Adenovirally transferred p16INK4/CDKN2 and p53 genes cooperate to induce apoptotic tumor cell death.

Repression of cell cycle progression by tumor suppressors might provide a means for tumor therapy. Here we demonstrate that ectopic overexpression of the p16INK4/CDKN2 tumor suppressor from an adenovirus vector in various cell lines results in block of cell division and, subsequently, in a gradual reduction of the levels of the product of retinoblastoma susceptibility gene, pRb. Overexpression of p53 and p16INK4/CDKN2, but not p53 on its own, induces apoptotic death only in tumor cells. Simultaneous adenoviral transfer of p16 and p53 genes leads to inhibition of tumor growth in nude mice. These results suggest that combined delivery of two cooperating genes like p16 and p53 could be the basis for the development of a new strategy for cancer gene therapy.

Drug-activated multiple pathways of defensin mRNA regulation in HL-60 cells are defined by reversed roles of participating protein kinases.

Defensin transcription in HL-60 promyelocytic leukemia cells is greatly enhanced during retinoic acid (RA)-induced differentiation. We have probed this regulatory pathway by selective modulation of various kinase activities. Induction was potentiated by elevated cAMP and attenuated by protein kinase C inhibition, entirely correlated to enhanced or blocked morphological differentiation, respectively. Yet, defensin mRNA was also induced in undifferentiated HL-60 cells, but not in others, by cAMP alone. By contrast, modulators that cooperated with RA had adverse effects on the normal capacity of dimethyl sulfoxide to up regulate these transcripts as well. Thus, defensin mRNA accumulation can be selectively uncoupled from maturation stage; and transcript levels may be regulated by multiple pathways, each independently acted upon by different chemical inducers.

Clinical experience with implantable atrial and combined atrioventricular defibrillators.

UNLABELLED: The high prevalence of atrial fibrillation (AF) and its clinical complications, the poor efficacy of medical therapy for preventing recurrences, and dissatisfaction with alternative modes of therapy stimulated interest in implantable atrial and combined atrioventricular defibrillators. In a multicenter study, the safety and efficacy of a stand alone implantable atrial defibrillator, the Metrix system, were evaluated. The device was implanted in 51 patients with highly symptomatic episodes of AF refractory to pharmacological treatment. During a follow-up of 9 months, 96% of 227 spontaneous AF episodes were successfully converted to sinus rhythm in 41 patients. In 62 episodes (27%), several shocks and/or additional drug treatment were required to maintain stable sinus rhythm because of early recurrences of AF. A total of 3719 shocks were delivered and no induction of ventricular proarrhythmia or inaccurately synchronized shocks occurred. The AF detection algorithm exhibited a 100% specificity for the recognition of sinus rhythm and a 92.3% sensitivity for the detection of AF. The combined atrioventricular defibrillator, Jewel AF 7250, was evaluated in a multicenter, randomized, cross-over trial. The primary study objectives included: overall safety as determined by complications-free survival at 6 months, efficacy of tiered atrial pacing and defibrillation therapies for termination of spontaneous atrial tachycardias (AT) and AF, and relative sensitivity of a new dual-chamber detection algorithm. The device was implanted in 211 patients with either a history of ventricular tachyarrhythmias (VT/VF) alone or with a history of both AT/AF and VT/VF. During a mean follow-up of 4.5 months, it has been shown that the Jewel AF is safe and effective in treating atrial and ventricular tachyarrhythmias. Pace termination of 85% of AT episodes were achieved with painless delivery of antitachycardia pacing; additional 35% of AT episodes were terminated by high frequency burst pacing. CONCLUSIONS: The stand alone implantable atrial defibrillator may be safe and clinically useful in selected patients for the treatment of highly symptomatic, drug resistant recurrences of AF. The combined atrioventricular defibrillator may be particularly indicated in patients presenting with both a history of atrial and ventricular tachyarrhythmias.

[p16 and p53 genes transferred with the help of adenovirus to induce apoptic tumor cell death]. / p16- og p53-generne overført ved hjaelp af adenovirus samarbejder for at inducere apoptotisk tumorcelledød.

Based on studies of the cell cycle in tissue cultures of cancer cell lines and cells from normal tissue interference in the regulatory network of pRB/cdk4/cyclin D1/p16 in combination with the tumour suppressor gene p53 was investigated. It was shown that overexpression of p53 and p16, but not p53 on its own, induced apoptotic cell death only in tumour cells. Gene transfer of the same two genes to tumours transplanted subcutaneously in mice also caused fast regression of some of the tumours.

The retinoblastoma protein: a master regulator of cell cycle, differentiation and apoptosis.

The retinoblastoma susceptibility gene is a tumour suppressor and its product retinoblastoma protein (pRb) has been known for 10 years as a repressor of progression towards S phase. Its major activity was supposed to be sequestration or inactivation of the transcription factor E2F which is required for activation of S phase genes. However, within recent years growing evidence has been accumulating for a more general function of pRb at both the transcriptional level and the cellular level. pRb not only regulates the activity of certain protein-encoding genes but also the activity of RNA polymerase pol I and pol III transcription. This protein appears to be the major player in a regulatory circuit in the late G1 phase, the so-called restriction point. Moreover, it is involved in regulating an elusive switch point between cell cycle, differentiation and apoptosis. Here, it seems to cooperate with another major tumour suppressor, p53. Thus, pRb sits at the interface of the most important cell-regulatory processes and therefore deserves close attention by specialists from different fields of research. This review provides an introduction to the complex functions of pRb.

Cross-linked amino acids in the protein pairs L3-L19 and L23-L29 of Bacillus stearothermophilus ribosomes after treatment with diepoxybutane.

Treatment of native 50 S ribosomal subunits of Bacillus stearothermophilus with the homobifunctional cross-linking reagent diepoxybutane generated two cross-linked protein pairs, L3-L19 and L23-L29, which were isolated and identified. The analysis of the cross-linking sites at the amino acid level in both protein pairs is presented. Using a combination of sequence analysis and mass spectrometry it could be demonstrated that His-28 in protein L3 and the N-terminal amino acids Met-1, His-2, and His-3 in protein L19 are involved in forming the cross-link L3-L19. Within the pair L23-L29 Met-1 in protein L23 and Lys-4 in protein L29 were identified as cross-linking sites employing a similar approach. Comparison of our data with results derived from other cross-linking experiments showed that in general the structural organization of the ribosomes in eubacteria (the Gram-positive B. stearothermophilus and the Gram-negative Escherichia coli) has been conserved to quite an extent during evolution but that the fine structures differ slightly. By mass spectrometry the specificity of diepoxybutane and its cleaving mechanism using sodium periodate could be examined. In addition the complete amino acid sequence of protein L19 of B. stearothermophilus has been determined and revealed 58% identical amino acid residues to the homologous E. coli protein L19.

Primary structures of ribosomal proteins L3 and L4 from Bacillus stearothermophilus.

Ribosomal proteins L3 and L4 were purified to homogeneity from total protein isolated from the 50S subunit of Bacillus stearothermophilus by reversed-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences of both proteins were determined by automated N-terminal sequence analysis and sequencing of internal peptides. Using oligonucleotides deduced from the N-terminal region of protein L3 as hybridization probes, a DNA fragment coding for proteins L3, L4 and the N-terminal part of protein L23 has been identified, cloned and sequenced. The organization of the genes is identical to that found in the S10 operon of Escherichia coli. Comparison of the sequences of proteins L3 and L4 with those of other organisms revealed that all proteins of the L3 family are highly conserved. On the other hand, the archaebacterial L4 proteins show no significant sequence similarity to the E. coli L4 protein whereas the L4 protein of B. stearothermophilus is significantly similar to all of the L4 proteins and thus justifies the membership of all the L4 proteins in one protein family. The results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes and possible functional domains of proteins L3 and L4.

Homology and distribution of CO dehydrogenase structural genes in carboxydotrophic bacteria.

The 17 (S), 30 (M) and 87 kDa (L) subunits of CO dehydrogenases from the CO-oxidizing bacteria Pseudomonas carboxydoflava, Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans OM5 were isolated and purified. The N-terminal sequences of same subunits from different bacteria showed distinct homologies. Dot blot hybridization employing oligonucleotide probes derived from the sequences of the S-subunit of P. carboxydovorans OM5 and the M-subunit of P. carboxydohydrogena and DNA of the plasmid-containing CO-oxidizing bacteria Alcaligenes carboxydus, Azomonas B1, P. carboxydoflava, P. carboxydovorans OM2, OM4 and OM5 indicated that all genes encoding these subunits reside on plasmids. That in P. carboxydovorans OM5 CO dehydrogenase structural genes are located entirely on plasmid pHCG3 was evident from the absence of hybridization employing DNA from the cured mutant strain OM5-12. CO dehydrogenase structural genes could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, P. carboxydohydrogena and P. carboxydovorans OM3. There was no example of a plasmid-harboring carboxydotrophic bacterium that did not carry CO dehydrogenase structural genes on the plasmid. The N-terminal sequences of CO dehydrogenase structural genes were found to be conserved among carboxydotrophic bacteria of distinct taxonomic position, independent of the presence of plasmids. It is discussed whether this might be the consequence of horizontal gene transfer.

Distinct temporal patterns of defensin mRNA regulation during drug-induced differentiation of human myeloid leukemia cells.

Defensins are microbicidal peptides and the principal constituents of neutrophil primary granules. They are presumed to play a prominent role in innate host defenses. We examined defensin mRNA levels during drug-induced differentiation of the promyelocytic leukemia cell line, HL-60. Transcription was restricted to promyelocyte, myelocyte, and very early metamyelocyte stages of the granulocytic pathway. Complete downregulation occurred during late granulocytic maturation or early during phorbol ester-promoted differentiation along the monocyte/macrophage lineage. Retinoic acid (RA) was the strongest inducer of defensin mRNA accumulation, even at doses too low to effect morphologic changes; the initial (first 48 hours), gradual increase resulted from transcriptional activation and was enhanced by granulocyte colony-stimulating factor. In contrast, addition of hybrid polar compounds led to a transient, drug-specific downregulation within the same time period, apparently by means of selectively induced, biphasic degradation of transcripts. Subsequent increase in transcript levels was faster and more pronounced with hexamethylene bisacetamide, relative to dimethyl sulfoxide (DMSO). DMSO-promoted effects were strikingly different in serum-free medium or in the presence of the tyrosine kinase inhibitor, genistein. Under these conditions, and although differentiation was unaffected, early defensin mRNA downregulation was final. The effect did not occur with RA and expression of other myeloid-specific genes was also unchanged. Addition of selected cytokines caused a similar "dip," only at earlier times and uncoupled from differentiation. Tumor necrosis factor-alpha markedly induced defensin levels after 2 days in previously untreated HL-60 cells, but inhibited expression in RA-differentiated cells. These results begin to detail a complex regulation of defensin mRNA synthesis with both spatial and temporal control elements, and a unique modulation by chemical agents, cytokines, and serum-factors.

Computed tomographic demonstration of cervical abscess and jugular vein thrombosis. A complication of intravenous drug abuse in the neck.

Cervical abscess and internal jugular vein thrombosis occurred in a 36-year-old man with a history of drug abuse. Computed tomography (CT) and angiography correctly identified the pathologic abnormalities that were surgically confirmed. We stress the use of CT in the evaluation of this condition.

[A new dual-chamber defibrillation system: duration, frequency and circadian variation of recurrent supraventricular tachyarrhythmias]. / Ein neuartiges Zwei-Kammer-Defibrillations-system: Dauer, Frequenz und zirkadiane Variation supraventrikulärer Tachyarrhythmierezidive.

BACKGROUND: The treatment of concomitant atrial tachyarrhythmias in patients with malignant ventricular tachyarrhythmias is a major challenge for new defibrillator devices. Atrial fibrillation is not only responsible for inappropriate ventricular therapies, but also reduced left ventricular performance, especially in patients with heart failure and severely depressed left ventricular function. Furthermore, it is a strong risk factor for the development of thromboembolism. NEW SYSTEM: A new dual-chamber implantable defibrillator is capable of tiered atrial therapies for both regular and irregular atrial tachyarrhythmias. In first investigations a high sensitivity and specificity could be shown as well as a promising therapy efficacy of atrial antitachycardia ramp and burst pacing for the treatment of atrial tachycardias. Atrial ramp pacing has shown to be successful for regular atrial tachyarrhythmias in up to 60 to 70% of all episodes. The results have supported a programming of a high first shock energy for treatment of atrial fibrillation. The incidence of atrial fibrillation in patients with a history of atrial fibrillation or without is much higher in the present investigated patient populations than expected. CONCLUSION: The more complicated and subtle new dual-chamber detection algorithm has proven to be safe and effective both for the detection of ventricular tachycardia but also in terms of an increase of specificity and a reduction of inappropriate ventricular therapies for atrial tachyarrhythmias.

Potential device interaction of a dual chamber implantable cardioverter defibrillator in a patient with continuous spinal cord stimulation.

Spinal cord or thalamic deep brain stimulation with a pacemaker is becoming more important in the treatment of drug refractory pain due to peripheral vascular disease, angina pectoris and intractable tremor in patients with neurologic disorders such as Parkinson's disease. An additional indication for a cardiac pacemaker or implantable cardioverter defibrillator raises concerns about possible interactions between the implanted electrical devices. We report on a patient with existing spinal cord stimulation who survived sudden cardiac death and received a dual chamber cardioverter defibrillator capable of delivering tiered therapies in both the atrium and ventricle.

Specific considerations with the automatic implantable atrial defibrillator.

INTRODUCTION: Internal atrial defibrillation has been evaluated as an alternative approach to the external technique for more than two decades. Previous studies in animals and humans have shown that internal atrial defibrillation is feasible with relatively low energies. The promising results achieved with internal atrial defibrillation have facilitated the development of an implantable atrial defibrillator (IAD). METHODS AND RESULTS: For any new therapy, it is imperative to demonstrate safety, efficacy, tolerability with improvement in quality of life, and cost-effectiveness compared with therapeutic options already available. Maintenance of sinus rhythm or prolonged duration in arrhythmia-free intervals should be demonstrated clearly with an IAD. Initial clinical experience with the Metrix system indicates stable atrial defibrillation thresholds, appropriate R wave synchronization markers, no shock-induced ventricular proarrhythmia, and excellent detection of atrial fibrillation (AF) with a specificity of 100%. Ventricular proarrhythmia has not been reported for correctly R wave synchronized low-energy shocks when closely coupled to RR intervals, and long-short cycles are avoided. CONCLUSION: Preliminary experience with the Metrix system suggests that the IAD may offer a therapeutic alternative for a subgroup of patients with drug-refractory, symptomatic, long-lasting, and infrequent episodes of AF. Further efforts must be undertaken to reduce the patient discomfort associated with internal atrial defibrillation in an attempt to make this new therapy acceptable to a larger patient population with AF.

[Cardiac resynchronization therapy by biventricular pacing. How many patients with left ventricular dysfunction are eligible?]. / Elektrische Resynchronisation mittels biventrikulärer Stimulation - Wie viele Patienten mit einer linksventrikulären Dysfunktion kommen hierfür in Frage? -

BACKGROUND AND OBJECTIVE: Cardiac resynchronization therapy by multisite biventricular pacing presents an additive therapeutic option in the treatment of severe congestive heart failure. The objective of the study was to evaluate how many patients with left ventricular dysfunction may potentially benefit from this therapy. METHODS: A total of 975 patients were screened for the prevalence of left ventricular dysfunction. Patients with a left ventricular ejection fraction (LVEF) <45 % were included into the investigation. Potential benefit of biventricular pacing was presumed in the presence of: LVEF < 35 %, severe heart failure (NHYA class III or IV), intrinsic left bundle branch block pattern with QRS interval > 150 ms and the absence of atrial fibrillation in the last 3 months before study inclusion. RESULTS: In 203 patients (168 male, 35 female, mean age: 64 +/- 11) an LVEF <45 % was found. A total of 12 of these patients (6 %) or 12 of 113 patients (11 %) with an LVEF <35 % were identified as appropiate candidates for biventricular resynchronization therapy. CONCLUSIONS: Cardiac biventricular pacing currently serves as a therapeutic option for a relatively small subgroup of patients with left ventricular dysfunction. Focusing on estimations that the incidence of heart failure in Germany amounts to more than 100.000 cases per year our results suggest that after all more than 6.000 patients per year may potentially benefit from electric resynchronization therapy. This number may increase substantially if prospective studies can prove that patients with heart failure and atrial fibrillation or left ventricular conduction delay due to univentricular pacing also benefit from cardiac resynchronization therapy.

Complement activation by recombinant adenoviruses.

Recombinant adenoviruses are currently the most important vector system in gene therapy. Adenoviruses frequently cause upper respiratory tract infections in humans and anti-adenoviral antibodies are found in 35-70% of the population. Therefore in the majority of potential patients receiving adenoviral gene therapy, the contact of virus particles and blood will lead to the formation of antigen-antibody complexes. These complexes have the ability to induce inflammatory reactions via an activation of the complement system. We have determined the level of C3a (the most reactive complement component) generated in isolated citrate plasma of healthy individuals after challenge with recombinant and wild-type adenoviruses in amounts corresponding to virus blood levels to be expected in patients during adenoviral gene therapy. All plasma samples containing anti-adenoviral antibodies showed a substantial, dose-dependent generation of C3a. A virus plasma level of about 7.5 x 10(9) particles/ml (which was calculated to be the highest blood level reached during clinical trials in the past) induced an average release of about 3000 ng/ml C3a (baseline levels <140 ng/ml). Analyzing the nature of anti-adenoviral antibodies showed, that not only antibodies with neutralizing properties (anti-Ad5), but also non-neutralizing anti-adenoviral antibodies are capable of complement activation. This study suggests that complement activation can be ignored in local low-dose applications of recombinant adenoviruses, but warrants attention after systemic application of large viral quantities. In clinical protocols aiming at systemic virus application, measures for monitoring and controlling the complement system should be included on a regular basis.

Titer determination of Ad5 in blood: a cautionary note.

Recombinant adenoviruses are presently the most efficient in vivo gene transfer system available. Targeting single organs or large tumors by adenoviral vectors requires an intravascular route of application. During the first pass of viral particles through the vascular bed of the target tissue, virus uptake is not quantitative and indefinite amounts of particles leak into circulation. To determine the amount of leaking particles and to calculate organ-specific uptake (in-/outflow ratio), it is necessary to titrate virus particles directly in blood. In preclinical and clinical trials titration is currently mostly done with blood plasma instead of full blood. However, this technique provides valid results only as long as there is no affinity between adenovirus particles and erythrocytes. In this study we demonstrate that Ad5 particles, as mostly employed for gene therapy, have a strong affinity to human erythrocytes. At 60 min after coincubation of human erythrocytes and Ad5 particles, more than 98% of the particles are attached to the surface of erythrocytes. Therefore, ignoring the amount of red cell bound particles by performing titration in plasma leads to severe miscalculation of organ-specific transfer rates or virus circulation half-life. The biological impact of an increased affinity between virus particles and erythrocytes will be discussed.