Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Differences have been shown between HLA characteristics of patients with different courses of alveolar echinococcosis (AE). Notably the HLA B8, DR3, DQ2 haplotype was associated with more severe forms of this granulomatous parasitic disease. We compared IL-10, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) secretion by peripheral blood mononuclear cells (PBMC) isolated from eight HLA-DR3+, DQ2+, B8+ AE patients and from 10 HLA-DR3-, DQ2-, B8- patients after non-specific mitogenic and specific Echinococcus multilocularis antigenic in vitro stimulation. PBMC from seven HLA-DR3+, DQ2+, B8+ healthy subjects and nine HLA-DR3-, DQ2-, B8- subjects were also studied as controls. PBMC from AE patients with HLA DR3+, DQ2+ haplotype secreted higher levels of IL-10 without any stimulation and after specific antigenic stimulation than did patients without this haplotype. Higher levels of IL-5 and IFN-gamma were also produced by these patients' PBMC after stimulation with non-purified parasitic antigenic preparations; however, the specific alkaline phosphatase antigen extracted from E. multilocularis induced only Th2-type cytokine secretion. A spontaneous secretion of TNF by HLA DR3+, DQ2+ B8+ AE patients was also found. These results suggest that HLA characteristics of the host can influence immune-mediated mechanisms, and thus the course of AE in humans; specific antigenic components of E. multilocularis could contribute to the preferential Th2-type cytokine production favoured by the genetic background of the host.
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PMID:Resistance/susceptibility to Echinococcus multilocularis infection and cytokine profile in humans. II. Influence of the HLA B8, DR3, DQ2 haplotype. 1097 16

We developed a simple method for determining the relative amount of alpha-smooth muscle actin (alphaSMA) produced in fibroblasts. The principle of the method is based on an enzyme immunoassay (EIA) for alphaSMA in microcultured fibroblasts. The optimized protocol of the assay is as follows. Human fibroblasts were cultured with transforming growth factor beta1 (TGFbeta1) in a microtiter plate and directly immobilized on the plate. The alphaSMA produced was labeled and subjected to indirect enzyme immunoassay using alkaline phosphatase, and optical density was measured. Semiquantitativeness was confirmed using various numbers of cells in which alphaSMA production was induced by treatment with TGFbeta1. The assay simply demonstrated that interferon-gamma (INF-gamma) inhibited the production of alphaSMA in an established cell line and that in primary cultured cells originated from the contractile nodule. Since the assay is simple and semi-quantitative, it is useful for elucidating the mechanism of contractile diseases and screening a large number of substances that have an inhibitory effect on the change in activity of myofibroblasts.
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PMID:Demonstration of downregulation of alpha-smooth muscle actin in interferon-gamma-treated myofibroblast by a novel cell-capture enzyme immunoassay. 1135 89

Calcitriol has shown a benefit in various small uncontrolled studies of ex vivo immune function. We hypothesized that paricalcitol, a new vitamin D derivative, will have a positive effect on the immune system with minimal adverse effects on calcium homeostasis. Thirty-one hemodialysis patients not administered vitamin D because of low intact parathyroid hormone (PTH) levels were randomized to placebo or 4 microg of paricalcitol intravenously with the hemodialysis session three times weekly for 12 weeks. Effects on in vivo and ex vivo assessments of immune function were evaluated. All patients achieved the target dose of paricalcitol. Twenty patients were anergic at the start of the study; 4 of 11 patients in the paricalcitol group and 0 of 9 patients in the placebo group converted to reactive (P = 0.09). The in vivo response to standard hepatitis B booster vaccine and in vitro proliferation and release of interleukin-2 (IL-2), IL-6, tumor necrosis factor-alpha, and interferon-gamma from stimulated lymphocytes were not different between the groups. In contrast to clinical immune effects, paricalcitol increased serum calcium levels and decreased PTH and bone alkaline phosphatase levels (all P < 0.05). However, hypercalcemia was infrequent. In vitro experiments showed that paricalcitol led to greater dose-dependent thymidine uptake than calcitriol in lymphocytes isolated from either dialysis patients or control subjects. Paricalcitol has a tendency toward improving delayed hypersensitivity reactions, but did not have other proimmune effects. However, as expected, paricalcitol had significant effects on calcium homeostasis compared with placebo. Thus, patients with low PTH levels are unlikely to experience the proimmune effects of vitamin D therapy without more profound and potentially adverse oversuppression of PTH.
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PMID:A placebo-controlled trial to evaluate immunomodulatory effects of paricalcitol. 1157 83

We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.
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PMID:Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues. 1192 70

Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-gamma (IFN-gamma) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-gamma and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-gamma and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of beta-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-alpha (TNF-alpha) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-alpha and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-gamma and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-alpha and OSM.
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PMID:Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages. 1211 16

Granulocyte colony-stimulating factor (G-CSF) has been shown to affect the biochemical markers of bone metabolism, including serum bone alkaline phosphatase (BALP), serum osteocalcin, and urine deoxypyridinoline. To determine the association between bone resorption and formation and the G-CSF-induced mobilization of peripheral blood stem cells (PBSC), we examined these markers during mobilization in 19 healthy donors. The average (+/- SEM) serum BALP level before treatment was 81.6 +/- 17.0 IU/dL, and the level increased significantly to 117.7 +/- 15.8 IU/dL on day 5 of G-CSF administration (P < .0001). The urine deoxypyridinoline level before treatment was 12.3 +/- 2.4 nmol/mmol creatinine, and this level also increased significantly to 19.4 +/- 3.0 nmol/mmol creatinine on day 5 of G-CSF administration (P < .0001). In contrast, the average level of serum osteocalcin significantly decreased from 8.07 +/- 2.88 ng/mL to 1.53 +/- 0.18 ng/mL on day 5 (P = .0353). During G-CSF administration, we also studied the serum levels of various cytokines (IL-1beta, osteoclastogenesis inhibitory factor [OCIF], IL-6, tumor necrosis factor alpha, transforming growth factor beta, interferon-gamma, macrophage colony-stimulating factor) related to bone metabolism. Only the kinetics of OCIF were significantly affected. The serum level of OCIF increased immediately after the start of G-CSF administration and remained high during G-CSF administration. These results demonstrate that high-dose G-CSF affects bone metabolism and that OCIF may play a role in bone metabolism. Consistent with the notion that G-CSF affects bone metabolism, a significant correlation was observed between CD34+ cell yield and the increase in urine deoxypyridinoline but not for the changes in serum BALP and osteocalcin levels. This result suggests that bone resorption is either directly or indirectly related to the mobilization of PBSC by G-CSF.
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PMID:Effect of granulocyte colony-stimulating factor on bone metabolism during peripheral blood stem cell mobilization. 1256 3

Although lipid-lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose-dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon-gamma, 1alpha,25-dihydroxyvitamin D3, tumor necrosis factor-alpha, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y-27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.
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PMID:Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators. 1538 84

A dual-color enzyme-linked immunospot (ELISPOT) assay enabled us to analyze three types of cytokine-secreting cells simultaneously. T helper (Th) cells can be subdivided into at least two distinct functional subsets based on their cytokine secretion profiles. The first type of clones (Th1) produces interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), but not IL-4 or IL-5. The second type of clones (Th2) produces IL-4 and IL-5, but not IL-2 or IFN-gamma. Furthermore, the presence of the third type (Th0) of cell, which is a precursor of Th1 or Th2 cells, has been demonstrated to produce both Th1- and Th2-type cytokines. The dual-color ELISPOT assay was developed to differentiate these three subtypes of Th cells in an identical well. In the system, the red spots corresponding to IL-2-secreting cells (Th1) were developed with horseradish peroxidase and aminoethylcarbazole/H(2)O(2). The light-blue spots corresponding to IL-4-secreting cells (Th2) were developed with alkaline phosphatase and Vector blue (chromogenic substrate for alkaline phosphatase). The mixed-colored (indigo) spots corresponding to both types of cytokine-secreting cells (Th0-cells) were developed with both chromogenic substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in a murine spleen cell or human peripheral mononuclear cell preparation.
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PMID:[Development of the method for analysis of the cytokine balance shift associated with diseases]. 1580 81

Salivary glands are an accessible organ for gene therapy, enabling expression of recombinant proteins for both exocrine and endocrine secretion. Lentivirus-based vectors have many advantages for gene therapy, including their ability to infect nondividing cells and to stably integrate into the host genome, enabling long-term transgene expression without eliciting an inflammatory immune response. In the present study, murine salivary glands were inoculated with feline immunodeficiency virus (FIV)-based lentiviral vectors expressing various reporter genes. Luciferase expression was observed as early as 24 h posttransduction, peaked at 17-21 days, and remained stable for more than 80 days. Staining with X-gal suggested that mucous acinar cells were effectively transduced. FIV vector transduction with the secreted alkaline phosphatase gene increased serum levels in treated animals for up to 45 days, and the FIV vector harboring the interferon-gamma (IFN-gamma) expression cassette induced an increase in IFN-gamma serum levels as well as in the supernatant of salivary gland explant cultures. These results demonstrate that the transduction of salivary glands with nonprimate lentiviral vectors may provide a novel and highly effective vehicle for long-term endocrine transgene expression.
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PMID:Prolonged transgene expression in murine salivary glands following non-primate lentiviral vector transduction. 1596 29

An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.
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PMID:Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis. 1619 41


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