Quality of IL-3 and G-CSF-mobilized peripheral blood stem cells in patients with early chronic phase CML.

Coexistence of Philadelphia chromosome (Ph)-negative, primitive hematopoietic progenitor cells with their malignant counterparts in chronic myelogenous leukemia (CML) has been reported. As most of the Ph-negative progenitor cells do not express the HLA-DR antigen, selection of them might be possible. Peripheral blood progenitor cells (PBPC) from eight early chronic phase (CML) patients were mobilized by ICE chemotherapy followed by simultaneous administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human interleukin 3 (rhIL-3). PBPCs were collected by leukapheresis in the early phase of hematopoietic recovery after chemotherapy, CD34 selected and cultured in vitro. The content of Ph chromosome-positive cells in leukapheresis products as well as after CD34 enrichment and after in vitro culture was analyzed by interphase fluorescence in situ hybridization (FISH) and RT-PCR. The percentage of Ph chromosome-positive PBPC was reduced after each purification step in almost all samples. A substantial number of PBPC samples were negative for the bcr/abl mRNA rearrangement as analyzed by RT-PCR. The present study demonstrates the feasibility of mobilizing Ph-negative PBPC during the early phase of hematopoietic recovery after ICE chemotherapy and simultaneous administration of rhIL-3 and rhG-CSF.

Local inflammation and devascularization--in vivo mechanisms of the "bystander effect" in VPC-mediated HSV-Tk/GCV gene therapy for human malignant glioma.

Somatic gene therapy with the herpes simplex virus type I thymidine kinase gene/ganciclovir (HSV-Tk/GCV) system and murine retroviral vector producer cells (VPCs) was introduced as a new adjuvant treatment modality to treat tumor bulk and to prevent tumor recurrence in patients harboring malignant glioma. The single-center experience after treatment of 27 patients undergoing tumor resection followed by intracerebral VPC injection for HSV-Tk suicide gene therapy will be presented focused on findings of systematic and close MRI follow-up and a few histological specimens. The data indicate that hemorrhagic necrosis due to endothelial cell transfection mediated vessel necrosis and that local inflammatory immune response occurs frequently after gene therapy. These phenomena seem to be specific because none of the patients of a control group showed any similar features. The prognosis (time to progression, survival) of the patients with "bystander effects" after gene therapy was better, but compared to those patients without bystander effects, they were also privileged by a favorable constellation of prognostic factors. Therefore, the appearance of these neuroradiologic features cannot serve as an indicator for treatment effectiveness and outcome.

Lack of efficacy of recombinant human interleukin-6 in patients with advanced renal cell cancer: results of a phase II study.

The present phase II study was undertaken to assess antitumoral activity, safety and tolerability of recombinant human interleukin-6 (rh IL-6) in patients with advanced renal cell cancer. Rh IL-6 was administered as a daily subcutaneous injection at a fixed dose of 150 micrograms/day for a maximum of 42 consecutive days. 12 patients with metastatic renal cell cancer without previous immunotherapy were enrolled and were evaluated for response. No objective clinical responses were observed in the trial. Toxicity was moderate and reversible and mainly comprised fever, influenza-like symptoms, fatigue and moderate hepatotoxicity. Anaemia, leucocytosis, thrombocytosis and induction of an acute phase response were observed in most patients. In conclusion, prolonged subcutaneous administration of rh IL-6 on an outpatient basis is safe and feasible. However, rh IL-6 exhibited no antitumoral activity in patients with metastastic renal cell cancer. Profound regulatory effects on haematopoiesis and inflammatory response of rh IL-6 were observed.

The calcium antagonist isradipine in the therapy of hypertension. A double-blind crossover comparison with nifedipine.

The calcium antagonists of the dihydropyridine group, isradipine and nifedipine, were compared in 64 patients with mild to moderate hypertension (diastolic blood pressure 95 to 110mm Hg). A 2-week placebo run-in phase was followed by a double-blind crossover trial comprising two 3-week treatment periods with either calcium antagonist. The (fixed) dosages were isradipine 2.5mg twice daily and nifedipine retard 20mg twice daily. Blood pressure (systolic/diastolic) at baseline was 155/101mm Hg and decreased significantly by 14%/15% on isradipine and by 11%/12% on nifedipine (difference between treatments not significant). The drugs differed significantly with regard to incidence of adverse effects (mostly flushing and headache); the total rates were 16% on isradipine and 36% on nifedipine. At the end of the trial, patients were asked which drug or treatment phase they preferred. Isradipine was preferred by 50% of patients; only 20% preferred nifedipine. Thus, it is concluded that isradipine, administered in an equally effective antihypertensive dosage regimen is superior to nifedipine with regard to the incidence of adverse effects, resulting in greater patient satisfaction with treatment.

Recombinant human granulocyte and granulocyte-macrophage colony-stimulating factor (G-CSF and GM-CSF) administered following cytotoxic chemotherapy have a similar ability to mobilize peripheral blood stem cells.

The availability of hematopoietic growth factors has greatly facilitated the mobilization and collection of peripheral blood stem cells (PBSC). It was the aim of this double-blind study to compare the PBSC-mobilizing efficacy of recombinant human G-CSF and GM-CSF when administered post-chemotherapy. Twenty-six patients with relapsed Hodgkin's disease were included in the study. Their median age was 31 years (range, 22-59) and 14 patients were males and 12 were females. Patients were pretreated with a median of eight cycles of cytotoxic chemotherapy, while 18 patients had undergone extended field irradiation. The patients received dexamethasone 24 mg days 1-7, melphalan 30 mg/m2 day 3, BCNU 60 mg/m2 day 3, etoposide 75 mg/m2 days 4-7, Ara-C 100 mg/m2 twice daily days 4-7 (Dexa-BEAM). Twelve patients were randomized to receive 5/microg/kg/day G-CSF and 14 patients to receive 5 microg/kg/day GM-CSF, both administered subcutaneously starting on day 1 after the end of Dexa-BEAM. Primary endpoints of the study were the number of CD34+ cells harvested per kg body weight on the occasion of six consecutive leukaphereses and the time needed for hematological reconstitution following autografting. Twenty-one patients completed PBSC collection, and six patients of the G-CSF group and nine of the GM-CSF group were autografted. No difference was observed with respect to the median yield of CFU-GM and CD34+ cells: 32.5 x 10(4)/kg vs 31.3 x 10(4)/kg CFU-GM, and 7.6 x 10(6)/kg vs 5.6 x 10(6)/kg CD34+ cells, for G-CSF and GM-CSF, respectively (U test, P= 0.837 and 0.696). High-dose chemotherapy consisted of cyclophosphamide 1.7 g/m2 days 1-4, BCNU 150 mg/m2 days 1-4, etoposide 400 mg/m2 days 1-4. All patients transplanted with more than 5 x 10(6) CD34+ cells/kg had a rapid platelet recovery (20 x 10(9)/l) between 6 and 11 days and neutrophil recovery (0.5 x 10(9)/1) between 9 and 16 days, while patients transplanted with less than 5 x 10(6)/kg had a delayed reconstitution, regardless of the kind of growth factor used for PBSC mobilization. In conclusion, our data indicate that in patients with Hodgkin's disease G-CSF and GM-CSF given after salvage chemotherapy appear to be not different in their ability to mobilize PBSC resulting in a similar time needed for hematological reconstitution when autografted following high-dose therapy.

Effect of short-term antihypertensive therapy on left ventricular wall tension. A double-blind comparison of isradipine and nifedipine.

The comparative efficacy of the calcium antagonists isradipine and nifedipine in reducing left ventricular peak systolic wall tension was assessed in 25 patients with essential hypertension (20 men, 5 women; mean age: 49 years). After 2 weeks of treatment with either isradipine (2.5 mg twice daily) or slow-release nifedipine (20 mg twice daily), blood pressure was similarly reduced in both groups of patients whereas the thickness of the interventricular septum and left ventricular free wall did not change. Echocardiographic end-diastolic volume of the left ventricle showed no change whereas end-systolic volume significantly decreased with isradipine, but not with nifedipine retard. This led to a significant reduction in peak systolic wall tension in the isradipine group, but not in the nifedipine group. In conclusion, antihypertensive treatment with isradipine produces a reduction in peak systolic wall tension which is not seen with nifedipine, probably because of its negative inotropic effect.

MR imaging and single-photon emission CT findings after gene therapy for human glioblastoma.

BACKGROUND AND PURPOSE: Our goal was to evaluate MR imaging findings after local intracerebral gene therapy in patients with glioblastoma and differentiate postoperative contrast enhancement phenomena. METHODS: In all, 26 patients with supratentorial single lesion glioblastoma underwent tumor resection and intracerebral injection of murine retroviral vector-producer cells for gene therapy with the herpes simplex virus type I thymidine kinase gene/ganciclovir system. Serial contrast-enhanced MR studies were obtained before treatment and postoperatively on day 1 or 2; weeks 2, 4, 9, 13, 17, 25, and 33; and every 8 weeks thereafter. Iodomethyltyrosine single-photon emission CT (IMT-SPECT) investigations also were performed in selected cases. RESULTS: Twelve patients showed nontumorous enhancement of various intensities after treatment, arising within 18 to 72 hours and persisting at 3 to 10 months. It was characterized by a strong local enhancement up to 20 mm thick, which was initially nodular and later linear along the resection cavity wall and surrounded by massive perifocal edema. This "flare" enhancement had features that clearly differed from those of residual tumor enhancements and benign postsurgical enhancements. The IMT-SPECT investigations showed increased amino acid uptake in patients with enhancement from residual or relapsing tumor, but not in patients with flare. CONCLUSION: After local gene therapy, a unique dynamic, transient perifocal flare enhancement can occur on MR images. IMT-SPECT may help to differentiate between tumorous and nontumorous flare enhancements in patients with enhancing tissue on MR images after gene therapy for glioblastoma.

Membrane binding of bacteriophage SPP1 DNA.

A fast sedimenting complex was isolated from B. subtilis cells infected with bacteriophage SPP1 by renografin centrifugation. This complex was identified as membrane bound parental and replicating SPP1 DNA. Synthesis of SPP1 DNA takes place in close association with the membrane. This newly synthesized DNA is then released into the cytoplasm. During release, concatemeric SPP1 DNA is sized into monomeric DNA molecules.

Removal of symmetrical sequences from the origin region of plasmid R1 reduces its replication efficiency.

The R1 origin region contains many symmetrical DNA sequence elements which allow the formation of complex secondary structures. A 218-bp in vivo deletion in a cloned R1 origin fragment removes most of them. As this deletion was never observed in plasmids containing all R1 replication functions, it was introduced by BglI in vitro recombination into the ;basic replicon' of R1 cloned into pBR322. The recombinant plasmid with the 218-bp deletion and its derivatives unambiguously show that the deleted symmetrical elements are not absolutely essential for R1 replication as was previously assumed though they seem to determine a more efficient origin function. Likewise, a hypothetical protein of a mol. wt. of 14 000 daltons, the major part of which would be encoded by the deleted sequences, does not seem to be of particular importance for R1-specific replication. This is the first report of an alteration in the origin region of an IncFII plasmid which affects plasmid replication without abolishing it completely.

[Circadian antihypertensive effect of sustained-release isradipine in patients with essential hypertension in comparison to placebo]. / Zirkadiane antihypertensive Wirkung von Isradipin retard bei Patienten mit essentieller Hypertonie im Vergleich zu Plazebo.

In this multicenter, placebo-controlled study, 16 patients with mild to moderate essential hypertension were treated with 10 mg/day isradipine retard (PN 200-110, Lomir SRO, CAS 75695-93-1) for 3 weeks. The study started with a 2 week placebo wash out phase. 13 patients were randomised to an exclusive placebo therapy. After the placebo wash out phase, following the 1st medication in active therapy and after the end of therapy, 24-h blood pressure profiles were recorded. The profile under placebo on the 1st medication was separated by a one-week intervening placebo therapy for all patients. On active therapy, the systolic as well as the diastolic blood pressure (day time, night time and 24-h mean values) were significantly reduced. The antihypertensive effect of the active therapy became already manifest after the 1st medication and was augmented after 3 weeks of therapy. In the placebo group no parameter of the 24-h profiles changed significantly. The tolerability of treatment was excellent in 14 (87.5%) of the isradipine patients and in 10 (76.9%) of the placebo group. In one of 16 patients in the active group, adverse events (flush and ankle oedema) were observed. However, therapy could be continued. In one patient of the placebo group, oedema of the fingers was noticed, in another headache was documented. In the placebo group two patients discontinued the study due to inefficacy, in the isradipine group one patient for the same reason; a second patient was excluded from this group due to a concomitant disease unrelated to the study drug.

[Antihypertensive effect and adverse effects of isradipine in patients with sever hypertension. Results of an open multicenter study]. / Antihypertensive Wirkung und Verträglichkeit von Isradipin bei Patienten mit schwerer Hypertonie. Ergebnisse einer offenen multizentrischen Studie.

Antihypertensive Efficacy and Tolerability of Isradipine in Patients with Severe Hypertension/Results of an open multicenter study. In this open multicentre study 55 patients (mean age 51.2 years) with severe hypertension (diastolic blood pressure > 115 mmHg) were treated for seven weeks with the calcium antagonist of the dihydropyridine type isradipine (CAS 75695-93-1, Lomir). If necessary, metoprolol or enalapril were added to the regimen. Before inclusion into the active treatment phase, responsiveness of the patients to a single administration of isradipine (5 mg) was compared with placebo. Preexisting antihypertensive therapy (18 patients) was to be maintained during the study period. Blood pressure was recorded with an automatic device. During the 7-week period blood pressure decreased from 173.7/124.8 mmHg to 143.2/97.8 mmHg. Both the group with isradipine monotherapy (n = 32) and the combination group (n = 11) showed a significant reduction in systolic and diastolic blood pressure. Diastolic blood pressure response, defined as a decline of more than 15 mmHg, was noted in 87.5% (monotherapy) and 72.7% (combination group) of patients. On the whole, blood pressure was normalized in 27.9% of the participants. Nineteen patients experienced 43 adverse events, most of which were rated mild to moderate. Therapy was withdrawn in only one patient (due to ankle edema). The most frequent adverse event was headache (20.9%).

Specific labelling of replicating SPP1 DNA: analysis of viral DNA synthesis and identification of phage DNA-genes.

Specific labelling of replicating bacteriophage SPP1 DNA can be achieved by infection at nonpermissive temperature of a B. subtilis strain carrying the initation mutation dnaB ts134. Under these conditions host DNA synthesis is reduced by 90 to 95%. This technique was used to identify cistrons of SPP1 involved in phage DNA synthesis and to define intermediates in SPP1 replication.

Restriction endonuclease BglI as a tool for in vitro reconstruction and recombination of plasmid and bacteriophage genomes.

The restriction endonuclease BglI produces different individual fragment ends from different cut sites. This property has allowed us to reconstruct efficiently several commonly used plasmid and bacteriophage genomes and a number of recombinant plasmids containing up to seven BglI restriction sites from their constituent BglI fragments. It is demonstrated that in vitro reconstitution from BglI fragments can be used to create, in a simple way, recombinant DNA molecules by recombining in vitro BglI fragments from different mutated or otherwise related genomes. Further applications of the method are discussed.

Comparison of negative inotropic and vasodilator effects of isradipine and nifedipine after complete autonomic blockade in ischemic heart disease.

The cardiodepressant and vasodilator effects of an intravenous (i.v.) infusion of 0.5 mg isradipine or 2 mg nifedipine were interindividually (10 vs. 10 patients) compared in a double-blind study in patients with stable coronary heart disease. To minimize vasodilation-induced autonomic reflex mechanisms, which may counterbalance negative inotropic effects after acute administration of calcium antagonists, i.v. autonomic blockade was produced by 0.2 mg/kg propranolol and 0.04 mg/kg atropine. Systemic hemodynamics were measured before and 15 min after the end of the 15-min calcium antagonist infusion. After administration of both drugs, heart rate (HR) decreased similarly during the observation period (isradipine from 93 +/- 10 to 88 +/- 9 beats/min and nifedipine from 84 +/- 9 to 79 +/- 6 beats/min, both p less than or equal to 0.01). The reduction in total peripheral resistance (TPR) was significantly (p less than or equal to 0.01) greater after isradipine (from 1,376 +/- 285 to 1,002 +/- 224 dynes s cm-5) than after nifedipine (1,258 +/- 262 to 1,112 +/- 225 dynes s cm-5). Between the two drugs, the difference in the reduction of afterload independent dP/dt40, determined by tip-manometry, reached borderline significance (p = 0.08) (isradipine from 1,197 +/- 258 to 1,157 +/- 225 mm Hg/s, NS and nifedipine 1,228 +/- 226 to 1,109 +/- 227 mm Hg/s, p less than or equal to 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of DNA polymerase from the archaebacterium Sulfolobus acidocaldarius.

DNA polymerase has been purified about 25,000-fold from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. On SDS-PAGE the enzyme was observed to have a molecular weight of 100 kDa and to be about 90% pure. The native molecular weight was 108 kDa indicating that the enzyme is composed of a single polypeptide. Activity gel analysis showed an active polypeptide of about 100 kDa. Under conditions promoting proteolysis this polypeptide was degraded to a slightly smaller form of 98 kDa. The enzyme has been characterized in respect to optimal assay conditions, template specificity, sensitivity to inhibitors and associated nuclease activities. The high temperature optimum of 65 degrees C should be emphasized. No substantial similarities have been found with other prokaryotic and eukaryotic DNA polymerases, although the enzyme bears certain resemblances to prokaryotic non-replicative polymerases.

[Circadian antihypertensive action and tolerability of a sustained-release form of isradipine in an intra-individual comparison with nitrendipine]. / Zirkadiane antihypertensive Wirkung und Verträglichkeit einer retardierten Form von Isradipin im intraindividuellen Vergleich mit Nitrendipin.

Antihypertensive efficacy and tolerability of a 4-week treatment each with the modified release formulation of the calcium antagonist isradipine (5 mg; Lomir SRO, CAS 75695-93-1) were compared with those of nitrendipine (20 mg) (both with morning intake) in 51 patients with mild to moderate hypertension using a double-blind, intraindividual crossover study. Blood pressure was measured over 24 h at the end of a 2-week placebo phase and after both treatment phases by means of a continuous ambulatory recording device. Upon statistical evaluation of all patients with 3 complete 24-h profiles (n = 44) and combined analysis of data from same treatments the following 24-h mean values were obtained: blood pressure (syst./diast.) was lowered from 151/98 mmHg to 141/91 mmHg by isradipine retard (IS) and to 141/92 mmHg by nitrendipine (NI), whereas heart rate remained nearly unchanged (78 vs 79 beats/min on both therapies). The 24-h profiles differed significantly between placebo and both therapies, the profile as a whole was more even on IS. Starting from a day-time mean value (6:00 a.m.-10:00 p.m.) on placebo of 155/102 mmHg blood pressure was reduced by IS to 143/94 mmHg and by NI to 144/95 mmHg; the corresponding night-time mean values were; placebo 138/85 mmHg, IS 132/82 mmHg, NI 134/83 mmHg. If one compares the area under the blood pressure curves during the hours from 6 p.m. to 12 p.m. significant differences (2p = 0.0128) were found for systolic pressure and borderline significance (2p = 0.0668) for diastolic differences in favour of IS.

Calcium antagonists as first-line antihypertensive agents: a placebo-controlled, comparative trial of isradipine and nifedipine.

The new calcium antagonist isradipine was compared with nifedipine retard in a multicenter, double-blind, placebo-controlled, randomized study involving 159 patients with mild hypertension. A 2-week run-in period was followed by a 6-week course of treatment with the possibility of dose doubling after 3 weeks, depending on blood pressure (BP) response (target diastolic BP less than 90 mm Hg). Systolic and diastolic BPs were reduced by isradipine (mean dose of 3.6 mg daily) from 151/101 to 136/89 mm Hg, by nifedipine (mean dose of 50 mg daily) from 155/101 to 144/90 mm Hg, and by placebo from 155/101 to 154/99 mm Hg. Normalization rates were 64% with isradipine, 56% with nifedipine, and 16% with placebo. Adverse events consisted mainly of flushing, headache, edema, and dizziness. Altogether, 8 patients receiving isradipine experienced adverse events in comparison to 21 taking nifedipine and 4 taking placebo. The superior tolerability of isradipine was paralleled by a significant improvement in the subjective well-being of the patients as assessed by the von Zerssen questionnaire (List of Complaints). With nifedipine and placebo, no statistically significant improvement was observed.

Efficacy and tolerability of the new calcium antagonist isradipine in essential hypertension.

The antihypertensive efficacy and tolerability of the new calcium antagonist isradipine was assessed in 86 hypertensive patients who had pretreatment diastolic blood pressures (DBP) greater than or equal to 105 mm Hg and who were randomly allocated to a double-blind comparison of three different dosage regimens: 1.25 mg, 2.5 mg, and 5 mg b.i.d., and placebo. A 2-week run-in period was followed by a 4-week course of treatment. Isradipine reduced systolic and diastolic blood pressures dose-dependently; the normalization rate (DBP less than or equal to 90 mm Hg) was 5% with placebo and 29, 55, and 64% with isradipine 1.25, 2.5, and 5 mg b.i.d., respectively. The proportion of patients experiencing at least a 10 mm Hg reduction in sitting DBP was 29, 67, 86, and 91%, respectively. All three dosages proved to be significantly effective compared to placebo. Neither heart rate nor blood pressure regulation in orthostasis were influenced. The main side effects were headache, dizziness, and flushing; isradipine 1.25 and 2.5 mg b.i.d. were well tolerated (not significantly different from placebo). In conclusion, isradipine 2.5 mg b.i.d. appears to be the potential dose of first choice, exhibiting a favorable benefit-risk profile.

The effect of recombinant human granulocyte macrophage colony stimulating factor after chemotherapy of various tumors.

Recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) has been successfully used in different clinical settings. We evaluated the tolerability of rhGM-CSF treatment in addition to its efficacy in preventing myelosuppression and reducing infectious complications after standard-dose chemotherapy of various tumors. Of the patient group analyzed (n = 308), 75% had solid tumors and 25% had hematological malignancies. In 27.9% of these patients an infection occurred after the first cycle of chemotherapy and between 8.2 and 19.0% in later cycles with a mean duration of fever (above 38.5 degrees C) of 3.6 days. Treatment with rhGM-CSF was well-tolerated. After the completion of treatment, the investigators assessed the efficacy of rhGM-CSF in 83.7% of the patients as 'very good' or 'good', and as 'moderate' or 'bad' in only 11.5% of patients as well as the tolerability as 'very good' or 'good' in 87.0% of the patients and in 9.4% 'moderate' or 'bad'. We conclude that rhGM-CSF proved to be an effective and well-tolerated tool in preventing myelosuppression and infectious complications after standard-dose chemotherapy of various tumors.

Evolution of catalytic and regulatory sites in phosphorylases.

Glycogen phosphorylase (E.C.2.4.1.1) was the first enzyme shown to be regulated by allosteric effectors and by protein phosphorylation. Transcriptional control of bacterial phosphorylases further extends the range of regulatory mechanisms by which phosphorylases contribute to the control of carbohydrate metabolism. Despite their regulatory differences, all known phosphorylases share catalytic and structural properties and a strongly conserved pyridoxal-5'-phosphate binding site; this makes phosphorylases highly attractive for investigations into the evolution of regulatory mechanisms. The primary and tertiary structure of rabbit muscle phosphorylase has been determined completely. Recently, comparable amino acid sequences from plants and bacteria have been resolved. Here we report the sequence of 687 amino acids of Escherichia coli maltodextrin phosphorylase, deduced from a cloned malP gene sequence. Alignment of animal and bacterial phosphorylase sequences shows strong homology (48%) throughout 91% of the polypeptide chain enclosing the extrinsic catalytic region. Within this region, structural homology identifies a presumed phosphate-binding site from which the allosteric 5' AMP binding site of rabbit muscle phosphorylase might have developed. From the decreased alignment at the N-terminus and the presence of additional residues compared with bacterial phosphorylases, we conclude that the regulatory sequences that also carry the phosphorylation site in the muscle enzyme were joined to a presumed ancestral precursor gene by gene fusion after separation of the eukaryotic and prokaryotic lines of descent.