Chemoprevention of spontaneous tumorigenesis in nullizygous p53- deficient mice by dehydroepiandrosterone and its analog 16alpha-fluoro- 5-androsten-17-one

Transgenic mice with both alleles of the p53 tumor suppressor gene product 'knocked out' by gene targeting are susceptible to early development of tumors, chiefly lymphomas and sarcomas. Compared with the control group, administration of dehydroepiandrosterone (DHEA) at 0.3% of the diet to male p53-deficient mice extended their lifespan by delaying death due to neoplasms (from 105 to 166 days on study, P = 0.002), primarily by suppressing lymphoblastic lymphoma (from 45 to 6% of neoplastic deaths, P = 0.010). Treatment with a synthetic DHEA analog, 16alpha-fluoro-5-androsten-17-one (compound 8354), at 0.15% of the diet also increased lifespan, to 140 days for mice that developed tumors (P = 0.037). The effects of these steroids on lifespan and tumor development did not appear to be strongly related to inhibition of food consumption and weight gain, in that a group pair-fed with control diet to the reduced food consumption of the DHEA-treated group developed and died of the same types of neoplasms at the same rate as the controls fed ad libitum. The chemopreventive effect of these steroids has been proposed to be due to suppression of DNA synthesis by inhibition of glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of the pentose phosphate pathway. Although DHEA and its analog are strong non- competitive inhibitors of this enzyme in vitro, treatment with DHEA did not deplete cellular nucleotide pools in the liver, as would have been predicted. The chemopreventive effect of DHEA in this model may be due to steroid-induced thymic atrophy and suppression of T cell lymphoma, permitting these mice to survive long enough to develop tumors with longer latency.      

Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes

Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using ¹²⁵I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (–/+/–)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(–/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.     

Staphylococcus aureus small colony variants, electron transport and persistent infections

Staphylococcus aureus can mutate to form a sub-population of bacteria known as small colony variants (SCVs). These bacteria have a characteristic phenotype defined by slow growth, the lack of pigment, an altered pattern of carbohydrate utilization, and a reduction in toxin production. This complex phenotype can be explained by deficiencies in electron transport. In clinical isolates, the most common mutations that affect electron transport are in the operons encoding menaquinone or heme biosynthesis. These isolates are responsible for persistent antibiotic resistant infections. The clinical presentation of these infections is readily explained by a reduction electron transport. SCVs survive within host cells, increasing the instances of recurrent infections and have a novel mechanism of resistance based upon their altered trans-membrane potential. Additionally, SCVs provide a connection between energy metabolism and toxin production. This link may operate through the bacteria responding to altered levels of NADH and ATP. A more complete understanding of these signaling pathways may provide new targets for the development of drugs to ameliorate staphylococcal virulence and disease.     

High dose ifosfamide in combination with etoposide and epirubicin followed by autologous stem cell transplantation in the treatment of relapsed/refractory Hodgkin's disease: a report on toxicity and efficacy

Patients with Hodgkin's disease (HD) refractory to first line chemotherapy and those who have rapid or multiple relapses have a very poor prognosis. With the increasing use of hybrid chemotherapy these patients will have been exposed to many of the drugs active in HD so it is important to develop salvage regimens that are novel and demonstrate activity in this group of patients. We report the use of a continuous high dose infusion of ?fosfamide at a dose of 9g/m(2) over 3 days in combination with etoposide and epirubicin followed by autologous stem cell transplant with either BEAM or Melphalan/VP16 conditioning in this difficult group. Forty six patients (28M:18F) with a median age of 28 years (range 13-45) were treated. Overall 39 out of 46 (85%) patients responded to treatment, with 17 achieving complete remission and 11 a good partial remission; 28 proceeded to autologous bone marrow/stem cell transplantation. In total, 23 patients are alive and in continuous remission with a follow up of between 12 and 61 months. Median overall survival for the whole group is 36 months. Haematological toxicity, particularly neutropenia (WHO grade IV), was observed in all cases but improved over the 3 courses of treatment in all patients. Non-haematological toxicity was not a major problem; no significant cardiac, hepatic, renal, pulmonary or neuro toxicity was observed and there were no deaths on treatment. This regime shows promise in patients with difficult Hodgkin's disease and warrants further study.     

Staphylocidal action of thrombin-induced platelet microbicidal protein is not solely dependent on transmembrane potential.

Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic, antimicrobial peptide released from rabbit platelets when stimulated with thrombin. We studied the relationship between staphylococcal transmembrane potential (delta psi) and tPMP staphylocidal activity. A genetically related pair of Staphylococcus aureus strains, 6850 and JB1, which differ in delta psi generation (-143 and -97 mV, respectively) were used. Mutant JB-1 was substantially less susceptible to tPMP than the parental strain, 6850. Menadione supplementation, which normalized the delta psi of strain JB-1, did not restore JB-1 tPMP susceptibility. These findings suggest that the staphylocidal activities of tPMP require factors other than or in addition to an intact delta psi.     

Flow pattern analysis in the abdominal aorta with velocity-encoded cine MR imaging

The sites of deposition of atherosclerotic plaque on the aortic wall are considered to be influenced by secondary and retrograde flow patterns that cause regions of altered shear stress. To detect secondary flow patterns and areas of retrograde flow in the abdominal aorta, velocity-encoded cine (VEC) magnetic resonance (MR) imaging was performed at five different levels of the abdominal aorta in nine healthy volunteers. Net retrograde flow (expressed as a percentage of antegrade flow) increased from proximal to distal levels and was maximal (13.8% ± 11.8) just distal to the origin of the renal arteries. An increase in the duration of retrograde flow over the cardiac cycle was observed from proximal to distal levels. Whereas retrograde flow was present at end systole and early diastole in each volunteer at every level, the duration and amount of retrograde flow during diastole showed high interindividual variation. Such differences suggest the possibility of variable vascular geometric risk factors in the population for the development of atherosclerotic plaque. The location of retrograde flow in the abdominal aorta demonstrated in vivo with VEC MR imaging was close to that obtained with in vitro flow visualization studies in models of the abdominal aorta.