Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes.

We have examined several variables inherent in aerosolizing cationic lipid:DNA complexes using a jet nebulizer and thereby have optimized the delivery of functional complexes. Maximal aerosol transfer efficiency of cationic lipid:pDNA complexes was quantitated and shown to require the presence of at least 25 mM NaCL as an excipient. This is possibly related to effects on the measured zeta potentials of the complex, which indicate that the complexes are more highly charged in solutions of physiological ionic strength than in solutions of low ionic strength. Inclusion of saline also resulted in retention of the starting lipid to plasmid DNA (pDNA) ratio following nebulization. These data were used to design in vitro aerosolization experiments with tissue culture cells that resulted in the identification of a cationic lipid:pDNA ratio of 0.75:1 (mol:mol) as being optimal for aerosolization. This formulation largely protected pDNA from shear degradation during nebulization and produced a respirable aerosol droplet size (1-3 microns). It was tested further in a mouse model and shown to result in the dose-dependent transfection of mouse lungs, generating the equivalent of several picograms of reporter gene activity per mouse lung. The results of these experiments have provided a set of optimal conditions for nebulizing cationic lipid:pDNA complexes that can be used as a starting point for the further evaluation of aerosol delivery of these nonviral gene delivery vectors in vivo.

Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung.

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.

A comparison of direct immunofluorescence, shell vial culture, and conventional cell culture for the rapid detection of influenza A and B.

Direct immunofluorescence (FA) and shell vial contrifugation cultures (SVCs) were compared with conventional tube cultures for the rapid detection of influenza A and B by using a commercial antibody. Of the 439 specimens tested, 82 were positive by conventional culture (CC). The direct smear prepared from pelleted cells or direct swab material exhibited positive fluorescence in only seven (8.5%) of these cases, whereas the SVC was positive in 30 (37%). The SVC method detected 12 additional positive isolates that were not recovered in CC. The mean time to isolation in CC was 3.6 days for influenza A and 4.3 days for influenza B. The use of SVC provided more rapid results (36-48 hr). The FA method, although more rapid, may be of limited sensitivity and difficult to interpret depending on the quality of the specimen. The results indicate that SVC complements conventional culture in the rapid detection of influenza and can detect infections that may be missed in conventional tubes, but should not be used to the exclusion of conventional culture.

Comparison of enzyme immunoassay, shell vial culture, and conventional cell culture for the rapid detection of herpes simplex virus.

Specimens submitted for the detection of herpes simplex virus (HSV) were inoculated into conventional cell-culture tubes and fresh MRC-5 shell vials. The shell vial centrifugation cultures (SVCs) were examined at 16 hr postinoculation for HSV by using type-specific monoclonal antibodies (SVC-FA); they were also analyzed for HSV antigen by using an enzyme-linked immunoassay (SVC-ELISA). Mink Lung (ML) and rhabdomyosarcoma (RD) cells were used in the cell-culture tubes. Of 182 specimens, 35 (19%) were positive in cell-culture tubes, 16 (9%) were positive by SVC-ELISA, and 22 (12%) were positive SVC-FA. All specimens that were positive by SVC-ELISA, SVC-FA, and in culture tubes displayed cytopathic effect (CPE) at 16 hr. Those specimens that had a negative ELISA and/or FA result and were positive in culture were evaluated for the time in which it took to detect CPE. At 16 hr, 48% of the positive tubes were detected; at 40 hr (second day), 83% of the positive tubes were detected; and by the third day, 94% were detected. The RD cell line displayed CPE at the same time or earlier than ML cells did in 92% of the positive cases. The sensitivity of the SVC-ELISA at 16 hr was 46% with 100% specificity. The sensitivity of the SVC-FA at 16 hr was 63% with 99% specificity. Given the increased sensitivity, rapid display of CPE, and reduced cost and handling time of cell cultures, our laboratory found that rapid SVC-ELISA and SVC-FA procedures for HSV detection have no clinical or laboratory advantage.

Cationic lipid structure and formulation considerations for optimal gene transfection of the lung.

Enhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid:plasmid DNA (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanolamine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphatidylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid:pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.

The history of DBCP from a judicial perspective.

The efforts of workers in less-developed countries who have been exposed to 1,2-dibromo-3-chloropropane (DBCP) to obtain redress through the courts for damages suffered from these exposures are reported. The authors, who are lawyers, have represented more than 26,000 such workers. Evidence of the culpability of the U.S. manufacturers and the corporate users of DBCP, particularly Standard Fruit Company in Costa Rica, is presented. The damaged-worker plaintiffs are stymied by the application by the U.S. judicial system of forum non conveniens, which works in the defendants' favor by shunting the cases back to the plaintiffs' home countries, where the judicial systems are inadequate to deal with such cases and unlikely to be able to enforce judgments against the defendants.

NAD+ and nicotinamide: sex differences in cerebral ischemia.

BACKGROUND: Previous literature suggests that cell death pathways activated after cerebral ischemia differ between the sexes. While caspase-dependent mechanisms predominate in the female brain, caspase-independent cell death induced by the activation of poly(ADP-ribose) polymerase (PARP) predominates in the male brain. PARP-1 gene deletion decreases infarction volume in the male brain, but paradoxically increases damage in PARP-1 knockout females. PURPOSE: This study examined stroke-induced changes in NAD+, a key energy molecule involved in PARP-1 activation in both sexes. METHODS: Mice were subjected to middle cerebral artery occlusion and NAD+ levels were assessed. Caspase-3 activity and nuclear translocation were assessed 6h after ischemia. In additional cohorts, Nicotinamide (500 mg/kg i.p.) a precursor of NAD+ or vehicle was administered and infarction volume was measured 24h after ischemia. RESULTS: Males have higher baseline NAD+ levels than females. Significant stroke-induced NAD+ depletion occurred in males and ovariectomized females but not in intact females. PARP-1 deletion prevented the stroke-induced loss in NAD+ in males, but worsened NAD+ loss in PARP-1 deficient females. Preventing NAD+ loss with nicotinamide reduced infarct in wild-type males and PARP-1 knockout mice of both sexes, with no effect in WT females. Caspase-3 activity was significantly increased in PARP-1 knockout females compared to males and wild-type females, this was reversed with nicotinamide. CONCLUSIONS: Sex differences exist in baseline and stroke-induced NAD+ levels. Nicotinamide protected males and PARP knockout mice, but had minimal effects in the wild-type female brain. This may be secondary to differences in energy metabolism between the sexes.

Evaluation of direct immunofluorescence, enzyme immunoassay, centrifugation culture, and conventional culture for the detection of respiratory syncytial virus.

Four methods of detecting respiratory syncytial virus (RSV) from clinical specimens were evaluated. A total of 410 specimens consisting of nasopharyngeal washes, aspirates, and swabs were simultaneously tested for the presence of RSV by direct immunofluorescence assay (DFA), enzyme immunoassay (EIA) (Kallestad Pathfinder), shell vial centrifugation culture (SVC), and conventional culture. DFA identified 146 (83%) of the 175 positive cases, EIA detected 153 (87%), SVC detected 127 (73%), and conventional culture detected 70 (40%). Conventional culture isolated an additional 19 respiratory viruses other than RSV. DFA and EIA were able to detect nonviable virus not isolated by a culture method, and SVC isolated low-titer virus not detected by conventional culture. DFA and EIA gave similar results; however, the EIA system was less dependent on technical expertise. The use of SVC enhanced the conventional culture system with 63 RSV isolates not recovered from the tube culture. We recommend complementary use of both culture and nonculture methods in the detection of RSV.

Presumptive identification of enteroviruses with RD, HEp-2, and RMK cell lines.

The limited supply of the Lim Benyesh-Melnick antiserum pools for the typing of enteroviruses has made this test inappropriate for routine use in most clinical laboratories. We studied the correlation between the enterovirus groups and the cell lines on which they displayed cytopathic effect in order to make identifications without using the neutralization test. This study indicated that a presumptive identification of the enterovirus group could be made on the basis of characteristic cytopathic effect displayed after passage into rhabdomyosarcoma (RD), HEp-2, and primary rhesus monkey kidney (RMK) cells. Echoviruses and group A coxsackieviruses could be isolated in RD and RMK cells but not HEp-2 cells. Group B coxsackievirus could be isolated in RMK and HEp-2 cells but not RD cells. Poliovirus could be isolated in all three cell lines. We recommend the use of these cell lines to make presumptive enterovirus group identifications for routine viral isolates.

Rapid isolation of herpes simplex virus by using mink lung and rhabdomyosarcoma cell cultures.

Highly sensitive and rapid results can be obtained by isolating herpes simplex virus from clinical specimens in simple cell culture with rhabdomyosarcoma (RD) cells. In this study, 3,186 clinical specimens were inoculated into locally produced, equivalent-age RD and mink lung (ML) cells. Of 727 positive isolates, all (100%) were isolated from RD cells and only 691 (95%) were isolated from ML cells. Furthermore, 162 of the positive isolates (22%) were isolated in RD cells earlier than in ML cells. RD cells are continuous and can be cultivated in house without decreasing sensitivity as the passage number increases. They produce a highly distinguishable cytopathic effect in response to herpes simplex virus and maintain intense confirmatory staining patterns.