Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel.

Samples of AISI 316L austenitic stainless steel were subjected either to grinding and polishing procedure, or to grinding and then low temperature glow-discharge nitriding treatment, or to grinding, nitriding and subsequently coating with collagen-I. Nitrided samples, even if only ground, show a higher corrosion resistance in PBS solution, in comparison with ground and polished AISI 316L. Biocompatibility was evaluated in vitro by incubating the samples with either peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC), tested separately or in co-culture. HUVEC-PBMC co-culture and co-incubation of HUVEC with PBMC culture medium, after the previous incubation of PBMC with metallic samples, allowed to determine whether the incubation of PBMC with the different samples might affect HUVEC behaviour. Many biological parameters were considered: cell proliferation, release of cytokines, matrix metalloproteinases (MMPs) and sICAM-1, gelatinolytic activity of MMPs, and ICAM-1 protein expression. Nitriding treatment, with or without collagen coating of the samples, is able to ameliorate some of the biological parameters taken into account. The obtained results point out that biocompatibility may be successfully tested in vitro, using cultures of normal human cells, as blood and endothelial cells, but more than one cell line should be used, separately or in co-culture, and different parameters should be determined, in particular those correlated with inflammatory phenomena.

In vitro response of human peripheral blood mononuclear cells to AISI 316L austenitic stainless steel subjected to nitriding and collagen coating treatments.

Surface modification treatments can be used to improve the biocompatibility of austenitic stainless steels. In the present research two different modifications of AISI 316L stainless steel were considered, low temperature nitriding and collagen-I coating, applied as single treatment or in conjunction. Low temperature nitriding produced modified surface layers consisting mainly of S phase, which enhanced corrosion resistance in PBS solution. Biocompatibility was assessed using human peripheral blood mononuclear cells (PBMC) in culture. Proliferation, lactate dehydrogenase (LDH) levels, release of cytokines (TNF-α, IL-1ß, IL-12, IL-10), secretion of metalloproteinase (MMP)-9 and its inhibitor TIMP-1, and the gelatinolytic activity of MMP-9 were determined. While the 48-h incubation of PBMC with all the sample types did not negatively influence cell proliferation, LDH and MMP-9 levels, suggesting therefore a good biocompatibility, the release of the pro-inflammatory cytokines was always remarkable when compared to that of control cells. However, in the presence of the nitrided and collagen coated samples, the release of the pro-inflammatory cytokine IL-1ß decreased, while that of the anti-inflammatory cytokine IL-10 increased, in comparison with the untreated AISI 316L samples. Our results suggest that some biological parameters were ameliorated by these surface treatments of AISI 316L.

Role of vitamin D derivatives in intestinal tissue of patients with inflammatory bowel diseases.

BACKGROUND AND AIM: The adhesion molecule expression and matrix metalloproteinases (MMPs) are proposed to be major factors for intestinal injury mediated by T cells in (IBD) and are up-regulated in intestinal mucosa of IBD patients. To investigate the effect of vitamin D derivatives on adhesion molecules and MMPs in colonic biopsies of IBD patients. METHODS: Biopsies from inflamed and non-inflamed tract of terminal ileum and colon and PBMC from the same IBD patients were cultured with or without vitamin D derivatives. MMP activity and adhesion molecule levels were determined. RESULTS: 1,25(OH)2D3 and ZK 191784 significantly decrease ICAM-1 protein levels in the biopsies obtained only from the inflamed region of intestine of UC patients, while MAdCAM-1 levels decrease in the presence of 1,25(OH)2D3 in the non-inflamed region, and, in the presence of ZK, in the inflamed one. In CD patients 1,25(OH)2D3 and ZK decrease ICAM-1 and MAdCAM-1 in the biopsies obtained from the non-inflamed and inflamed regions, with the exception of ICAM-1 in the inflamed region in the presence of 1,25(OH)2D3. The expression of MMP-9, MMP-2, and MMP-3 decreases in the presence of vitamin D derivatives in UC and CD with the exception of 1,25(OH)2D3 that does not affect the levels of MMP-9 and MMP-2 in CD. Vitamin D derivatives always affect MMP-9, MMP-2 and ICAM-1 in PBMC of UC and CD patients. CONCLUSIONS: Based on the increased expression of ICAM-1, MAdCAM-1 and MMP-2,-9,-3 in IBD, our study suggests that vitamin D derivatives may be effective in the management of these diseases.

The novel vitamin D analog ZK191784 inhibits prostate cancer cell invasion.

BACKGROUND: Low serum levels of 1,25(OH)(2)D(3) (1,25D), have been associated with aggressive biologic behavior of prostate cancer (PCa). In the present study, we examined the effects of 1,25D and its novel, low-calcemic analog ZK191784 (ZK) on matrix metalloproteinases (MMPs), as well as on intercellular adhesion molecule-1 (ICAM-1) protein levels in human PCa cell lines LNCaP and DU-145. MATERIALS AND METHODS: Cells were incubated with either vehicle (control), 1,25D or ZK. MMP-2 and MMP-9 activity was determined by gelatin zymography, while ICAM-1 levels were assessed by Western blot analysis and immunocytochemistry. RESULTS: Compared to the controls, 1,25D and ZK caused a marked dose-dependent decrease in the gelatinolytic activity of the MMPs under study, particularly when ZK was used. Likewise, ICAM-1 was down-regulated in the cells incubated with 1,25D or ZK. CONCLUSION: Vitamin D analogs appear to be involved in the regulation of extracellular MMP activity and membrane adhesion molecule expression. Further studies, both in vitro and in vivo, are needed to define their role as potential therapeutic tools.

Down-regulation of adhesion molecules and matrix metalloproteinases by ZK 156979 in inflammatory bowel diseases.

Intracellular adhesion molecules and matrix metalloproteinases (MMPs) are up-regulated in intestinal mucosa of patients with inflammatory bowel diseases (IBD), i.e. ulcerative colitis (UC) or Crohn's disease (CD). Our aim was to verify whether the vitamin D analogue ZK 156979 (ZK) down-regulates adhesion molecules, and decreases MMPs production by PBMC of IBD patients. ICAM-1 and LFA-1 levels increase, when PBMC were incubated with PHA or LPS or TNF-alpha, and decrease when these substances were used in combination with ZK. MMPs activity increases incubating the cells with PHA or LPS or TNF-alpha. MMP-9 decreases when ZK was used in association, while MMP-2 decreases only when ZK was used in combination with anti-TNF-alpha. Our results suggest that the down-regulation of ICAM-1 and LFA-1 on PBMC and the inhibition of MMP-9 activity by ZK could provide a potential role of this low calcemic vitamin D derivative in future strategies in IBD therapy.

In vitro biocompatibility evaluation of surface-modified titanium alloys.

The present work is aimed to evaluate the effects of a surface modification process on the biocompatibility of three vanadium-free titanium alloys with biomedical applications interest. Chemical composition of alloys investigated, in weight %, were Ti-7Nb-6Al, Ti-13Nb-13Zr, and Ti-15Zr-4Nb. An easy and economic method intended to improve the biocompatibiblity of these materials consists in a simple thermal treatment at high temperature, 750 degrees C, in air for different times. The significance of modification of the surface properties to the biological response was studied putting in contact both untreated and thermally treated alloys with human cells in culture, Human Umbilical Vein Endothelial Cells (HUVEC) and Human Peripheral Blood Mononuclear Cells (PBMC). The TNF-alpha release data indicate that thermal treatment improves the biological response of the alloys. The notable enhancement of the surface roughness upon oxidation could be related with the observed reduction of the TNF-alpha levels for treated alloys. A different behavior of the two cell lines may be observed, when adhesion molecules (ICAM-1 and VCAM-1 in HUVEC, ICAM-1, and LFA-1 in PBMC) were determined, PBMC being more sensitive than HUVEC to the contact with the samples. The data also distinguish surface composition and corrosion resistance as significant parameters for the biological response.

Vitamin D derivatives induce apoptosis and downregulate ICAM-1 levels in peripheral blood mononuclear cells of inflammatory bowel disease patients.

BACKGROUND: Lymphocytes are crucial in the pathogenesis of inflammatory bowel disease (IBD) and are an important target for drug development. Our aim was to verify whether 2 vitamin D derivatives, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and EB 1089, could induce cell apoptosis and affect cell-cell interaction by regulating adhesion molecule levels. METHODS: Peripheral blood mononuclear cell (PBMC) proliferation was studied by [3H]thymidine incorporation and apoptosis was determined using an enzyme-linked immunosorbent assay (ELISA) kit. (Poly(ADP-ribose)polymerase (PARP) cleavage, caspase-3, and ICAM-1 protein levels were determined by Western blot analysis. RESULTS: Our results indicate that 1,25(OH)2D3 or EB 1089 or anti-TNF-alpha (infliximab) induce apoptosis in PBMC obtained from healthy subjects. In IBD patients apoptosis is induced by vitamin D derivatives and by anti-TNF-alpha only in CD patients. Caspase-3 activation and PARP cleavage are registered when PBMC were treated with vitamin D derivatives. ICAM-1 levels remarkably increase when PBMC was incubated with lipopolysaccharide (LPS) or TNF-alpha. The treatment with the vitamin D derivatives, alone or in combination with LPS or TNF-alpha, significantly decreases ICAM-1 levels both in healthy subjects and IBD patients. In HUVEC cocultured with PBMC, previously incubated with LPS or TNF-alpha associated with 1,25(OH)2D3, ICAM-1 levels decrease both in healthy subjects and IBD patients. CONCLUSIONS: 1,25(OH)2D3 and EB 1089 inhibit PBMC proliferation, induce apoptosis in PBMC of healthy subjects and IBD patients, and affect ICAM-1 expression on PBMC and on HUVEC cocultured with PBMC, suggesting that the ICAM-1 downregulation could provide a new target for controlling the recruitment of leukocytes at the sites of inflammation in IBD.

The Vitamin D analogue TX 527 blocks NF-kappaB activation in peripheral blood mononuclear cells of patients with Crohn's disease.

Crohn's disease (CD) is an inflammatory disease characterized by the activation of the immune system in the gut. Since tumor necrosis factor (TNF-alpha) plays an important role in the initiation and perpetuation of intestinal inflammation in CD, we investigated whether TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)], a Vitamin D analogue, could affect peripheral blood mononuclear cells (PBMC) proliferation and exert an immunosuppressive effect on TNF-alpha production in CD patients, and whether this immunosuppressive action could be mediated by NF-kappaB down-regulation. TX 527 significantly decreased cell proliferation and TNF-alpha levels. On activation, NF-kappaB, rapidly released from its cytoplasmatic inhibitor (IKB-alpha), transmigrates into the nucleus and binds to DNA response elements in gene promoter regions. The activation of NF-kappaB, stimulated by TNF-alpha, and its nuclear translocation together with the degradation of IKB-alpha were blocked by TX 527. At the same time, NF-kappaB protein levels present in cytoplasmic extracts decreased in the presence of TNF-alpha and increased when PBMC were incubated with TX 527. The results of our studies indicate that TX 527 inhibits TNF-alpha mediated effects on PBMC and the activation of NF-kappaB and that its action is mediated by Vitamin D receptor (VDR), which is activated when the cells are stimulated with TX 527.

Interaction among vitamin D(3) analogue KH 1060, TNF-alpha, and vitamin D receptor protein in peripheral blood mononuclear cells of inflammatory bowel disease patients.

BACKGROUND: The active form of vitamin D, 1,25(OH)(2)D(3), exerts important effects on proliferation and differentiation of many cell types, and immunoregulatory activities in particular on T cell-mediated immunity. AIM: The aim of this study was to investigate whether KH 1060, a vitamin D analogue, could decrease tumor necrosis factor-alpha (TNF-alpha) levels in patients with inflammatory bowel disease (IBD). METHODS: PBMC proliferation was determined by [(3)H]thymidine incorporation. TNF-alpha levels were measured by ELISA kit; VDR, Bcl-2 and Bax protein levels with Western blot analysis. RESULTS: KH 1060 inhibited PBMC proliferation and decreased TNF-alpha levels in IBD patients and this effect was synergistic with anti-TNF-alpha. VDR protein levels were significantly increased by PBMC treatment with KH 1060 or anti-TNF-alpha or their combination in ulcerative colitis (UC) patients, and decreased in Crohn's disease (CD) patients, treating the cells with KH 1060. In UC patients an increase in Bcl-2 and Bax levels was observed incubating, PBMC with KH 1060 or anti-TNF-alpha or their combination. In CD patients a slight decrease in Bcl-2 levels was registered when anti-TNF alone or in association with KH 1060 was used. Bax protein levels were slightly increased in the presence of KH 1060 alone or in combination with anti-TNF. CONCLUSION: This study shows that KH 1060 acts as an immunomodulator on PBMC, acting as TNF-alpha inhibitor. This finding provides strong evidence that vitamin D status could be an important regulator of immunity IBD.

1,25-Dihydroxyvitamin D3 inhibits tumor necrosis factor-alpha-induced adhesion molecule expression in endothelial cells.

This study tested the hypothesis that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)(2)D(3) for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) or 2 ng/ml interleukin-1alpha (IL-1alpha). 1,25(OH)(2)D(3) did not affect HUVEC viability and proliferation, while TNF-alpha, alone or in combination with the hormone, significantly inhibited HUVEC viability. [(3)H]thymidine incorporation in HUVEC treated with TNF-alpha or IL-1alpha significantly decreased, in the absence or in the presence of the hormone, while the levels of vitamin D receptor markedly increased in the presence of 1,25(OH)(2)D(3) alone or associated with TNF-alpha or IL-1alpha, in comparison to the control. The noteworthy increase in protein levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha was significantly decreased after incubation of the cells with 1,25(OH)(2)D(3), this effect not being seen on E-selectin expression. Neither apoptosis nor nuclear translocation of NF-kappaB, induced in HUVEC by TNF-alpha was influenced by 1,25(OH)(2)D(3) treatment.

In vitro interaction between surface-treated Ti-6Al-4V titanium alloy and human peripheral blood mononuclear cells.

The Ti-6Al-4V titanium alloy is widely employed as an implant material. The effects of Ti-6Al-4V samples, tested in both an untreated state and one in which the samples were subjected to a glow-discharge treatment performed with the use of air, on human peripheral blood mononuclear cells (PBMC) were studied. Apoptosis, undetectable after 24-h contact of PBMC with the two sample types, is induced after 48 h by treated samples, and, after 48 h, but in the presence of 1.5 microg/mL PHA, by both sample types. The expression of intercellular adhesion molecule-1 (ICAM-1) always increases, in comparison with control, in PBMC put in contact with the two sample types. In the same way, a remarkable increase in tumor necrosis-alpha (TNF-alpha) release in the culture medium is registered, when PBMC are put in contact with the two sample types for 24 and 48 h. Human umbilical-vein endothelial cells (HUVEC) cocultured for 48 h with PBMC, previously incubated with the two sample types for 24 h, show an increase in ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) protein expression in comparison with control (HUVEC cocultured with control PBMC), indicating that inflammatory phenomena might occur. Taken together, these results suggest that, although plasma-treated titanium alloy shows a better biocompatibility in comparison with the untreated one, attention must be paid to the careful control of the first signs of inflammation.

Biochemical effects of KH 1060 and anti-TNF monoclonal antibody on human peripheral blood mononuclear cells.

The aim of this study was to investigate whether the vitamin D analogue KH 1060 could exert a suppressive action on Tumor necrosis factor-alpha (TNF-alpha). The chimeric anti-TNF-alpha monoclonal antibody (anti-TNF), alone or in combination with KH 1060, was also used. KH 1060 (0.01, 0.1, 1 nM) significantly inhibited cell proliferation, determined after 5 days by [3H]thymidine incorporation, when peripheral blood mononuclear cells (PBMC), obtained from healthy subjects, were stimulated with phytohaemagglutinin (PHA) and incubated for 24 h in the absence and in the presence of lipopolysaccharide (LPS). In the same experimental conditions, anti-TNF exerted a significant inhibition on PBMC proliferation, at the lowest doses (0.001, 0.01 microg/ml) in the absence of LPS, and at 0.001, 1, 10 microg/ml in its presence. A synergistic inhibition was registered combining KH 1060 and anti-TNF, at well-defined concentrations. 0.1 nM KH 1060 produced a significant decrease in TNF-alpha levels, determined by ELISA, although less remarkable than in the presence of anti-TNF. This decrease was synergistic, associating 0.1 nM KH 1060 and 0.1 microg/ml anti-TNF. VDR protein levels were increased by 0.1 nM KH 1060, 0.1 microg/ml anti-TNF or their combination. The protein levels of two oncogenes, Bax and Bcl-2, remained unchanged, when PBMC were incubated with KH 1060, anti-TNF or their combination in the absence of LPS, while, in its presence, an increase was registered. The demonstrated anti-TNF-alpha effect of KH 1060 may suggest for this compound an immunosuppressive action and the possibility to synergistically act with other drugs.

Effects of surface treatment of Ti-6Al-4V titanium alloy on biocompatibility in cultured human umbilical vein endothelial cells.

Among the titanium alloys employed as implant materials, the Ti-6Al-4V alloy is still widely used. Ti-6Al-4V titanium alloy samples, in untreated state and subjected to treatments in air by furnace or glow-discharge processes, were put in contact with human umbilical vein endothelial cells (HUVEC) in order to evaluate their effects on biocompatibility. In HUVEC kept for 48 h in the presence of the three sample types neither cell proliferation nor protein content nor lactate dehydrogenase release in the culture medium are affected, while apoptosis is induced after 48- and 96-h contact of the cells with the untreated sample type, and after 96-h contact with the plasma treated one, the furnace treated sample type being ineffective. The expression of two adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was also studied. The incubation of HUVEC with the three sample types for 48 or 96 h induces a significant increase in ICAM-1 protein levels, in comparison with control cells, while VCAM-1 expression is not detectable. In the same way, TNF-alpha release in the culture medium, assayed after 48- and 96-h contact of the cells with the three sample types, is significantly higher, in comparison with control, even if the highest values are registered in the presence of the untreated samples. Taken together, these data indicate that, although Ti-6Al-4V alloy samples, and in particular the treated ones, show a good biocompatibility, attention must be given to the first signs of inflammation.

Effect of anti-TNF therapy and vitamin D derivatives on the proliferation of peripheral blood mononuclear cells in Crohn's disease.

Infliximab treatment demonstrated clinical and endoscopic benefits in active refractory and fistulizing Crohn's disease. The aim of this research was to investigate the proliferative response of peripheral blood mononuclear cells (PBMC) obtained from patients with active and fistulizing Crohn's disease treated with infliximab therapy. PBMC proliferation and VDR protein levels were also studied when 1,25(OH)2D3 or its analogues (EB 1089, KH 1060) were added to cells cultures. At day 5 of culture, the proliferation of PBMC obtained from patients responsive to the therapy showed a remarkable decrease (about 60%) at T6 (after two infusions) with respect to T0 (before the first infusion). On the contrary, in the unresponsive patient, the proliferative response was four times higher at T6 in comparison with T0. Vitamin D derivatives induced a decrease in cell proliferation higher in responsive patients than in the unresponsive one. Increased VDR levels during therapy were registered only in the unresponsive patient. Our results indicate that PBMC proliferation and VDR expression may be useful indicators to predict the response of patients with Crohn's disease to the infliximab therapy.

Synergistic effect of vitamin D derivatives and retinoids on C2C12 skeletal muscle cells.

The response of C2C12 myoblasts to 1 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 100 nM retinoids (9-cis retinoic acid, all-trans retinoic acid) and to combination treatments, after 72 h incubation, was studied. The incubation with 1,25(OH)2D3 was ineffective on either cell proliferation or [3H]thymidine incorporation (expressed as DPM per cell) or protein content per cell. On the contrary, all the other treatments inhibited cell proliferation, this inhibition being synergistic when the vitamin D derivatives were combined with 9-cis or all-trans retinoic acid, and increased [3H]thymidine incorporation and protein content per cell. The levels of the VDR protein remarkably increased in comparison with control cells, except for the incubation with 9-cis retinoic acid. This increase was particularly accentuated in C2C12 cells treated with KH 1060 and 9-cis retinoic acid in combination. These results, taken together, suggest a role for vitamin D derivatives and retinoids on C2C12 cells.

Synergistic inhibitory effect of cyclosporin A and vitamin D derivatives on T-lymphocyte proliferation in active ulcerative colitis.

OBJECTIVE: 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the hormonal active form of vitamin D3, could represent a potentially therapeutic agent in autoimmune diseases. Cyclosporin A (CsA) shows immunoregulatory properties, which, in many respects, seem to be similar to those of 1,25(OH)2D3. Our aim was to investigate the possible synergistic effect exerted by CsA in combination with 1,25(OH)2D3 or its nonhypercalcemic analogues, EB 1089 and KH 1060, on the proliferative response of T lymphocytes obtained from active ulcerative colitis patients. METHODS: The T lymphocyte-enriched population was treated with phytohemagglutinin and CsA (doses from 1 ng to 1000 ng/ml) alone or in association with 1,25(OH)2D3 or EB 1089 or KH 1060 (0.1, 1, 10 nM final concentration). Cell proliferation was determined by [3H]thymidine incorporation and analyzed on day 5 of culture. RESULTS: After incubation with CsA, T lymphocyte proliferation was significantly inhibited in comparison with the vehicle-treated cultures. However, T lymphocytes from ulcerative colitis patients were significantly more sensitive to CsA than those from healthy controls. The inhibition in T lymphocyte proliferation, after treatment of the cultures with CsA associated with either 1,25(OH)2D3 or EB 1089 or KH 1060, was synergistic at well-defined concentrations. CONCLUSIONS: Taking into account the lowest CsA dose (1 ng/ml), the highest synergistic inhibition in the proliferation of T lymphocytes prepared from ulcerative colitis patients was found combining CsA and 10 nM of 1,25(OH)2D3 or 10 nM of EB 1089 or KH 1060 at the three concentrations. The results obtained, associating the lowest CsA dose and the lowest KH 1060 concentration, may suggest an alternative therapeutic approach in these patients, reducing the dose, and consequently the toxicity, of CsA.