Induction of cellular glutathione and glutathione S-transferase by 3H-1,2-dithiole-3-thione in rat aortic smooth muscle A10 cells: protection against acrolein-induced toxicity

There is increasing evidence that aldehydes, including acrolein generated endogenously during the degradation process of biological molecules or the metabolism of foreign chemicals may be involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Because glutathione (GSH) and GSH S-transferase (GST) are a major cellular defense against the toxic effects of reactive aldehydes, in this study we have characterized the inducibility of GSH and GST by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) and their protective effects against acrolein-induced toxicity in rat aortic smooth muscle A10 cells. Incubation of rat aortic A10 cells with micromolar concentrations of D3T resulted in a concentration- and time-dependent induction of both GSH and GST. Treatment of A10 cells with D3T also led to induction of gamma-glutamylcysteine synthetase, the key enzyme involved in GSH biosynthesis. Notably, the levels of GSH and GST remained higher than basal levels 72 h after removal of D3T from the culture media. To examine the protective effects of D3T-induced GSH and GST against reactive aldehyde-mediated toxicity, A10 cells were pretreated with D3T and then exposed to acrolein. Pretreatment of A10 cells with D3T resulted in a marked decrease of acrolein-induced toxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and morphological changes. To further demonstrate the involvement of GSH and GST in protecting against acrolein-induced toxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of cellular GSH by BSO or inhibition of cellular GST by sulfasalazine led to a marked potentiation of acrolein-induced toxicity in A10 cells. Furthermore, co-treatment of cells with BSO was found to greatly abolish the protective effects of D3T on acrolein-induced toxicity. Taken together, our results demonstrate for the first time that both GSH and GST in aortic smooth muscle cells can be induced by D3T, and that this increased cellular defense affords great protection against reactive aldehyde-induced cardiovascular cell injury.     

The role of chemically induced glutathione and glutathione S-transferase in protecting against 4-hydroxy-2-nonenal-mediated cytotoxicity in vascular smooth muscle cells

4-Hydroxy-2-nonenal (HNE) has been suggested to contribute to the pathogenesis of atherosclerosis. One of the major metabolic transformation pathways of HNE involves conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST). In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat aortic smooth muscle A10 cells. Incubation of A10 cells with D3T resulted in a marked concentration-dependent induction of both GSH and GST. The induction of cellular GST by D3T also exhibited a time-dependent response. Pretreatment of A10 cells with D3T led to a dramatic decrease of HNE-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay and scanning electron microscopy. Incubation of A10 cells with HNE for 0.5 h and 1 h resulted in a significant depletion of cellular GSH, which preceded the decrease of cell viability. To further demonstrate the involvement of GSH and GST in protecting against HNE-induced cytotoxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of GSH by BSO or inhibition of GST by sulfasalazine caused great potentiation of HNE-mediated cytotoxicity. Moreover, cotreatment of A10 cells with BSO was found to completely block the D3T-mediated GSH induction and to largely reverse the cytoprotective effects of D3T on HNE-induced toxicity. Taken together, this study demonstrates that D3T can induce both GSH and GST in aortic smooth muscle cells, and that the D3T-augmented cellular defenses afford a marked protection against HNE-induced vascular cell injury.     

Arrhythmogenic peroxynitrite-induced alterations in mammalian heart contractility and its prevention with quercetin-filled liposomes

The aim of the present study was to evaluate the effects of quercetin-filled phosphatidylcholine liposomes (PCLs) on peroxynitrite (ONOO⁻)-induced cardiac arrhythmias. Experiments were done using different experimental models, including isolated rat papillary muscle, Langendorff perfused rat hearts, and anesthetized animals. Being exogenously applied in a concentration greater than 50 μM, ONOO⁻, caused inhibition of isometric twitch amplitude in isolated papillary muscles and led to an appearance of arrhythmias. Decomposed ONOO⁻ had no similar effects and reversibly increased twitch amplitude. Authentic nitric oxide (NO, 100 μM) did not produce arrhythmias and had no significant effect on twitch amplitude. Verapamil and ruthenium red were with-out effect on ONOO⁻-induced arrhythmias, whereas tetrodotoxin and nicorandil effectively prevented arrhythmias development. Ouabain increased the arrhythmogenic effect of ONOO⁻. ONOO⁻ significantly decreased coronary perfusion pressure (CPP) and mean left-ventricular pressure (MLVP) in the Langendorff perfused rat heart and produced severe arrhythmias. Authentic nitric oxide (NO) decreased CPP and MLVP insignificantly and resulted in a low incidence of arrhythmias. The NO donor SIN-1 in doses greater than 50 μM led to the appearance of low-incidence arrhythmias in anesthetized rats. Intraventricular injection of ONOO⁻ promotes the appearance of a high incidence of arrhythmias in anesthetized rats and decreased MLVP. PCLs filled with the antioxidant quercetin restored normal cardiac contractility in both isolated tissues and anesthetizes animals. In conclusion, we hypothesized that ONOO⁻, but not its decomposed products, can initiate membrane lipid peroxidation and damage the phospholipid environment of ionic channels in myocardial cell plasma membranes inducing abnormal cardiac action potentials, arrhythmogenesis, and contractile dysfunction. Quercetin-filled PCL provide reliable protection against peroxynitrite-induced myocardial injury in isolated cardiac tissues and anesthetized animals primarily as a result of the decomposition of endogenously formed ONOO⁻.     

The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O₂^•−) though the rate of reaction of O₂^•− with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O₂^•−. We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O₂^•− scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O₂^•−. In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O₂^•− as compared to cytosols from untreated cells. This increased ability to scavenge O₂^•− was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O₂^•− generator further confirmed the ability of NQO1 from HASMCs to scavenge O₂^•−. The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O₂^•− scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O₂^•− scavenger.     

Long-term culture of leukemic bone marrow primary cells in biomimetic osteoblast niche

We constructed a “biomimetic osteoblast niche” with bio-derived bone as a scaffold, on which we seeded marrow mesenchymal stem cells (MSCs) from CML patients, and induced the MSCs to differentiate into osteoblasts. Bone marrow mononuclear cells from CML patients were cultured in the biomimetic niche (3D culture system) or a 2D culture system with the induced MSCs/osteoblasts as a feeder cell layer for 2 and 5 weeks without adding exogenous cytokines. Cultured cells were analyzed regarding their phenotypes and functions using flow cytometry, colony-forming unit (CFU) assay and long-term culture-initiating cells (LTC-IC) assay. All cultured and colony cells in the LTC-IC assay were collected and analyzed by fluorescent in situ hybridization to identify Ph (bcr/abl)-positive cells. Our results showed that all parameters were higher in the 3D than in the 2D system, either at 2 or 5 weeks, i.e., regarding the number of CD34⁺ cells (8,277.00 vs. 4,490.75 or 2,276.75 vs. 786.00 per well on average, respectively), number of CD34⁺/CD38⁻ cells (1,207.50 vs. 474.25 or 497.25 vs. 114.25 per well on average, respectively), numbers of CFUs (103.33 vs. 79 or 47.0 vs. 21.67/10⁵ MNCs; 189.33 vs. 131.00 or 10.33 vs. 3.67 per well on average, respectively), frequency of LTC-ICs (2.23 × 10⁻⁵ vs. 1.40 × 10⁻⁵ or 1.86 × 10⁻⁵ vs. 0.64 × 10⁻⁵, respectively) and number of remaining LTC-ICs (2.80 vs. 2.03 or 0.46 vs. 0.07 per well on average, respectively). The Ph (bcr/abl)-positive cell fraction was reduced in both systems during culture, but in the 3D system, it was not as rapid as in the 2D system and showed a leukemic predominance. In conclusion, our “biomimetic osteoblast niche” might provide a more adaptive microenvironment for leukemic stem/progenitor cell growth. The biological characteristics of leukemic stem/progenitor cells were partially maintained. It was suggested that the 3D biomimetic niche might be a new tool for studying the behaviors of leukemic hematopoietic stem cells/hematopoietic progenitor cells in vitro.     

1α,25-Dihydroxyvitamin D(3) down-regulates pleiotrophin messenger RNA expression in osteoblast-like cells

Pleiotrophin (PTN)[heparin-binding-growth-associated molecule (HB-GAM), heparin-binding neurite-promoting factor (HBNF)] is a recently identified polypeptide that stimulates growth of fibroblasts and enhances neurite extension. PTN is expressed in many tissues but relatively high level of expression has been observed in brain and bone. We examined hormonal regulation of PTN mRNA expression in several osteoblast-like cell lines including MC3T3-E1 and ROS17/2.8. The levels of PTN mRNA in these cells was significantly reduced by treatment with 10⁻⁸ m 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) for 24 h. However, PTN mRNA levels were increased when the non-osteoblastic cell line, ROS 25/1, was treated with 1,25(OH)₂D₃. These effects were observed in a dose-dependent manner in a dose range between 10⁻¹¹ m to 10⁻⁸ m. This effect was specific to 1,25(OH)₂D₃, since PTN mRNA levels were not affected by other steroids such as retinoic acid and dexamethasone in MC3T3-E1 or ROS17/2.8 cells. Similar 1,25(OH)₂D₃ down-regulation of PTN mRNA was also observed in primary cultures of osteoblast-enriched fetal rat calvaria cells as well as cultures of MC3T3-E1 and ROS17/2.8 cells. These observations suggest that PTN expression in osteoblasts is regulated by the calcitropic hormone, 1,25(OH)₂D₃, and that PTN may play a role in vitamin D-dependent regulation of bone metabolism.     

Oxidative Inactivation of Surfactants

Reactive oxygen species (ROS) may play an important role in the chronic pulmonary morbidity of preterm infants. We therefore studied the magnitude and mechanisms of oxidative inactivation of a natural lung surfactant (NLS) and of two surfactants used for treatment of respiratory distress syndrome, beractant and KL₄ surfactant (KL₄). Incubation with Fenton reagents, 2-4 mM peroxynitrite (ONOO⁻) or 0.5 mM hypochlorous acid (OCl⁻), resulted in an increased minimum surface tension (MST) of all surfactants; the order of effect on MST was beractant > KL₄ > NLS. After incubation with Fenton reagents, NLS contained a higher concentration of conjugated dienes (p < 0.01) but lower concentration of malondialdehyde (p < 0.001) than beractant. Protein carbonyl concentrations after treatment with Fenton reagents were higher in NLS and KL₄ than in beractant (p < 0.05). Surface area cycling for 24 h with 2 mM ONOO⁻ or 0.5 mM OCl⁻ caused both beractant and KL₄ to increase the proportion of light subtypes from 8–10% to 26–29%; with Fenton reagents, there was disappearance of the light subtype and formation of ultraheavy subtype 74–91% with poor MST. Natural and therapeutic surfactants differ markedly in their sensitivity to ROS, which may be important for surfactants in therapeutic use because oxidative inactivation may limit their effect. Oxidation of natural surfactant may result in reduced function and contribute to chronic lung disease. Accepted for publication: 8 January 1999     

Antioxidant Function of Ambroxol in Mononuclear and Polymorphonuclear Cells in Vitro

This study quantifies the antioxidant function of ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine) in vitro. Polymorphonuclear cells (PMN) and mononuclear cells were isolated from the blood of healthy volunteers (n= 46) to determine reactive oxygen species (ROS) by luminol-enhanced chemiluminescence. Ambroxol or the controls N-acetylcysteine (NAC), nacystelyn (NAL), glutathione (GSH), superoxide dismutase (SOD), catalase, and the combination of SOD/catalase were incubated for 1 or 2 h with zymosan-activated cells in vitro using concentrations ranging from 10⁻⁶ to 10⁻³ mol/liter. Reduction of ROS-mediated luminescence was similar within the cell types. Ambroxol (10⁻⁴ mol/liter) reduced ROS about 75% (1-h incubation) and 98% (2-h incubation), respectively (p < 0.001). SOD and SOD/catalase, but not the H₂O₂-catalyzing substances (NAC, NAL, GSH, and catalase), reduced cellular ROS. This indicates that inflammatory cells predominantly generate O⁻ ₂, which can be scavenged by ambroxol. The antioxidant function of ambroxol with increasing incubation time suggests additional cellular antiinflammatory properties of this substance. Our results indicate that good antioxidant function of ambroxol is related mainly to direct scavenger function of reactive oxygen metabolites such as O⁻ ₂. However, an antioxidative effect of ambroxol may also be associated with the reduction of prooxidative metabolism in inflammatory cells. Concluding from this observation, and because of the well known high affinity of ambroxol for lung tissue, ambroxol may be an alternative in antioxidant augmentation therapy, particularly in pulmonary diseases characterized by an overburden of toxic oxygen metabolites. Accepted for publication: 5 December 1996     

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3

To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10⁻¹⁰–10⁻⁸ M 1,25-(OH)₂D₃ or 10⁻⁹–10⁻⁷ M 24,25-(OH₂)D₃, and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [³⁵S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)₂D₃ caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)₂D₃ responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)₂D₃; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)₂D₃. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification.     

T cells cooperate with passive antibody to modify Cryptococcus neoformans infection in mice

Cryptococcus neoformans is an encapsulated fungus that is a major cause of meningitis in patients with AIDS. In immunocompetent mice, administration of IgG1 mAb protects against cryptococcal infection, whereas administration of IgG3 is not protective and can accelerate the infection. In beige mice with impaired natural killer cell function, the effects of IgG1 and IgG3 are similar to those observed in immunocompetent mice, suggesting that natural killer cells are not crucial for antibody-mediated modulation of cryptococcal infection. In mice lacking CD4⁺ T cells, IgG1 is not protective and IgG3 accelerates infection, indicating that CD4⁺ T cells are required for antibody-mediated protection. In mice lacking CD8⁺ T cells, both IgG1 and IgG3 antibodies prolong survival, indicating that acceleration of the disease process by IgG3 involves CD8⁺ T cells. Both IgG1-mediated protection and IgG3-mediated acceleration of infection require interferon γ. These results reveal a functional dependence of passively administered antibody on cellular immunity in cryptococcal infection in mice and have implications for antibody-based therapies in humans in the setting of CD4⁺ lymphopenia.     

Nitric oxide–mediated chondrocyte cell death requires the generation of additional reactive oxygen species

Objective

Chondrocyte cell death, possibly related to increased production of endogenous nitric oxide (NO), has been observed during the pathogenesis of osteoarthritis and rheumatoid arthritis. The purpose of this study was to investigate the potential role of NO in causing chondrocyte cell death and to determine the contribution of other reactive oxygen species (ROS).

Methods

Cell death and cytotoxicity were evaluated in human articular chondrocytes in response to various NO donor compounds with and without agents that stimulate or inhibit the production of additional ROS using both the alginate bead and the monolayer culture systems. Cell death was quantified by a total cell count with fluorescent labels, and cytotoxicity was measured as a function of cellular NADH‐ and NADPH‐dependent dehydrogenase activity. To determine if the redox status of the chondrocyte could influence the observed effect of NO, cells were preincubated for 24 hours in L‐cystine– and glutathione (GSH)–depleted media to reduce intracellular GSH levels, a major defense mechanism against oxidative stress. Apoptosis was analyzed with the quantification of histone‐associated DNA fragments.

Results

Treatment of chondrocytes with peroxynitrite (ONOO⁻), 3‐morpholinosydnonimine (SIN‐1), and sodium nitroprusside (SNP) resulted in apoptotic cell death at concentrations of 0.5 mM, 1.0 mM, and 0.5 mM, respectively. In contrast, treatment of chondrocytes with diazeniumdiolates (or the “NOC” compounds, NOC‐5 and NOC‐12) at concentrations as high as 2.0 mM did not cause cell death. Furthermore, NOC‐5 and NOC‐12, at all concentrations tested (0.125–2.0 mM), could prevent cell death caused by oxidative stress. Selective ROS scavengers protected against cell death caused by either SIN‐1 or ONOO⁻; however, no protection could be afforded against the cytotoxicity of SNP with any of the ROS scavengers tested.

Conclusion

These results show that NO by itself is not cytotoxic to cultured chondrocytes and can even be protective under certain conditions of oxidative stress. Chondrocyte cell death from NO occurs under conditions where other ROS are also generated.

     

Stathmin levels in growth plate chondrocytes are modulated by vitamin D3 metabolites and transforming growth factor-beta1 and are associated with proliferation

Stathmin is a highly conserved, phosphorylated cytosolic protein that is found at decreased levels in all cells as they become more terminally differentiated, or when they decrease in their rate of proliferation. This study examined the hypothesis that stathmin levels in growth plate chondrocytes decreases as endochondral maturation increases. To test this hypothesis, we used a costochondral growth plate chondrocyte cell culture model. Cells derived from the resting zone (RC) express twice as much stathmin mRNA in culture and have twice as much stathmin protein as cells derived from the post proliferative growth zone ([GC];prehypertrophic and upper hypertrophic cell zones). Stathmin levels in vivo were assessed by immunohistochemistry. To assess the effects of agents that modulate proliferation and differentiation, RC and GC chondrocytes were cultured in the presence of 10⁻¹⁰ to 10⁻⁸ M 1α,25-(OH)₂D₃, which regulates proliferation in both cell types but affects differentiation of only GC cells, or 10⁻⁹ to 10⁻⁷ M 24R,25-(OH)₂D₃, which regulates differentiation and maturation of RC cells, but decreases proliferation of GC cells. In addition, RC cells were treated with 0.44 or 0.88 ng/mL of recombinant human transforming growth factor β1 (rhTGF-β1), which stimulates proliferation of RC cells and regulates proteoglycan production, but not alkaline phosphatase activity. Stathmin protein levels were determined using quantitative immunoblots, with recombinant human stathmin as a standard. The results show that stathmin levels are associated with proliferation. Proliferating chondrocytes in vivo exhibited higher levels of immunoreactive stathmin than either RC or GC cells in the growth plate. In culture, 1α,25-(OH)₂D₃ caused a dose-dependent de-crease in stathmin in RC and GC cells within 24 h. 24 R, 25-(OH)₂D₃ also reduced stathmin levels in GC cells within 24 h but only affected RC cells after prolonged exposures (96 h), at which time RC cells express a GC-like phenotype. rhTGF-β1 caused an increase in stathmin levels in RC cells. Stathmin levels are sensitive to protein kinase C (PKC) in other cells. Inhibition of PKC with chelerythrine had no effect on the response of RC cells to 1α,25-(OH)₂D₃ but it blocked the effect of rhTGF-β1, indicating that decreases in stathmin by vitamin D₃ metabolites may not be modulated by PKC, whereas increases in stathmin via rhTGF-β1 may be regulated via a PKC-dependent mechanism. These results support the hypothesis that constitutively expressed levels of stathmin are related to cell maturation state and that they are modulated by factors that regulate proliferation.     

Adoptive cell therapy using antigen-specific CD4<Superscript>−</Superscript>CD8<Superscript>−</Superscript> T regulatory cells to prevent autoimmune diabetes and promote islet allograft survival in NOD mice

Aims/hypothesis  

A new differentiation pathway for CD4⁻CD8⁻ (DN) T cells has recently been identified that exhibits the potent function of peripheral converted DN T cells in suppressing immune responses and provides the potential to treat autoimmune diseases. The aim of this study was to determine if the DN T cells converted from CD4⁺ T cells of NOD mice retain the antigen-specific regulatory capacity and prevent autoimmune diabetes in vivo. We also sought to determine if the combination of DN T cells with rapamycin promotes islet allograft survival in autoimmune diabetic NOD recipients.     

Demonstration of Low-Regulatory CD25High+CD4+ and High-Pro-inflammatory CD28−CD4+ T-Cell Subsets in Patients with Ulcerative Colitis: Modified by Selective Granulocyte and Monocyte Adsorption Apheresis

Low-CD25^High+CD4⁺, a subset of regulatory CD25⁺CD4⁺ T cells and high-inflammatory CD28⁻CD4⁺ T cells can exacerbate ulcerative colitis (UC). This study sought to investigate the frequency of CD25^High+CD4⁺ and CD28⁻CD4⁺ T cells in patients with UC and the changes in these cells during Adacolumn granulocyte and monocyte adsorption apheresis (GMA). Subjects were 12 patients with active UC, 11 with quiescent UC, and 14 healthy volunteers (HVs). The mean clinical activity index was 15.7 ± 2.2 in active UC and 4.5 ± 1.1 in quiescent UC. Peripheral blood samples were stained with CD4, CD25, and CD28 antibodies for flow cytometry. Patients with active UC received GMA and blood samples were examined before and after the first GMA session. Patients with active UC (P < 0.04) or quiescent UC (P < 0.02) had a higher percentage of CD28⁻D4⁺T cells compared with HVs, while the percentage of CD28⁺CD4⁺ T cells was lower in both UC groups compared with HVs (P = 0.03 and P < 0.02). Patients with active UC had a lower percentage of CD25^High+CD4⁺T cells compared with quiescent UC patients (P < 0.001). A significant increase in CD25^High+CD4⁺ T cells was associated with GMA (P < 0.03). Low CD25^High+CD4⁺ and high CD28⁻CD4⁺ are prominent features in UC. The increase in CD25^High+CD4⁺ T cells induced by GMA should contribute to improved immune function. Additional studies are warranted, since a low frequency of CD25^High+CD4⁺ ₋ and a high frequency of CD28⁻CD4⁺ ₋ expressing T cells might be a predictor of clinical response to GMA.     

Klotho protein activates the PKC pathway in the kidney and testis and suppresses 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression

Homozygous Klotho mutant (kl ^−/−) mice exhibit a variety of phenotypes resembling human aging, including arteriosclerosis, infertility, skin atrophy, osteoporosis, and short life span. Calcium abnormality, one of the phenotypes in kl ^−/− mice, is thought to be due to the elevated gene expression of 25-hydroxyvitamin D₃ 1α-hydroxylase in the kidney. We studied 25-hydroxyvitamin D₃ 1α-hydroxylase gene expression using a Klotho plasmid that we had previously constructed for Klotho protein production. It was found that Klotho protein medium upregulated cAMP and the PKC pathway, and suppressed 25-hydroxyvitamin D₃ 1α-hydroxylase in kidney cells. However, both cAMP and PKC are known to elevate 25-hydroxyvitamin D₃ 1α-hydroxylase gene expression, therefore, another unknown calcium regulation pathway using Klotho protein medium might exist. Furthermore, we found that activation of the PKC pathway by Klotho was observed only in the kidney and testis, where the Klotho gene is expressed, although activation of the cAMP pathway was observed in any kind of cell. These data suggest that calcium regulation through 25-hydroxyvitamin D₃ 1α-hydroxylase by Klotho depends on non-cAMP and a non-PKC pathway and that the Klotho protein may have different signaling pathways, depending on the Klotho gene expression in different cells and organs.     

Peroxynitrite-induced changes of thiol groups in human blood platelets

In this study we investigated sulfhydryl and disulfide metabolism in blood platelets treated with peroxynitrite (ONOO^?) at concentrations of 10, 100, and 1000?µM. In platelets incubated with peroxynitrite, a decrease of both protein and low molecular weight thiols (glutathione, cysteine and cysteinylglycine) was observed. The reduction of platelet free thiols caused by ONOO^? was dose dependent. We found that peroxynitrite induces the oxidation of platelet free SH-groups. Platelets contain a large fraction of glutathione in reduced form (GSH). We showed that after ONOO^? treatment, GSH/GSSG ratio in platelets was distinctly decreased. The presented results suggest that oxidation of thiols and depletion of GSH in platelets caused by peroxynitrite may be responsible for cytotoxic effects of this compound.     

Deficiency in TIMP-3 increases cardiac rupture and mortality post-myocardial infarction via EGFR signaling: beneficial effects of cetuximab

Cardiac rupture is a fatal complication of myocardial infarction (MI); however, its underlying molecular mechanisms are not fully understood. This study investigated the role of tissue inhibitor of metalloproteinase-3 (TIMP-3)/matrix metalloproteinase (MMP)/epidermal growth factor (EGF)/transforming growth factor (TGF)-β1 pathway in infarct healing and effects of cetuximab on cardiac rupture after MI. Induction of MI was achieved by left coronary artery ligation in wild-type (WT) and TIMP-3^−/− mice. TIMP-3 deficiency resulted in a fourfold increase in cardiac rupture and 50% decrease in survival after MI. Hydroxyproline content, collagen synthesis and myofibroblast cell number in the infarct region, and the force required to induce rupture of the infarct scar were significantly decreased, while MMP activity was increased in TIMP-3^−/− mice. EGF proteins were increased by threefold in TIMP-3^−/− mice following MI, while TGF-β1 mRNA levels were decreased by 68%. Cell proliferation of cultured adult cardiac myofibroblasts was significantly decreased in TIMP-3^−/− compared to WT myofibroblasts. EGF treatment significantly decreased collagen synthesis and TGF-β1 expression. Conversely, TGF-β1 treatment increased collagen synthesis in cardiac myofibroblasts. Treatment with cetuximab significantly decreased the incidence of cardiac rupture and improved survival post-MI in TIMP-3^−/− mice. We conclude that deficiency in TIMP-3 increases cardiac rupture post-MI via EGF/epidermal growth factor receptor (EGFR) signaling which downregulates TGF-β1 expression and collagen synthesis. Inhibition of EGFR by cetuximab protects against cardiac rupture and improves survival post-MI.     

NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

Human aging is characterized by changes in the immune system which have a profound impact on the T-cell compartment. These changes are more frequently found in CD8+ T cells, and there are not well-defined markers of differentiation in the CD4+ subset. Typical features of cell immunosenescence are characteristics of pathologies in which the aberrant expression of NKG2D in CD4+ T cells has been described. To evaluate a possible age-related expression of NKG2D in CD4+ T cells, we compared their percentage in peripheral blood from 100 elderly and 50 young adults. The median percentage of CD4+ NKG2D+ in elders was 5.3% (interquartile range (IR): 8.74%) versus 1.4% (IR: 1.7%) in young subjects (p < 0.3 × 10⁻¹⁰). CD28 expression distinguished two subsets of CD4+ NKG2D+ cells with distinct functional properties and differentiation status. CD28+ cells showed an immature phenotype associated with high frequencies of CD45RA and CD31. However, most of the NKG2D+ cells belonged to the CD28^null compartment and shared their phenotypical properties. NKG2D+ cells represented a more advanced stage of maturation and exhibited greater response to CMV (5.3 ± 3.1% versus 3.4 ± 2%, p = 0.037), higher production of IFN-γ (40.56 ± 13.7% versus 24 ± 8.8%, p = 0.015), lower activation threshold and reduced TREC content. Moreover, the frequency of the CD4+ NKG2D+ subset was clearly related to the status of the T cells. Higher frequencies of the NKG2D+ subset were accompanied with a gradual decrease of NAIVE and central memory cells, but also with a higher level of more differentiated subsets of CD4+ T cells. In conclusion, CD4+ NKG2D+ represent a subset of highly differentiated T cells which characterizes the senescence of the immune system.     

Hypocalcemia reduces insulin turnover but not insulin-mediated glucose metabolism in piglets

Disturbances of glucose metabolism similar to type II diabetes are often accompanied by low extra cellular calcium concentrations, as shown in periparturient ruminants. Hypocalcemia reduces insulin secretion and possibly endogenous glucose production, but whether hypocalcemia also affects peripheral actions of insulin is not clear. The problem was investigated with piglets in this study. Insulinmediated turnover of glucose and the clearance of insulin were measured in 7 control piglets (+D) and in 10 piglets with inherited calcitriol deficiency (−D). The measurements involved continuous intravenous infusions of insulin (1, 2, 5, 7.5 and 10 mU·kg⁻¹·min⁻¹) and glucose to maintain a glucose concentration of 7 mmol · l⁻¹ (combined insulin and glucose clamps). Both groups of piglets were studied under normo- and hypocalcemic conditions. During hypocalcemia and with equal insulin infusion rates, −D and +D piglets attained significantly higher (28%–133%) steady state insulin concentrations in plasma than during normocalcemia. This showed that the clearance rates of insulin during hypocalcemia were 48% lower in +D and 20% lower in −D piglets than during normocalcemia. With equivalent insulin infusion rates +D piglets developed significantly higher (15%–48%) steady state insulin concentrations in plasma than −D piglets, indicating that the clearance rate of insulin was higher in the calcitriol-deficient state. The relationship between insulin concentration in plasma and glucose turnover (measured with [3-³H]glucose) was not influenced by the calcemic and vitamin D status of the piglets. This indicated that the peripheral action of insulin on glucose metabolism was not influenced by the extracellular calcium concentration and vitamin D status. Received: March 2001 / Accepted in revised form: July 2002 Correspondence to C. Schlumbohm