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The widespread environmental pollutant 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) is a non-dioxin-like toxicant. It is a potential carcinogen compound able to induce gap junction (GJ) intercellular communication impairment, probably the first non-genomic event leading to tumor promotion. Although PCBs have been known for many years, the molecular mode of PCB153 action is still unclear. Recent studies from our research group have shown that the toxicant elicits a transient modulation of connexin (Cx) 43-formed GJs in hepatic stem-like WB-F344 cells involving sphingosine 1-phosphate (S1P) path. Taking into account that other strictly related bioactive sphingolipids, such as ceramide (Cer), may have different effects from S1P, here we aim to clarify the signaling paths engaged by PCB153 in the control of GJs, focusing primarily on the role of Cer. Accordingly, we have achieved a combined biomolecular and electrophysiological analysis of GJs in cultured WB-F344 cells treated with PCB153 at different time points. We have found that the toxicant elicited a time-dependent regulation of GJs formed by different Cx isoforms, through a transient modulation of Cer/Cer kinase (CerK) axis and, in turn, of protein phosphatase 2A (PP2A). Our new findings demonstrate the existence of a specific molecular mechanism downstream to Cer, which distinctly affects the voltage-dependent and -independent GJs in liver stem-like cells, and open new opportunities for the identification of additional potential targets of these environmental toxicants. Supplementary Information The online version contains supplementary material available at 10.1007/s11010-021-04135-z.

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.

The biocompatibility of austenitic stainless steels can be improved by means of surface engineering techniques. In the present research it was investigated if low temperature nitrided AISI 316L austenitic stainless steel may be a suitable substrate for bioactive protein coating consisting of collagen-I. The biocompatibility of surface modified alloy was studied using as experimental model endothelial cells (human umbilical vein endothelial cells) in culture. Low temperature nitriding produces modified surface layers consisting mainly of S phase, the supersaturated interstitial solid solution of nitrogen in the austenite lattice, which allows to enhance surface microhardness and corrosion resistance in PBS solution. The nitriding treatment seems to promote the coating with collagen-I, without chemical coupling agents, in respect of the untreated alloy. For biocompatibility studies, proliferation, lactate dehydrogenase levels and secretion of two metalloproteinases (MMP-2 and MMP-9) were determined. Experimental results suggest that the collagen protection may be favourable for endothelial cell proliferation and for the control of MMP-2 release.

Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ), acts on a variety of tissues including the nervous system, where the hormone plays an important role as neuroprotective, antiproliferating and differentiating agent. Sphingolipids are bioactive lipids that play critical and complex roles in regulating cell fate. In the present paper we have investigated whether sphingolipids are involved in the protective action of 1,25(OH) 2 D 3. We have found that 1,25(OH) 2 D 3 prevents amyloid-β peptide (Aβ(1−42)) cytotoxicity both in differentiated SH-SY5Y human neuroblastoma cells and in vivo . In differentiated SH-SY5Y cells, Aβ(1−42) strongly reduces the sphingosine-1-phosphate (S1P)/ceramide (Cer) ratio while 1,25(OH) 2 D 3 partially reverts this effect. 1,25(OH) 2 D 3 reverts also the Aβ(1−42)-induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH) 2 D 3 and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH) 2 D 3 prevents the reduction of S1P1 expression promoted by Aβ(1−42) and thereby it modulates the downstream signaling leading to ER stress damage (p38MAPK/ATF4). Similar effects were observed by using ZK191784. In addition, chronic treatment with 1,25(OH) 2 D 3 protects from aggregated Aβ(1−42)-induced damage in the CA1 region of the rat hippocampus and promotes cell proliferation in the hippocampal dentate gyrus of adult mice. In conclusion, these results represent the first evidence of the role of 1,25(OH) 2 D 3 and its structural analogue ZK191784 in counteracting the Aβ(1−42) peptide-induced toxicity through the modulation of S1P/S1P1/p38MAPK/ATF4 pathway in differentiated SH-SY5Y cells.

Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)

1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)2D3 as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [3H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)2D3 and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)2D3 receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)2D3 supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)2D3 and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.

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.

Transient receptor potential canonical (TRPC) channels provide cation and Ca2+ entry pathways, which have important regulatory roles in many physio-pathological processes, including muscle dystrophy. However, the mechanisms of activation of these channels remain poorly understood. Using siRNA, we provide the first experimental evidence that TRPC channel 1 (TRPC1), besides acting as a store-operated channel, represents an essential component of stretch-activated channels in C2C12 skeletal myoblasts, as assayed by whole-cell patch-clamp and atomic force microscopic pulling. The channel's activity and stretch-induced Ca2+ influx were modulated by sphingosine 1-phosphate (S1P), a bioactive lipid involved in satellite cell biology and tissue regeneration. We also found that TRPC1 was functionally assembled in lipid rafts, as shown by the fact that cholesterol depletion resulted in the reduction of transmembrane ion current and conductance. Association between TRPC1 and lipid rafts was increased by formation of stress fibres, which was elicited by S1P and abolished by treatment with the actin-disrupting dihydrocytochalasin B, suggesting a role for cytoskeleton in TRPC1 membrane recruitment. Moreover, TRPC1 expression was significantly upregulated during myogenesis, especially in the presence of S1P, implicating a crucial role for TRPC1 in myoblast differentiation. Collectively, these findings may offer new tools for understanding the role of TRPC1 and sphingolipid signalling in skeletal muscle regeneration and provide new therapeutic approaches for skeletal muscle disorders.

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.

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43Δ130–136reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

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.

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.

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-1β, 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-1β 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.

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.

1α,25-Dihydroxyvitamin D3 (1,25(OH)₂D₃), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)₂D₃ as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [³H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)₂D₃ and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)₂D₃ receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)₂D₃ supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)₂D₃ and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.

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.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.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.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.

In the present work, X-rayphotoelectronspectroscopy (XPS) was used to study the surface chemical composition of three alloys for biomedical applications: Ti–7Nb–6Al, Ti–13Nb–13Zr and Ti–15Zr–4Nb. The surface of these alloys was modified by annealing in air at 750 °C for different times with the aim of developing an oxide thick layer on top. The evolution of surface composition with annealing time was studied by XPS, and compared with the composition of the native oxide layer present on the samples before annealing. Two different oxidation trends were observed depending on the alloying elements and their corresponding diffusion kinetics, which give rise to different chemical species at the topmost layers. These results were linked with an evaluation of the biological response of the alloys by bringing them in contact with human peripheral blood mononuclear cells (PBMC)., This work has been partially supported by the Spanish MEC under Projects MAT-2008-01497/NAN, MAT-2007-66719-C03-03, MAT-2006-13348 and CSD2007-00041, and by grants from Italian MIUR.

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°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-α 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-α 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., MIUR and Cassa di Risparmio di Firenze. Project CSD2007‐00041 of the Spanish MEC.

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca(2+)-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.

The effects of AISI 316L austenitic stainless steel, tested in untreated state or subjected to glow-discharge nitriding (at 10 or 20 hPa) and nitriding + post-oxidizing treatments, on human umbilical vein endothelial cells (HUVEC) and on peripheral blood mononuclear cells (PBMC) were evaluated. All the treated samples showed a better corrosion resistance in PBS and higher surface hardness in comparison with the untreated alloy. In HUVEC put in contact for 72 h with the sample types, proliferation and apoptosis decreased and increased, respectively, in the presence of the nitrided + post-oxidized samples, while only slight differences in cytokine (TNF-alpha, IL-6, and TGF-beta1) release were registered. Intercellular adhesion molecule-1 (ICAM-1) increased in HUVEC incubated with all the treated samples, while vascular cell adhesion molecule-1 (VCAM-1) and E-selectin increased in the presence of all the sample types. PBMC incubated for 48 h with the samples showed a decrease in proliferation and an increase in apoptosis in the presence of the untreated samples and the nitrided + post-oxidized ones. All the sample types induced a remarkable increase in TNF-alpha and IL-6 release in PBMC culture medium, while only the untreated sample and the nitrided at 10 hPa induced an increase in ICAM-1 expression. In HUVEC cocultured with PBMC, previously put in contact with the treated AISI 316L samples, increased levels of ICAM-1 were detected. In HUVEC coincubated with the culture medium of PBMC, previously put in contact with the samples under study, a noteworthy increase in ICAM-1, VCAM-1, and E-selectin levels was always registered, with the exception of VCAM-1, which was not affected by the untreated sample. In conclusion, even if the treated samples do not show a marked increase in biocompatibility in comparison with the untreated alloy, their higher corrosion resistance may suggest a better performance as the contact with physiological environment becomes longer.

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.

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.

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-α) levels in patients with inflammatory bowel disease (IBD). Methods PBMC proliferation was determined by [ 3 H]thymidine incorporation. TNF-α 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-α levels in IBD patients and this effect was synergistic with anti-TNF-α. VDR protein levels were significantly increased by PBMC treatment with KH 1060 or anti-TNF-α 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-α 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-α inhibitor. This finding provides strong evidence that vitamin D status could be an important regulator of immunity IBD.

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 μg/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-α) 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. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005

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.