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M Christ

Publications and source records attributed to M Christ.

At least 55 records · Page 3Linked to original sources
PubMed1999

Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: requirement for E4 ORF3.

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022-2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

Adenoviridae↗
PubMed1999

Specific progesterone binding to a membrane protein and related nongenomic effects on Ca2+-fluxes in sperm.

Rapid, nongenomic effects of steroids are supposed to be transmitted by membrane receptors unrelated to the classic intracellular steroid receptors. In this context, a putative progesterone membrane binding protein (mPR) has been identified, recently. Here we show that expression of mPR-cDNA in CHO cells leads to increased microsomal progesterone binding. This result is mirrored by effects of an antibody raised against the recombinant E. coli mPR which suppressed the rapid progesterone-initiated Ca2+ increase in sperm. Our results support the assumption that mPR represents the first steroid membrane receptor or a part of it involved in rapid, nongenomic steroid signalling.

Animals↗
PubMed1999

Short term cardiovascular effects of aldosterone in healthy male volunteers.

Clinical evidence of rapid, nongenomic aldosterone effects in the cardiovascular system has been provided by clinical studies; an increase in systemic vascular resistance (SVR) was shown by invasive techniques within 3 min after injection of aldosterone. Here, we study the dose dependency and the later course of the rapid aldosterone effects by noninvasive techniques. In 12 healthy male volunteers, SVR and heart rate variability were determined by impedance cardiography and digital electrocardiography, respectively, for 8 h after the injection of 0.05 or 0.5 mg aldosterone in a double blind, placebo-controlled, 3-fold cross-over study. No significant differences were observed for baseline values among the three treatments. The area under the curve of SVR during the first 45 min after injection was significantly different between the periods with the highest areas under the curve seen after the injection of 0.5 mg aldosterone (mean +/- SD, 40.4 +/- 12.8 vs. 36.8 +/- 10.3 for 0.05 mg aldosterone and 36.8 +/- 10.4 for placebo; P = 0.05). Individual comparisons showed significant differences at 6 and 30 min between placebo and the 0.5 mg aldosterone period (P < 0.05), with values for the 0.05 mg aldosterone period similar to those for the placebo period. From 330-390 min, opposite changes occurred; SVR was depressed during the 0.05 mg (P < 0.05) and 0.5 mg aldosterone periods compared with that during the placebo period. These delayed effects may reflect an increased vagal tone in the aldosterone groups, as demonstrated by higher values of the time domain parameter of heart rate variability pNN50. This study provides further evidence for clinically detectable rapid cardiovascular aldosterone effects in vivo obtained by noninvasive techniques. The data are consistent with the view of aldosterone as a rapid modulator of cardiovascular responses acting through nongenomic mechanisms.

Adult↗
PubMed1999

Rapid aldosterone effects on tyrosine phosphorylation in vascular smooth muscle cells.

Non-genomic aldosterone effects are characterized by their rapid onset, their specificity for mineralocorticoids and their insensitivity both to the mineralocorticoid type 1 receptor antagonist spironolactone and to the inhibitors of transcription and translation, cycloheximide and actinomycin D. The aim of the present study was to further characterize the second messenger system involved in the non-genomic pathway of aldosterone with particular emphasis on protein phosphorylation. The rapid increase of free intracellular calcium by aldosterone in VSMC is sensitive to genistein, so that tyrosine kinase activity appears likely to be involved in the signaling pathway. Here, the effect of 100 nmol/l aldosterone (10 min.) on tyrosine protein-phosphorylation was determined in VSMC. Our findings show that aldosterone (100 nmol/l) in combination with shear stress as additional stimulus induces a rapid (within 10 min.) small but consistent increase in tyrosine-phosphorylation compared with aldosterone or shear stress alone. Immunoprecipitation of the MAPK-isoforms ERK 1 and ERK 2 showed an increased phosphorylation after 3 and 5 min.

Aldosterone↗
PubMed1998

Cardiovascular steroid actions: swift swallows or sluggish snails?

Steroid actions on the vascular wall have been thought to depend on direct, genomic mechanisms being characterized by a considerable delay and on secondary events, including changes of coagulation, plasma lipids, and renal electrolyte and volume regulation. Recently, rapid effects of steroids on the vascular wall have been reported being clearly incompatible with the classical theory of genomic steroid action. As these effects occur in classical target tissues for genomic steroid action, and modulation of intracellular signaling has been shown to influence genomic steroid action, a two-step model of steroid action was developed integrating both genomic and nongenomic aspects and their possible interaction. This review summarizes recent studies on both types of direct, vascular steroid actions, the swift and the sluggish ones, and discusses the role of these actions in regulation of circulatory homeostasis and their potential therapeutic implications.

Adrenal Cortex Hormones↗
PubMed1998

Levonorgestrel capsule implants in the United States: a 5-year study.

OBJECTIVE: To measure effectiveness, adverse event experience, and acceptability of the Food and Drug Administration-approved variant of levonorgestrel capsule implants in the United States through 5 years and to examine determinants of these outcomes. METHODS: In a prospective, multicenter study, 511 sexually active women selecting contraceptive implants were monitored four times in the 1st year, then semiannually through 5 years. Adverse events were elicited by query and physical examination, and their incidence was measured. Lifetable analyses computed pregnancy and other discontinuation rates. Cox regression models examined effects of age, parity, and preadmission desire for more children on continuation. Removal times were analyzed by analysis of variance. RESULTS: Three pregnancies occurred, yielding a 5-year cumulative rate of 1.3+/-0.8 per 100 users, an average annual rate of three per 1000 women, and an ectopic pregnancy rate of 0.6 per 1000 woman years. No pregnancies occurred to women weighing less than 79 kg. Prolonged or irregular menstrual bleeding, followed distantly by headache, weight gain, and mood changes, was the most frequent medical conditions leading to removal. Weight gain averaged 1 kg per year. Each annual continuation rate was above 80 per 100, for a cumulative 5-year rate of 39 per 100. Continuation was age-dependent, with younger women (younger than 25 years at entry) having lower 5-year continuation rates than older subjects (P < .01). Tissue trauma from deeply placed or poorly aligned implants or severe reactions to local anesthetic affected subjects in 3.1% of removals (nine cases). CONCLUSION: As measured by annual continuation rates of 80 per 100 or higher and annual pregnancy rates below one per 100, implant contraception in the United States was found to be highly acceptable and effective, year after year, regardless of the woman's age or family formation status. The cumulative 5-year pregnancy rate, 1.3 per 100, is comparable to that of tubal ligation.

Adult↗
PubMed1998

Nongenomic steroid actions: completing the puzzle. Aldosterone as an example.

In the common unidimensional theory of steroid action, steroids bind to intracellular receptors and modulate nuclear transcription and thus protein synthesis. These genomic steroid effects, being characterized by their delayed onset of action and their dependence on transcription and protein synthesis, have been known for several decades. In contrast, very rapid actions of steroids, which are considered to be of nongenomic origin, have been recognized more widely and characterized in detail only during the past ten years. Specific rapid effects of steroids and related hormones like vitamin D3 and thyroid hormones on cellular function involve a conventional second messenger cascade which in most cases includes phospholipase C, phosphoinositide turnover, intracellular pH and intracellular calcium ([Ca2+]i), and protein kinase C. Furthermore, binding sites in membranes have been characterized exposing binding features compatible with an involvement in rapid-steroid signaling. Characteristics of putative membrane receptors are completely different from those of classic intracellular steroid receptors; this also includes the inability of classic steroid receptor antagonists to inhibit those rapid nongenomic steroid actions. The physiological and pathophysiological relevance of these effects is still largely unclear, but their existence has been proven recently even under in vivo conditions. New drugs modulating nongenomic steroid actions may find applications in various areas such as the cardiovascular and central nervous systems, infertility and electrolyte homeostasis. This short review focuses mainly on the nongenomic actions of aldosterone and their cardiovascular implications.

Aldosterone↗
PubMed1998

In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted.

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Adenovirus E1 Proteins↗
PubMed1998

Rapid cardiovascular action of aldosterone in man.

Rapid nongenomic in vitro effects of aldosterone have been demonstrated recently in cultured vascular smooth muscle and endothelial cells. But there is, as yet, little evidence for corresponding in vivo effects. The present study thus investigates the rapid nongenomic effects of aldosterone on human cardiovascular function. In a double-blind placebo-controlled randomized parallel trial on 17 patients with suspected coronary heart disease, the effect of 1 mg aldosterone iv on cardiovascular function was assessed during cardiac catheterization. Hemodynamic parameters (such as heart rate, left ventricular and atrial pressures, arterial pressures, vascular resistances, and cardiac output) were measured before and 3 and 10 min after administration of aldosterone or placebo. Significant changes were found for systemic vascular resistance, cardiac output, and cardiac index, compared with the placebo group (Wilcoxon test, P < 0.02-0.05). The effect of aldosterone dissipated within 10 min. The results are in line with the in vitro data cited above and consistent with earlier findings on acute cardiovascular effects of aldosterone, which have now been confirmed and extended by contemporary techniques. The hypotheses of rapid nongenomic in vivo effects of aldosterone are further substantiated by this study.

Aldosterone↗
PubMed1997

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response.

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Adenoviruses, Human↗
PubMed1997

[Genetic therapy for hemophiliacs--therapeutic potential and technological limits].

Defects in the genes encoding the human coagulation factor VIII (hFVIII) and IX (hFIX) result in life-threatening haemorrhages and severe arthropathies. While haemophiliacs are currently treated by blood-derived factors or recombinant hFVIII and hFIX, a number of recent technical advances make the prospect of using gene therapy to treat such genetic diseases a realistic goal. Several gene therapy strategies have therefore been developed and evaluated in recent years. Most of the initial protocols were ex vivo gene transfer approaches in which the target cells (fibroblasts, keratinocytes, myoblasts, ...) were expanded and genetically-engineered in the laboratory and then implanted in the host. However, the complexity of most ex vivo gene therapy strategies, together with the disappointing results obtained in various animal models stimulated the development of more direct in vivo gene therapy protocols. In aiming to establish such an in vivo gene transfer protocol for haemophilia B, we constructed and tested in vitro and in vivo various recombinant adenovirus vectors expressing human FIX. Intravenous administration of this vector into various strains of immunocompetent and immunodeficient mice led to an efficient hFIX gene transfer in liver and lung. As a consequence, the hFIX protein was correctly produced and secreted at high levels in the blood of the treated animals. However, expression was transient in all immunocompetent mice, except surprisingly in C57B1/6 animals. A systematic molecular and immunological analysis allowed us to identify the parameters that prevent the long-term in vivo expression of the human molecule and to improve the current adenovirus vectors.

Adenoviridae↗
PubMed1997

Adenovirus-mediated gene transfer: influence of transgene, mouse strain and type of immune response on persistence of transgene expression.

E1-deleted adenovirus (Ad) vectors expressing the human coagulation factor IX (hFIX) or the bacterial beta-galactosidase (lacZ) were injected intravenously into various strains of immunocompetent (C57BI/6, BALB/c, CD1, CBA/J, C3H) and immunodeficient (BALB/c-nu/nu, C57BI/6-nu/nu, SCID, NIH-bg-nu-xid) mice. Regular analysis of mouse sera and tissues showed a persistent expression of both transgenes in immunodeficient mice, while detection diminished very rapidly in immunocompetent mice. The mechanisms responsible for the transient detection of the two transgenes were however not identical. Rapid decline of lacZ expression was correlated with a rapid decrease of viral DNA sequences, and consequently to the induction of a cellular immune response to the lacZ antigen. In contrast, absence of detectable levels of serum hFIX in immunocompetent animals was not associated with a loss of viral DNA but was strictly correlated with the induction of anti-hFIX antibodies. Surprisingly, anti-hFIX antibodies were never detected in C57BI/6 mice, leading to prolonged detection of hFIX. These results suggest that cellular immunity to viral antigens plays a minor role in the early extinction of transgene expression and illustrate the influence of the cellular (eg lacZ) or humoral (eg hFIX) immunity to transgene-encoded products on the persistence of transgene expression.

Adenoviridae↗
PubMed1997

Nongenomic effects of aldosterone on intracellular calcium in porcine endothelial cells.

Rapid in vitro effects of aldosterone on intracellular electrolytes, cell volume, and the sodium-proton antiport have been described in human mononuclear leukocytes and vascular smooth muscle cells. In the present study, we demonstrate rapid aldosterone effects on free intracellular calcium as determined by fura 2 fluorometry in single porcine endothelial cells. After addition of 100 nmol/l aldosterone, cells respond with a sustained rise in free intracellular calcium by approximately 50% of initial levels within 1-5 min. Elevations are predominantly seen in the subplasmalemmal space. Effective half-maximal concentration values for aldosterone are approximately 1 pmol/l and for cortisol approximately 1 nmol/l. These effects are blunted in calcium-free medium and absent after pretreatment by thapsigargine. They remain unchanged by a >1,000-fold excess of spironolactone. These findings indicate the existence of a nongenomic pathway for aldosterone action in porcine endothelial cells and may be related to known rapid cardiovascular effects of aldosterone in vivo mediated through the baroreceptor reflex.

Aldosterone↗
PubMed1996

Nongenomic effects of aldosterone on intracellular pH in vascular smooth muscle cells.

The aim of the present study was to investigate rapid effects of aldosterone and other steroids on intracellular pH of vascular smooth muscle cells and to compare these effects with those of peptide hormones. After addition of 100 nmol/L aldosterone, initial acidification is followed by significant alkalinisation occurring within two minutes, while 1 mumol/L hydrocortison does not affect intracellular pH. The initial response to 100 nmol/L angiotensin II is similar; however, subsequent alkalinization is not seen for this agonist. PDGF induces an initial acidification followed by a minor recovery so that cells remain acidified for eight minutes. Both pH recovery after angiotensin II and alkalinization after aldosterone were blocked in sodium-free medium. These results demonstrate rapid effects of aldosterone on intracellular pH in vascular smooth muscle cells, which include final alkalinization not seen after angiotensin II or PDGF.

Aldosterone↗
PubMed1996

Spontaneous changes of heart rate, blood pressure, and ischemia-type ST-segment depressions in patients with hypertension without significant coronary artery disease: beneficial effects of beta-blockade.

In hypertensives, a reduced coronary flow reserve is observed and may contribute to angina pectoris and silent (painless) myocardial ischemia, which frequently occur in these patients even in the absence of coronary artery disease (CAD). To assess the frequency of ischemia-type ST-segment depressions in these patients and the influence of heart rate (HR) and blood pressure (BP) as major determinants of myocardial oxygen demand and to test the effects of beta-blocker therapy (10-20 mg betaxolol/day for 4 weeks) on these variables, simultaneous 24-h Holter and 24-h ambulatory BP monitoring was performed in 19 patients with hypertension (age, 43-71 years; nine women, 10 men) without CAD (stenosis < 50% in angiography). Before treatment, 25 periods of significant ST-segment depressions with a total duration of 470 min were observed in nine patients. ST-segment depressions were significantly correlated with preceding increases in HR and the rate-pressure product. The majority (79%) of episodes with ST-segment depression were clinically painless. In this open study, beta-blockade significantly decreased the number of episodes with ST-segment depressions to six in four of 15 patients and the total duration to 38 min (p < 0.05). The data demonstrate that HR seems to be associated with the development of ischemic ST-segment deviations in patients with hypertension without CAD. Antihypertensive therapy in these patients should target not only sufficient BP control, but also reduction of ischemic events.

Adrenergic beta-Antagonists↗
PubMed1995

Rapid aldosterone signaling in vascular smooth muscle cells: involvement of phospholipase C, diacylglycerol and protein kinase C alpha.

Rapid in vitro effects of aldosterone (ALDO) on intracellular sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in human mononuclear leukocytes and rat vascular smooth muscle cells (VSMC). These nongenomic effects are signaled through membrane receptors with a high affinity for aldosterone, but not for hydrocortisone. Effects of ALDO on the production of diacylglycerol (DAG) and protein kinase C alpha (PKC) were measured in VSCM by enzymatic assay and immunoblotting. DAG production was stimulated twofold by ALDO (> or = 1 nM) within 30 sec while hydrocortisone was inactive at concentrations of up to 1 microM. The inhibitors of phospholipase C, neomycin and U-73122 completely blocked this effect. PKC translocation from cytosol to membranes by ALDO occurred within 5 min, the extent of this effect was comparable to that of angiotensin II. These data demonstrate rapid intracellular signaling for ALDO in VSMC through phospholipase C, DAG and PKC in addition to calcium and inositol-1,4,5-trisphosphate as determined earlier.

Aldosterone↗
PubMed1995

Characterization and solubilization of novel aldosterone-binding proteins in porcine liver microsomes.

Using the radioligand [1,2,6,7-3H]aldosterone ([3H]aldosterone), specific binding sites for aldosterone were identified and characterized in microsomal preparations from porcine liver. The maximum binding capacity is approximately 700 fmol x mg-1 microsomal protein. The reversible binding of [3H]aldosterone was saturable and Scatchard analysis revealed two apparent dissociation constants (Kd), Kd1 < or = 11 nM and Kd2 = 118 nM. Binding was optimal at pH 7.2, thermolabile, and was reduced by more than 70% when membrane vesicles were pretreated with trypsin. Binding was selective for aldosterone with cortisol being a weak agonist at 1000-fold higher concentrations only. Among those detergents tested to optimize conditions for solubilization, n-octylglucoside (75 mM) was most favorable and solubilized 25% of the radioligand-binding protein complex in the undissociated form. These binding sites have unique pharmacological properties, which are similar to those found for aldosterone membrane binding in human lymphocytes and pig kidney, and for rapid aldosterone effects on sodium-proton exchange.

Aldosterone↗