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Use of the post-insertion technique to insert peptide ligands into pre-formed stealth liposomes with retention of binding activity and cytotoxicity.

PURPOSE: Simple methods for the large-scale manufacture of ligand-targeted liposomes will be needed if clinical trials are to proceed. We tested a recently developed technology for inserting peptide ligands into preformed Stealth liposomes. Antagonist G-targeted liposomes (PLG) were prepared and loaded with doxorubicin and their cellular association and cytotoxicity were evaluated using the human small cell lung cancer H69 cell line. METHODS: The hexapeptide antagonist G was covalently coupled via a thioether bond to the terminus of polyethylene glycol (PEG) in micelles formed from maleimide-derivatized poly(ethylene glycol) (Mr 2000) distearoylphosphatidylethanolamine followed by transfer into preformed liposomes during a one-step incubation. For cellular association, we used radiolabeled liposomes. Cytotoxicity was evaluated using the MTT in vitro proliferation assay. RESULTS: The postinsertion approach to the formation of peptide-targeted liposomes led to the production of PLG bearing a maximum of approximately 0.3 microg antagonist G/micromol phospholipid. These liposomes had increased cellular association to H69 cells relative to nontargeted liposomes and, when loaded with doxorubicin, they resulted in similar levels of cytotoxicity to those obtained by conventional coupling techniques. CONCLUSIONS: The postinsertion technique is a simple, effective means for the production of biologically active peptide-targeted liposomes.

Binding Sites↗

R-region cDNA inserts in retroviral vectors are compatible with virus replication and high-level protein synthesis from the insert.

Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by the retroviral promoter. However, the typical retroviral vector design places the cDNA downstream of the retroviral packaging signal and far from the retroviral promoter. In an attempt to improve protein production levels from cDNAs expressed in retroviral vectors, we inserted the MyoD or the purine nucleoside phosphorylase (PNP) cDNAs into the R regions of both retroviral LTRs, close to the retroviral promoter and just upstream of the polyadenylation signal present in each long terminal repeat (LTR). These R-region double-copy vectors could be produced in unrearranged form, although the titer was about seven-fold lower than that of typical vectors. R-region positioning of the MyoD cDNA resulted in five-fold higher MyoD expression compared to MyoD expression in a typical vector, whereas PNP expression was not improved. Thus, R-region double-copy vectors provide an alternative vector design that can improve protein expression from some cDNAs.

Animals↗

Transposition in Lactobacillus sakei: inactivation of a second lactocin S operon by the insertion of IS1520, a new member of the IS3 family of insertion sequences.

The analysis of spontaneous bacteriocin-negative mutants has led to the identification and characterization of a new, transpositionally active, insertion sequence of the IS3 family in the lactocin-S-producing Lactobacillus sakei strain L45. The element, which has been designated IS1520, is 1302 bp long with 10 bp perfect inverted repeat ends and generates direct repeats of a trinucleotide of target sequence upon transposition to the lactocin S locus. IS1520 encodes two consecutive, partially overlapping, major ORFs, which are frameshifted in a manner typical of the IS3 family. Despite a low overall DNA sequence similarity, the putative frameshifting region of IS1520 is highly similar to the corresponding region of IS1163, which is a related element previously shown to be active in L. sakei L45.

Amino Acid Sequence↗

Novel face-lift suspension suture and inserting instrument: use of large anchors knotted into a suture with attached needle and inserting device allowing for single entry point placement of suspension suture. Preliminary report of 20 cases with 6-to 12-month follow-up.

BACKGROUND: Various suspension suture techniques exist to elevate the mid-face, jowls, and neck. OBJECTIVE: To assess safety and efficacy of a new suspension suture and inserting instrument with both standard and minimal incision (no-skin-excision) face-lifts. METHODS: A new type of multianchor suspension suture assembled from commercially available 2-0 absorbable monofilament material, with 5 to 9 equally spaced knots through which are secured 7 to 9 mm bits of 0 thickness similar suture material, and an attached straightened needle, was used to elevate and suspend facial tissues to temporal or mastoid fascia. The suspension sutures are placed in the deep subcutaneous tissues, just above the superficial musculo aponeurotic system (SMAS), by use of a novel, blunt instrument, which does not require a second, distal exit point. The suspension suture distal end floats free. The proximal needle end is sutured to fascia. The suture was used on 20 patients. Fourteen of them underwent pure, "no-skin-excision," suspension lifts. Six had suspension suture elevation of the mid-face in conjunction with relatively conservative open lifts. Nine- to 12-month results were evaluated. RESULTS: With open face-lifts, 9- to 12-month results are excellent with significant persistence of the correction initially achieved. Resulting scars remained fine line. There were no complications. With pure suspension lifts, initial results were impressive. By 6 months, correction started to fade. By 12 months 100% of initial correction for jowls, and 80 to 100% for mid-face, appeared lost. Recovery time was 2 to 4 days. There were no significant complications. CONCLUSION: Large multianchor, absorbable monofilament sutures can safely and effectively enhance results of conservative lifts, with remarkable elevation of the mid-face not achievable with simple SMAS flaps. These suspension sutures can easily and safely achieve impressive, though relatively short-term results, with a minimal incision, "no-skin-excision" technique.

Female↗

Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis.

In this study we established the usefulness of DNA fingerprinting for the epidemiology of tuberculosis on the basis of the DNA polymorphism generated by the insertion sequence (IS) IS986. Although clinical isolates of Mycobacterium tuberculosis displayed a remarkably high degree of restriction fragment length polymorphism, we showed that transposition of this IS element is an extremely rare event in M. tuberculosis complex strains grown either in vitro or in vivo for long periods of time. The M. tuberculosis and Mycobacterium africanum strains tested in this study contained 6 to 17 IS copies. In the Mycobacterium bovis strains, the copy numbers ranged between 1 and 5, and all 27 M. bovis BCG strains investigated invariably contained a single IS copy. This copy was located at a unique chromosomal position, reinforcing the idea that the frequency of IS transposition is very low in M. tuberculosis complex strains. Various microepidemics are described in which each microepidemic corresponds to a particular fingerprint type. The extent of similarity between Dutch and African strains was quantitatively assessed by computer-assisted analysis of DNA fingerprints. The results indicate that M. tuberculosis strains from regions in central Africa, where tuberculosis is highly prevalent, are generally more related to each other than isolates from the Netherlands, where the transmission rate is low and where the majority of the tuberculosis cases are presumed to be the result of reactivation of previously contracted M. tuberculosis infections.

Africa↗

Experience of cardioverter-defibrillators inserted without thoracotomy: evaluation of transvenously inserted intracardiac leads alone or with a subcutaneous axillary patch.

OBJECTIVES: To compare the efficacy of a purely transvenous cardioverter-defibrillator (ICD) system with that of a system with a supplementary subcutaneous patch. To evaluate clinical follow up of these lead arrangements that do not require thoracotomy. DESIGN: A simplified defibrillation protocol to test two different lead arrangements during implantation, with routine clinical follow up after implantation. SETTING: Tertiary referral centre for treatment of arrhythmia. PATIENTS: 22 consecutive patients selected for implantation of an ICD because of life-threatening ventricular arrhythmias (ventricular fibrillation or sustained ventricular tachycardia) of whom 20 entered the test protocol. INTERVENTION: Implantation of an ICD with transvenously inserted intracardiac leads and a subcutaneous patch and assessment of effective defibrillation followed by testing of the purely transvenous approach. MAIN OUTCOME MEASURES: Reproducible conversion of ventricular fibrillation to sinus rhythm at a certain energy level, providing a safety margin of at least 10 J for both lead arrangements. Confirmation of efficacy during clinical follow up (mean 6 months). RESULTS: A transvenous lead system combined with a subcutaneous axillary patch was implanted in 20/22 patients and it provided adequate and acceptable energy levels. In 10/20 tested patients a purely transvenous lead configuration provided an acceptable safety margin as well. Nine patients had clinical recurrences: all these arrhythmias were successfully converted. CONCLUSION: A transvenous lead system was sufficient in 50% of the patients at the time of implantation. Data on long-term clinical follow up of this arrangement are not available. The approach without thoracotomy with a subcutaneous patch is feasible and effective in most patients selected for ICD treatment.

Adult↗

Peripherally inserted central catheters for parenteral nutrition: a comparison with centrally inserted catheters.

BACKGROUND: Central venous access is crucial for the provision of adequate parenteral nutrition (PN). The type of central venous access device (CVAD) has evolved over the past 10 years. The most recent trend has been to use peripherally inserted central catheters (PICCs). This development has occurred without controlled clinical trials. METHODS: Over a 10-year period, the nutrition support service at a single institution has prospectively collected data on CVADs used for providing PN. The types of CVAD used for providing PN were analyzed, and the major complications associated with CVADs, thrombosis and line sepsis, were compared over three different time periods: 1988-1989; 1992-1993; 1996-1997. In addition, complications associated with PICCs were compared with other CVADs. RESULTS: The following were the dominant CVADs over each of the three time periods: 1988-1989: tunneled catheters, 80%; 1992-1993: nontunneled catheters, 46%; and 1996-1997: PICCs, 48%. There was a decreased incidence of sepsis and pneumothorax in 1996-1997 and an increase in severed or leaking catheters and phlebitis. In a comparison of PICC and non-PICC catheters over the past 3 years, there was a trend toward decreasing sepsis with PICC catheters but an increase in malposition, inadvertent removal, and severed or leaking catheters. CONCLUSIONS: PICCs have replaced tunneled and nontunneled central catheters as the most commonly used CVAD for providing PN. PICCs do not result in increased line sepsis or thrombosis but have an increased incidence of local complications such as leaking catheters, phlebitis, and malposition.

Catheterization, Central Venous↗

Repeated postpericardiotomy syndrome following a temporary transvenous pacemaker insertion, a permanent transvenous pacemaker insertion and surgical pericardiotomy.

The postpericardiotomy syndrome is a well-known complication of opening and manipulating the pericardium. The occurrence of this syndrome following transvenous pacemaker insertion is very rare, and only 5 cases have been reported to date. The present patient repeated this syndrome 3 times in a short period following 3 different interventional techniques: a temporary transvenous pacemaker, a permanent transvenous pacemaker and surgical pericardiotomy.

Aged↗