, the latter approach demonstrated its superiority in terms of significantly increased patency of the grafts that underwent endothelialization several years earlier.

Regulation by PGE2 of IL-2, IL-3 and IFN production by cortico-resistant thymocytes.

We have investigated the role of prostaglandin E2 (PGE2) in the regulation of cytokine release (IL-2, IL-3 and IFN) by cortico-resistant thymocytes (CRT) stimulated or not through the T-cell antigen receptor by an anti-CD3 monoclonal antibody (mAb). CRT were found to spontaneously produce IL-2 and IL-3 on day 4 of culture, but not IFN. After activation with an anti-CD3 mAb, the maximal levels for IL-2 and IFN were observed on day 1 and for IL-3 on day 4. Addition of PGE2 inhibits IL-2 production and has no effect on IFN production. Indomethacin, an inhibitor of the cyclooxygenase pathway, enhanced both IL-2 and IFN production. In contrast, IL-3 secretion by anti-CD3 activated CRT was up-regulated by PGE2, and its level was decreased in the presence of indomethacin in both stimulated or unstimulated cells. As has been observed with PGE2, forskolin which activates adenylate cyclase increases the IL-3 level. Thus PGE2 may interfere in the process of thymocyte proliferation and/or differentiation by regulating differentially the interleukin production.

Effect of PGE2 on the cell surface molecule expression in PMA treated thymocytes.

PGE2 is produced by cells of the thymic microenvironment. The effects of PGE2 are mediated by cAMP through binding to its intracellular receptor protein kinase A (PKA). Phorbol 12-myristate 13-acetate (PMA) is known to modulate CD molecule expression on thymocytes, probably through activation of protein kinase C (PKC). We have hypothesized that cross-talk between these two signalling pathways may affect modulation of the CD molecules on the cell surface of thymocytes. For this purpose, we compare the effects of PMA alone or combined with PGE2 on CD3, CD4 and CD8 expression on mouse thymocytes by flow-cytometric analysis. PMA treatment almost completely abolished CD4 expression and slightly decreased CD3 and CD8 expression. PGE2 alone did not change the CD3, CD4 and CD8 molecule expression. Combined with PMA, PGE2 can overcome the decrease induced by PMA of the CD3 expression and partially reduced the disappearance of the CD4 molecule. On the other hand PGE2 accelerated the loss of CD8 molecule expression. These events occurred only in CD4+ CD8+ immature thymocytes. An analogue of cAMP (dibutyryl cAMP) mimics the effect of PGE2, but not Br-cGMP. This differential regulation by PGE2 of the CD molecule expression on immature thymocytes may provide additional evidence on the role of PGE2 during the process of thymic differentiation.

Detection of CD1 positive cells in the peripheral blood of patients suffering from cancer-associated malnutrition.

The immunological phenotyping of peripheral blood mononuclear cells (PBMC) was assayed in a series of 22 patients suffering from severe cancer-associated malnutrition. A marked decrease of the T-lymphocyte subsets (CD3+, CD4+, CD8+) and of the CD20+ B lymphocytes occurred; there was however an increased percentage of monocytes but their absolute number was normal. Interestingly, 5% of the PBMC expressed "activated T-cell antigens". The specificity of two different monoclonal antibodies (MoAbs) towards CD1 epitopes (OKT6 and D47) was assessed by indirect immunofluorescence (IIF): about 5% of CD1+ thymocytes were detected with no antigen (Ag) cross reactivity with the small subset of activated T cells. It is hypothesized that some relationship may exist between such cells and malnutrition and/or cancer.

Effect of prostaglandin E2 on cytotoxic activity and granzyme A protease release by murine adherent IL-2 activated killer cells.

The effects of prostaglandin E2 (PGE2) have been studied on a highly purified population of murine IL-2 activated killer cells obtained by selecting plastic-adherent splenocytes (AK cells) after incubation with high doses of recombinant IL-2. AK cells were highly cytotoxic for YAC-1 target cells. The cytotoxic activity was detectable at one hour after initiation of the cytotoxic assay and then increased with time. Cytotoxic activity of AK cells was inhibited by the addition of PGE2 or forskolin during the cytotoxic assay. When AK cells were generated in the presence of PGE2, the yielding cytotoxic activity was lower than the one expressed by "regular" AK cells but were insensitive to the inhibitory effect of PGE2 even if their lytic capability was still suppressed by forskolin. The presence of PGE2 during the AK cell culture had no effect on the cellular proliferation. Moreover, using tetrazolium-based colorimetric assay which reflects the cellular activation, it was observed that AK cells cultured in presence of PGE2 had an increased capacity to cleave the tetrazolium salt to formazan. Since the cytotoxic activity of killer cells is related to expression of serine esterase enzymes we evaluated the effects of PGE2 on serine esterase (Granzyme A) release after one hour of incubation of AK cells either alone or in presence of PGE2, YAC-1 cells or both. We observed that (i) AK cells spontaneously release granzyme A, (ii) the level of granzyme A was significantly increased when AK cells were incubated either with YAC-1 cells or PGE2 but did not change when YAC-1 cells and PGE2 were both associated with AK cells.

Natural killer activity, production of interferon-gamma and interleukin 2 in immunodeficient C57BL/6 mice injected with RadLV-Rs viral complex.

Injection of the RadLV-Rs, a viral complex originally obtained from radio-induced thymic lymphosarcomas, into C57BL/6 mice induces a massive enlargement of spleen and lymph nodes. T- and B cell-proliferating populations display severe deterioration of their immune functions, resulting in death of the animals within three months. In contrast, the experiments reported here indicate that in such animals the natural killer (NK) activity is increased for about 2 months after viral injection. Interleukin 2 production is dramatically decreased, whereas interferon-gamma production is increased to twice the control value and thereafter decreases concomitantly with NK activity. This suggests that in RadLV-Rs-injected mice, the high NK activity is related to interferon-gamma production.

Production of interleukin 3 by spleen cells in immunodeficient C57BL/6 mice after injection of RadLV-Rs viral complex.

Injection of RadLV-Rs viral complex to C57BL/6 mice results in massive enlargement of lymphoid organs. The polyclonal T and B populations which proliferate in spleen and lymph nodes display severely impaired immune functions. Several data suggest that development of this immunodeficiency syndrome is dependent on the presence of T cells whose functions are progressively altered. In contrast, the erythro-myeloid populations and the stem cell compartment are not deeply modified, suggesting that the production of the lymphokines involved in their regulation is not altered. Interleukin 3 (IL-3) is mainly involved in this regulation. Thus, the purpose of the present experiment was to evaluate the IL-3 production: in RadLV-Rs injected mice IL-3 production is decreased at the cellular level but if it is evaluated by taking into account the increase of spleen cellularity, it is initially decreased and later on, above the normal value.

Attachment of human endothelial cells to polyester vascular grafts: pre-coating with adhesive protein assemblies and resistance to short-term shear stress.

Cardiovascular prosthetic bypass grafts do not endothelialize spontaneously in humans, and so they pose a thrombotic risk. Seeding with cells improves their performance, particularly in small-caliber applications. Knitted tubular polyethylene-terephthalate (PET) vascular prostheses (6 mm) with commercial type I collagen (PET/Co) were modified in the lumen by the adsorption of laminin (LM), by coating with a fibrin network (Fb) or a combination of Fb and fibronectin (Fb/FN). Primary human saphenous vein endothelial cells were seeded (1.50 × 10(5)/cm2), cultured for 72 h and exposed to laminar shear stress 15 dyn/cm(2) for 40 and 120 min. The control static grafts were excluded from shearing. The cell adherence after 4 h on PET/Co, PET/Co +LM, PET/Co +Fb and PET/Co +Fb/FN was 22%, 30%, 19% and 27% of seeding, respectively. Compared to the static grafts, the cell density on PET/Co and PET/Co +LM dropped to 61% and 50%, respectively, after 120 min of flow. The cells on PET/Co +Fb and PET/Co +Fb/FN did not show any detachment during 2 h of shear stress. Pre-coating the clinically-used PET/Co vascular prosthesis with LM or Fb/FN adhesive protein assemblies promotes the adherence of endothelium. Cell retention under flow is improved particularly on fibrin-containing (Fb and Fb/FN) surfaces.

Whatever their differentiation status, human progenitor derived - or mature - endothelial cells induce osteoblastic differentiation of bone marrow stromal cells.

Association of the bone-forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co-culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular-like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co-cultured with human endothelial progenitor cells in order to assess whether progenitor-derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co-culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non-orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co-cultures were undertaken: HBMSCs-PDECs, HBMSCs-HUVECs, HOPs-PDECs, HOPs-HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co-culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real-time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono- and co-cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering.

Polyelectrolyte multilayer films allow seeded human progenitor-derived endothelial cells to remain functional under shear stress in vitro.

There is considerable interest in making multilayer films for various applications, among which are cell contacting biomaterials, allowing new opportunities to prepare functionalized biomaterials. In this study we have explored the capability of poly(sodium-4-styrene sulfonate)/poly(allylamine hydrochloride) polyelectrolyte multilayer films (PMFs) as functional coatings for human progenitor-derived endothelial cells (PDECs), since the latter are a potential source of endothelial-type cells to be used in bioartificial vascular substitutes. We performed investigations with PDECs derived from peripheral blood and characterized as endothelial cells. After forming a confluent monolayer on PMFs they were exposed to laminar pulsatile physiological shear stress. We investigated whether PDECs were able to withstand shear stress and to respond at the mRNA (microarray analysis) and protein levels (thrombomodulin and tissue factor functional activity), in comparison with collagen I and fibrin glue used as controls. After shear stress the PDECs remained spread on the substrates, with a resulting increase in the number of expressed genes. Considering the functional significance of our findings for the regulation of coagulation and fibrinolytic factors, mRNA tissue plasminogen activator and thrombomodulin, profibrinolytic and thrombin inhibiting respectively, were overexpressed in PDECs after 6h shear stress. von Willebrand factor showed down-regulation, while tissue factor was up-regulated. We can speculate that PMFs could favour anti-thrombogenic activity by PDECs because activated protein C generation, measuring thrombomodulin activity, was particularly high on PMFs, but unchanged after 6h shear stress. Thus, PMFs could represent suitable coatings able to provide functional surfaces for endothelialization with PDECs.

Heterogeneity of murine adherent interleukin-2-activated killer cells. Differential effect of prostaglandin E2 and forskolin.

We have studied the relationship between cytotoxic activity, size and granularity of murine interleukin-2-activated adherent killer cells issued from spleen cells cultured with high levels of IL-2. The effects of prostaglandin E2 (PGE2) and forskolin upon these cells were assessed. All adherent spleen cells obtained after 5 days of culture were large granular lymphocytes but presented a heterogeneity in size and granularity. After fractionation on a discontinuous-density Percoll gradient, four cellular subpopulations were isolated. Fluorescence-activated cell sorting analysis showed that cells of the lightest fraction (F1) were the largest, while the cells found in the heaviest fraction (F4) were much more granular than the cells collected in the two intermediate fractions (F2 and F3). The serine esterases level was higher in F4 than in unfractionated cells and diminished to about 40% in cells of fractions F2 and F3, which expressed a cytotoxic activity against YAC-1 cells higher than that in unfractionated cells or in F1 or F4, which presented the lowest cytotoxic activity. When AK cells were cultured for 48 h in the presence of either PGE2 or forskolin, which induce an intracellular increase of cAMP, we observed that PGE2 (1 microM) inhibited the cytotoxic activity, but surprisingly forskolin (2 microM) exerted a stimulating effect on the induction of cytotoxic activity. After fractionation on a discontinuous Percoll gradient we observed the same cellular distribution among PGE2 or forskolin-treated or -untreated cells, but PGE2 induced an increase of size and granularity. This effect of PGE2 was more potent on the cells collected in F4. However this variation of granularity was not associated with any variation in the serine esterase level. The cytotoxic activity of PGE2- or forskolin-treated cells did not present any significant variation relative to the control for cells collected in F2 and F3; on the other hand, forskolin-treated cells collected in F4 showed a significantly higher cytotoxicity than did the corresponding untreated or PGE2-treated cells.

Nitric oxide production in murine spleen cells: role of interferons and prostaglandin E(2) in the generation of cytotoxic activity.

The production of nitric oxide (NO) was measured in cultures of spleen cells stimulated by lipopolysaccharide (LPS), IL-2 or LPS + IL-2. We observed that NO synthesis is increased by IFN-gamma but inhibited by IFN-alpha/beta. This is not the case when IL-2 is present in the cultures, since interferons play a minor role in the regulation of the NO production. When IL-2 and LPS were associated in the cultures, the IFN-alpha/beta role seems more important than that of IFN-gamma. PGE(2) inhibits NO production in LPS supplemented cultures but has a slight effect in the presence of IL-2 and no effect with IL-2 + LPS. 3-isoButyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, induces a decrease of IFN production. In the presence of H-7, an inhibitor of protein kinase C (PKC), NO production is reduced when the cultures are supplemented by LPS or IL-2 but not when IL-2 and LPS are both added. H-7 also reduced IFN production. In the presence of N(G)-monomethyl-L-arginine (N-MMA), an inhibitor of NO synthesis, IFN production was increased, with no change in the cytotoxic activity. Hence, interferons regulate NO production by mouse spleen cells and, in return, NO modulates the generation of IFN.

Effect of BNU treatment on leukaemogenesis in lethally irradiated AKR mice restored with bone-marrow and spleen cells.

The leukaemogenic effect of N-butyl-N-nitrosourea (BNU) was studied in normal and thymectomized AKR mice which were lethally irradiated and restored with either bone-marrow (BM) or spleen cells from (AKR X AKR/T1ALD)F1 donors. In some instances T1ALD thymic cells were added to the restorative inoculum. It was possible to determine the origin of the leukemic cells by the metacentric marker chromosomes of T1ALD. The T- or B-cell characteristics were further ascertained by the cytotoxicity test for theta antigen and the EAC rosette test. All leukaemias whether thymic (TLS) or extra-thymic (ETL), developed from donor bone-marrow or spleen cells and never from the injected thymic cells. In non-thymectomized animals BNU increased the percentage of TLS and shortened their latency. Most of TLS which occurred after BNU treatment of BM-restored mice were theta-negative whereas the majority of TLS which occurred in controls and in spleen-restored animals were theta-positive. This suggests that during their maturation process BM-derived T precursors transit through a theta-negative compartment. This compartment does not reach a similar size during the maturation process of the spleen-derived precursors. Adding thymic cells to the restorative inoculum enhanced leukaemogenesis and suppressed theta-negative TLS in BM-restored mice. Thymectomized mice, restored either by BM or spleen, had a low incidence of ETL which was not significantly increased by BNU treatment except in the case of mice restored with spleen cells. The leukaemic cells of one ETL were theta-positive whereas all the other leukaemias had no detectable T or B marker. The percentage of ETL was higher in thymectomized mice treated with BNU alone than in those previously subjected to irradiation and restoration. These results strongly suggest that a theta-negative T precursor could be involved in extra-thymic leukaemogenesis but the possible involvement of a B precursor cannot be rule out unless experiments are carried out with specific markers of T- and B-cell sub-classes.

Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons.

Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic activity against the YAC-1 lymphoma cell line and to a lesser extent against P815 mastocytoma cells. The association of IL-2 and LPS had an additive effect on induction of cytotoxicity. The IL-2-induced cytotoxic activity lasted for the whole of the culture; however, the addition of LPS at the initiation of the culture increased the cytotoxic activity during its the early phase, the increment being followed by a fall of lytic activity after 72 h of culture. Assessment of interferon (IFN) in the culture supernatants showed (a) a production of IFN gamma by IL-2-supplemented cultures, (b) a more potent IFN production by cultures treated with IL-2 plus LPS (including 20% IFN alpha/beta, (c) and that indomethacin (1 microM) potentiated the effect of either IL-2 or LPS used alone but did not significantly increase the cytotoxic activity of cultures treated with IL-2 plus LPS (the one that produced a high level of IFN). When cultures were treated by an anti-IFN gamma antibody we observed no change in the cytotoxic activity; however, in the presence of anti-IFN alpha/beta serum the cytotoxic activity of cultures treated with IL-2 plus LPS was inhibited after 24 h but stimulated after 72 h. When cultures treated with IL-2 plus LPS were supplemented with both indomethacin and anti-IFN alpha/beta the cytotoxic activity assessed after 72 h of culture was maintained at the same level as that of IL-2-treated cultures, hence the fall after 72 h of the cytotoxicity of cultures initiated in the presence of LPS alone was affected by both the immune serum and the cyclooxygenase inhibitor. Altogether these data show that when splenocytes are cultured for more than 72 h in the presence of IL-2 and LPS their cytotoxic activity decreases, and it is likely that this diminution is linked to the endogenous production of prostaglandin E2 and INF alpha/beta.

Effect of PGE2 on thymocyte proliferation induced by Con A or IL-4 + PMA.

Prostaglandin E2 (PGE2) is known to inhibit peripheral T-lymphocyte and thymocyte proliferation activated by antigens, mitogens or anti-CD3 antibodies. In this study, we have investigated, the effect of PGE2 on thymocyte proliferation induced by the combination of IL-4 plus PMA. PGE2 inhibits the proliferation of thymocytes activated by ConA, whatever the culture period; in contrast PGE2 shifts the kinetics of thymocyte proliferation after stimulation by IL-4 plus PMA, but does not sustain the proliferation beyond day 3. This effect depends upon cell density, IL-4 concentration and on the time that PGE2 is added to the culture. By use of the cAMP inducer, forskolin, or a cAMP analog, db-cAMP, we observed the same results, PGE2 increases the proliferation of CD8+ corticoresistant thymocytes (CRT) activated by IL-4 plus PMA, but inhibits that of CD4+ CRT. These results suggest that PGE2 can regulate thymocyte proliferation differently according to the activation pathway and the thymic subpopulations.

Effect of LTB4 on the inhibition of natural cytotoxic activity by PGE2.

NK activity is regulated by arachidonic acid metabolites. More precisely PGE2 and LTB4 decreases and increases respectively non-MHC-restricted cytotoxicity in humans. We have observed similar data in mice since NK activity was inhibited by PGE2 (10(-6) to 10(-8) M) and enhanced by LTB4 (10(-8) to 10(-12) M). On the other hand when PGE2 and LTB4 were combined during the same assay the lysis percentage was smaller than the one which was induced by PGE2 alone. Because PGE2 increases intracellular cyclic AMP and that LTB4 augments cyclic GMP we used a cAMP inducer (forskolin) and a cGMP analogue (8 Br-cGMP) instead of eicosanoids and we observed similar data (i.e., a decrease of natural killing) as when PGE2 was combined with LTB4. When splenocytes are cultured for 1-4 days alone, cytotoxic activity decreases unless they are cultured in the presence of indomethacin. Cytotoxic activity of spleen cells cultured in the presence of PGE2 or LTB4 is respectively decreased or increased. However, splenocytes that were cultured alone for at least 24 hr were no longer sensitive to inhibition by PGE2 but were still PGE2-sensitive when cultured in the presence of LTB4.

Production of thymic cells by mouse spleen and bone marrow.

Karyotype difference between the AKR/TIALD strain (T1) that bears 2 metacentric markers, and the AKR strain (no marker) was used to follow the thymic repopulation of lethally irradiated (AKR X T1) F1 hybrids restored by AKR bone marrow (BM) or spleen cells. Eleven days following radiation exposure, 40-50% of the thymic cells were BM-derived in the mice restored with BM cells whereas spleen-derived cells remained below 10% in those restored with spleen cells. The thymic repopulation by spleen-derived elements was enhanced either by injecting a larger munber of spleen cells or by adding thymic cells to the spleen inoculum; however in both cases the appearance of the spleen-derived karyotypes still required a delay of about 11 days. The thymic cells could either recruit thymic precursor cells or trigger their multiplication. On the opposite, it has not been possible to demonstrate a favorable effect of the injected thymic cells on the repopulation of the thymus by BM-derived elements.