Maria Stio

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

This study tested the hypothesis that 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)2D3 for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor‐α (TNF‐α) or 2 ng/ml interleukin‐1α (IL‐1α). 1,25(OH)2D3 did not affect HUVEC viability and proliferation, while TNF‐α, alone or in combination with the hormone, significantly inhibited HUVEC viability. [3H]thymidine incorporation in HUVEC treated with TNF‐α or IL‐1α 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)2D3 alone or associated with TNF‐α or IL‐1α, 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‐α was significantly decreased after incubation of the cells with 1,25(OH)2D3, this effect not being seen on E‐selectin expression. Neither apoptosis nor nuclear translocation of NF‐κB, induced in HUVEC by TNF‐α was influenced by 1,25(OH)2D3 treatment.

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

Crohns 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.

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‐&agr; (infliximab) induce apoptosis in PBMC obtained from healthy subjects. In IBD patients apoptosis is induced by vitamin D derivatives and by anti‐TNF‐&agr; 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‐&agr;. The treatment with the vitamin D derivatives, alone or in combination with LPS or TNF‐&agr;, significantly decreases ICAM‐1 levels both in healthy subjects and IBD patients. In HUVEC cocultured with PBMC, previously incubated with LPS or TNF‐&agr; 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.

Age and GSH metabolism in rat cerebral cortex, as related to oxidative and energy parameters.

A comprehensive study on GSH metabolism in relation to some markers of oxidative and energy status in rat cerebral cortex as a function of age was performed. Reduced GSH, total GSH and the GSH Redox Index decreased both during growth (defined as the period between 1 and 5 months) and during aging (defined as the period between 5 and 27 months) while GSSG levels increased during the two periods, but most significantly during aging. Also GSH-associated enzymes and adenine-pyridine nucleotide levels show age characteristic changes. The obtained results suggest that decreases in oxidative and energy metabolism occur during aging. They probably contribute to decreases in the activity of the biosynthetic processes (i.e., NADP+(H) and GSH synthesis) and in the antioxidant capacity of the GSH system. However, the oxidative stress does not seem to be a typical characteristic of the aging period; as an oxidative status is present during the growth period too. Typical parameters of aging process are mainly the low levels of reduced GSH, total GSH and GSH Redox Index and the high levels of GSSG as well as the high levels of GSH peroxidase and GSH transferase and the low levels of gamma-glutamylcysteine synthetase.

Suppressive effect of 1,25-dihydroxyvitamin D3 and its analogues EB 1089 and KH 1060 on T lymphocyte proliferation in active ulcerative colitis

This study examined the effect exerted by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and two vitamin D analogues, EB 1089 and KH 1060, on the proliferation of T lymphocytes obtained from ulcerative colitis (UC) patients and healthy controls. The proliferative response of T lymphocytes to phytohaemagglutinin treatment was first analyzed on days three, five, and seven of culture. Cell proliferation was significantly lower in UC patients than that observed in healthy controls. The highest proliferation value, in either controls or patients, was registered on day five of culture. On day seven, a decrease in proliferation occurred, less evident in patients with respect to controls, whereas on day three, controls and patients showed the same proliferation value. The response of T lymphocytes of either healthy controls or UC patients to 1,25(OH)2D3, EB 1089, or KH 1060 was then investigated, treating the cells for three, five, and seven days with 10 nM vitamin D derivatives. In the presence of these compounds, cell proliferation was significantly inhibited in both groups, but on day seven, the inhibition of lymphocyte proliferation was remarkable in controls, whereas in patients it was similar to that registered on day five. The highest inhibition values were always obtained in the presence of KH 1060, and the time dependence was continuous in controls, but in the presence of EB 1089 only in patients. T lymphocytes prepared from healthy controls and UC patients were then cultured for five days in the presence of vitamin D derivatives at three different concentrations (0.1, 1, and 10 nM). In the two groups, a dose-dependent inhibition was registered in the presence of 1,25(OH)2D3 or EB 1089, while the inhibition of proliferation exerted by KH 1060 was not dose-dependent. The results obtained suggest an option for the use of the two non-hypercalcemic vitamin D analogues in the therapy of UC patients, perhaps in association with other immunosuppressive drugs.

Intestinal glutathione transport system: a possible detoxication role

The epithelium of the small intestine act by the formation of GSH-S-conjugation, as a first line of defence against various ingested toxic chemicals. GSH and GSH-dependent enzymes are present in the gastrointestinal wall. We and others have characterized the GSH-specific transport systems in intestinal brush-border and in basolateral membrane vesicles, in which gamma-glutamyltranspeptidase (gamma-GT) activity was inactivated by AT-125. In the present study we use inhibition experiments, kinetic studies, trans-stimulation of GSH uptake and HPLC determination to demonstrate (for the first time) that GSH and two GSH-S-conjugates (chosen as model compounds) share a common transport system. Plasma GSH-S-conjugates that may enter the intestinal cells via basolateral membrane, and GSH-S-conjugates that form in intestinal cells, may be eliminated directly by this GSH transporter across brush-border membranes or transported into lumen to the active site of gamma-GT; they are then further metabolized and excreted by various routes. This transport system may thus contribute to the intestinal detoxication role.

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 Crohns 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.

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells

This study examines the effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)(2)D(3) or its derivatives, but significantly decreased in the presence of the two retinoids (0.001--10 microM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060, and 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.

Relationship between age and GSH metabolism in synaptosomes of rat cerebral cortex

A comprehensive analysis on glutathione metabolism in rat cerebral cortex synaptosomes as a function of age was performed. All different glutathione system components (GSH, GSSG, total GSH, and GSH redox index) changed significantly only during aging. GSH, total GSH, and GSH redox index decreased by about 40%, 24%, and 52%, respectively, while GSSG showed a remarkable increase of about 60%. On the contrary, some GSH-related enzyme activities showed characteristic changes both during growth and aging. GSH peroxidase and GSH-S-transferase activities significantly increased both during growth and aging, GSH reductase and gamma-glutamylcysteine synthetase activities showed lower levels only during aging, while glucose-6-phosphate dehydrogenase activity did not change throughout the life of the rat. The results obtained suggest an increase of the oxidative status due to a reduced antioxidant capacity of the GSH system in the synaptosomal compartment during aging. The main cause of these metabolic modifications is a lowering of the rates of both GSSG reduction to GSH and GSH synthesis. Moreover, an irreversible loss of GSH as GSH-S-conjugates due to a high detoxification mechanism during aging is also possible. These alterations in glutathione metabolism, found mainly during aging in rat cerebral cortex synaptosomes may contribute to clarify some aspects of cerebral diseases.