Vacuum‐assisted closure has become a new technique in the challenging management of contaminated, acute, and chronic wounds. Although promising clinical results have been described, scientific proof ...to substantiate the mechanism of action of this therapy is scarce. In the present study, we examined whether the positive effect on wound healing found in vacuum‐assisted closure–treated wounds could be explained by an effect on the bacterial load. Fifty‐four patients who needed open wound management before surgical closure were included in this study. Wounds were randomized to either vacuum‐assisted closure therapy (n= 29) or treatment by conventional moist gauze therapy (n= 25). Healing was characterized by development of a clean granulating wound bed (“ready for surgical therapy”) and reduction of wound surface area. To quantify bacterial load, biopsies were collected. No significant difference was found in time needed to reach “ready for surgical therapy” comparing both therapies. Wound surface area reduction was significantly larger in vacuum‐assisted closure–treated wounds: 3.8 ± 0.5 percent/day (mean ± SEM) compared to conventional‐treated wounds (1.7 ± 0.6 percent/day; p < 0.05). The total quantitative bacterial load was generally stable in both therapies. However, nonfermentative gram negative bacilli showed a significant decrease in vacuum‐assisted closure–treated wounds (p < 0.05), whereas Staphylococcus aureus showed a significant increase in vacuum‐assisted closure–treated wounds (p < 0.05). In conclusion, this study shows a positive effect of vacuum‐assisted closure therapy on wound healing, expressed as a significant reduction of wound surface area. However, this could not be explained by a significant quantitative reduction of the bacterial load.
The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)2D3 increased VDR and calbindins in controls but not ...GHS rats. VDR half‐life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats.
Introduction: Genetic hypercalciuric stone‐forming (GHS) rats form calcium kidney stones from hypercalciuria arising from increased intestinal calcium absorption and bone resorption and decreased renal calcium reabsorption. Normal serum 1,25‐dihydroxyvitamin D3 ‘1,25(OH)2D3’ levels and increased vitamin D receptor (VDR) protein suggest that high rates of expression of vitamin D‐responsive genes may mediate the hypercalciuria. The mechanism of elevated VDR and state of receptor function are not known.
Materials and Methods: GHS and non‐stone‐forming control (NC) male rats (mean, 249 g), fed a normal calcium diet, were injected intraperitoneally with 1,25(OH)2D3 (30 ng/100 g BW) or vehicle 24 h before cycloheximide (6 mg/100 g, IP) and were killed 0–8 h afterward. Duodenal VDR was measured by ELISA and Western blot, and duodenal and kidney calbindins (9 and 28 kDa) were measured by Western blots.
Results and Conclusions: Duodenal VDR protein by Western blot was increased 2‐fold in GHS versus NC rats (633 ± 62 versus 388 ± 48 fmol/mg protein, n = 4, p < 0.02), and 1,25(OH)2D3 increased VDR and calbindins (9 and 28 kDa) further in NC but not GHS rats. Duodenal VDR half‐life was prolonged in GHS rats (2.59 ± 0.2 versus 1.81 ± 0.2 h, p < 0.001). 1,25(OH)2D3 prolonged duodenal VDR half‐life in NC rats to that of untreated GHS rats (2.59 ± 0.2 versus 2.83 ± 0.3 h, not significant). This study supports the hypothesis that prolongation of VDR half‐life increases VDR tissue levels and mediates increased VDR‐regulated genes that result in hypercalciuria through actions on vitamin D‐regulated calcium transport in intestine, bone, and kidney.
Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by ...large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, α enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.
There is considerable divergence in the sequences of steroid receptor response elements, including the vitamin D response elements (VDREs). Two major VDRE-containing and thus 1,25-dihydroxyvitamin ...D3(1,25-(OH)2D3)-regulated genes are the two non-collagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. We observed a stronger induction of osteopontin than osteocalcin mRNA expression by 1,25-(OH)2D3. Subsequently, we have shown that vitamin D receptor/retinoid X receptor α (VDR/RXRα) heterodimers bind more tightly to the osteopontin VDRE than to the osteocalcin VDRE. Studies using point mutants revealed that the internal dinucleotide at positions 3 and 4 of the proximal steroid half-element are most important for modulating the strength of receptor binding. In addition, studies with VDRE-driven luciferase reporter gene constructs revealed that the central dinucleotide influences the transactivation potential of VDR/RXRα with the same order of magnitude as that observed in the DNA binding studies. The synthetic vitamin D analog KH1060 is a more potent stimulator of transcription and inducer of VDRE binding of VDR/RXR in the presence of nuclear factors isolated from ROS 17/2.8 osteoblast-like cells than the natural ligand 1,25-(OH)2D3. Interestingly, however, KH1060 is comparable or even less potent than 1,25-(OH)2D3 in stimulating VDRE binding ofin vitro synthesized VDR/RXRα. Thus, the extent of 1,25-(OH)2D3- and KH1060-dependent binding of VDR/RXRα is specified by a central dinucleotide in the VDRE, and the ligand-induced effects on DNA binding are in part controlled by the cellular context of nuclear proteins.
Prostate-specific antigen (PSA) is considered as an important marker for prostate cancer. Regulation of PSA gene expression is mediated by androgens bound to androgen receptors via androgen response ...elements (AREs) in its promoter and far upstream enhancer regions. In addition, GATA proteins contribute to PSA gene transcription by interacting with GATA motifs present in the PSA enhancer sequence. The TRPS1 gene contains a single GATA zinc finger domain and not only binds to forward consensus GATA motifs but also to an inverse GATA motif overlapping the ARE III in the far upstream enhancer of the PSA gene. Overexpression of TRPS1 in androgen-dependent human LNCaP prostate cancer cells inhibited the transcription of a transiently transfected PSA enhancer/promoter-driven luciferase reporter construct. Furthermore, overexpression of TRPS1 reduced the androgen-induced endogenous PSA levels secreted in culture medium of LNCaP cells. Our results suggest a role of TRPS1 in androgen regulation of PSA gene expression.
The steroid hormone 1,25-dihydroxyvitamin D3 1,25-(OH)2D3 has potential to be used as an antitumor agent, but its clinical application is restricted by the strong calcemic activity. Therefore, new ...vitamin D3 analogues are developed with increased growth inhibitory and reduced calcemic activity. In the present study, we have examined the antiproliferative effects of four novel vitamin D3 analogues (CB966, EB1089, KH1060, and 22-oxa-calcitriol) on breast cancer cells, either alone or in combination with the antiestrogen tamoxifen. The estrogen-dependent ZR-75-1 and estrogen-responsive MCF-7 cell lines were used as a model. It was shown that, with EB1089 and KH1060, the same growth inhibitory effect as 1,25-(OH)2D3 could be reached at up to 100-fold lower concentrations, whereas CD966 and 22-oxa-calcitriol were nearly equipotent with 1,25-(OH)2D3. The growth inhibition by the vitamin D3 compounds could be augmented by combined treatment with tamoxifen. At the maximal effective concentrations of the vitamin D3 compounds, the effect of combined treatment was addictive (MCF-7 cells) or less than additive (ZR-75-1 cells). Tamoxifen increased the sensitivity of the cells to the vitamin D3 compounds 2- to 4000-fold, which was expressed by a shift to lower median effective concentration values. Thereby, the vitamin D3 compounds may be used at even lower dosages in combination therapy with tamoxifen. A major problem of tamoxifen therapy is the development of tamoxifen resistance. We have observed that tamoxifen-resistant clones of ZR-75-1 cells retain their response to the vitamin D3 compounds. Regulation of the growth-related oncogene c-myc (mRNA level) and the estrogen receptor (protein level) were studied but appeared not to be related to the antiproliferative action of the vitamin D3 compounds. Together, our data point to a potential benefit of combination therapy with 1,25-(OH)2D3 or vitamin D3 analogues and tamoxifen for the treatment of breast cancer.
24,25-Dihydroxyvitamin D3 and bone metabolism VAN LEEUWEN, Johannes P. T. M; VAN DEN BEMD, Gert-Jan C. M; VAN DRIEL, Marjolein ...
Steroids,
05/2001, Letnik:
66, Številka:
3-5
Journal Article
In the present study the involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast-like
cells and in the stimulation of in vitro bone resorption by ...1,25(OH)2D3 was examined. Incubation for 24 h with 1,25(OH)2D3
potently stimulated osteocalcin synthesis by ROS 17/2.8 cells. This stimulation was inhibited (30-70% inhibition) by 25 microM
of the protein kinase C (PKC) inhibitors 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG) and sphingosine without affecting basal
osteocalcin synthesis. 1,25(OH)2D3-stimulated osteocalcin secretion by nontransformed isolated fetal rat osteoblasts was also
inhibited (30-55%) by AMG. Also, AMG inhibited 10(-9) M 1,25(OH)2D3-induced up-regulation of vitamin D receptor in ROS 17/2.8
cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) did not cause an increase in osteocalcin secretion, while
only a small increase in cellular content of osteocalcin in ROS 17/2.8 cells was observed. Addition of PMA together with 1,25(OH)2D3
did not change the response to 1,25(OH)2D3. The PKC inhibitors were not toxic for the cells. 1,25(OH)2D3 did not stimulate
diacylglycerol production in ROS 17/2.8 cells up to 5 min after administration. However, 4- and 24-h incubation with 10 nM
1,25(OH)2D3 increased phorbol ester binding in ROS 17/2.8 cells. 1,25(OH)2D3 potently stimulated bone resorption after 3 and
6 days of culture in fetal mouse long bones and calvaria. Both the PKC inhibitors AMG (25 microM) and staurosporine (50 nM)
strongly inhibited (60-86% inhibition) 1,25(OH)2D3-stimulated bone resorption without affecting basal 45Ca release. These
effects were not due to a cytotoxic effect of both PKC inhibitors. Nor is it likely that the effects of AMG and staurosporine
are due to inhibition of cell proliferation as hydroxyurea did not affect 1,25(OH)2D3-stimulated bone resorption. The inhibition
of 1,25(OH)2D3-stimulated bone resorption by PKC inhibitors suggests that besides osteocalcin synthesis PKC is also involved
in other responses of 1,25(OH)2D3 in bone. 1,25(OH)2D3 does not directly activate PKC via an increase in diacylglycerol production
but more likely via an increase in PKC. Together, the present study demonstrates a functional involvement of PKC in the action
of 1,25(OH)2D3 in bone and bone cells which may have consequences for the development of 1,25(OH)2D3 analogs, e.g. with less
hypercalcemic and relatively more antiproliferative activity.
24,25-Dihydroxyvitamin D 3 and bone metabolism van Leeuwen, Johannes P.T.M.; van den Bemd, Gert-Jan C.M.; van Driel, Marjolein ...
Steroids,
03/2001, Letnik:
66, Številka:
3
Journal Article
Recenzirano
The 1α-hydroxylated metabolite of 25-hydroxyvitamin D
3, 1,25-dihydroxyvitamin D
3, is the biologically most active metabolite of vitamin D. The 24-hydroxylated metabolites were generally considered ...as degradation products of a catabolic pathway finally leading to excretion of calcitroic acid. Studies with analogues fluorinated at the C-24 position did not indicate a physiological function for 24R,25(OH)
2D
3. Nevertheless throughout the years various studies showed biologic effects of other metabolites than 1α,25(OH)
2D
3. In particular the metabolite 24R,25(OH)
2D
3 has been functionally analyzed, e.g. with respect to a role in normal chicken egg hatchability and effects on chondrocytes in the resting zone of cartilage. Numerous studies have shown the presence of the vitamin D receptor in bone cells and effects of 1α,25(OH)
2D
3 on bone and bone cells. Also for 24R,25(OH)
2D
3 studies have been performed focusing on effects on bone and bone cells. The purpose of this review is to summarize the data regarding 24R,25(OH)
2D
3 and bone and to evaluate its role in bone biology.
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