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.     

A non-heart-beating donor model to evaluate functional and morphologic outcomes in resuscitated pig hearts.

A non-heart-beating donor model was considered to examine whether pig hearts from the abattoir could be resuscitated by whole blood reperfusion. For preservation, machine perfusion using University of Wisconsin (UW) solution was compared with storage on ice. Nineteen hearts from abattoir pigs, harvested 25 +/- 3 min after exsanguination, were harvested and transported to the laboratory. Controls (n = 7) were immediately reperfused with homologous whole pig blood in an isolated heart model for 60 min with monitoring of left ventricular developed pressure (LVDP), contractility, and coronary flow. UW solution hearts (UW, n = 6) were perfused for 4 h with 10 degrees C cold UW solution before blood reperfusion. In the cold storage group (CS, n = 6), the organs were stored for an additional 4 h on ice before blood reperfusion. In all hearts, histology was performed after 60 min of blood reperfusion to evaluate myocardial reperfusion injury. All three groups showed significant increases in LVDP (p <.001), although this functional recovery was earliest in the control group and latest in the UW group. Significant declines were observed for both LVDP and contractility from the peak values in each group to the end of blood reperfusion. Coronary flow increased steadily over the time course for the UW group, whereas in the control and CS groups flow increased during the first 15 min of blood reperfusion and then decreased. In the UW and CS groups, there were significant positive correlations between coronary flow and LVDP (p <.001). Microscopic examination revealed no differences between the three groups. Thus, hearts from an abattoir with 25 min of warm ischemic time can be resuscitated. For storage of these organs, continuous machine perfusion with UW solution is superior to cold storage on ice.     

Delayed hepatic CT scanning: increased confidence and improved detection of hepatic metastases

Fifty oncologic patients with suspected hepatic metastases were prospectively evaluated by dynamic sequential hepatic computed tomography (DSHCT) and by delayed iodine hepatic computed tomography (DICT) scanning. DICT scanning was performed 4-6 hours following administration of 60 g of intravenous iodine. Both techniques were evaluated for lesion definition relative to the adjacent hepatic parenchyma and for numbers of metastases detected. Metastases were detected by both techniques in 26 patients. Fifteen patients (58%) had lesions better defined by DICT. DICT scanning detected more metastases in seven of these 15 patients. In eight patients (31%), there was no difference between the two techniques in numbers of masses detected or lesion definition. In three cases (11%), metastases were more confidently identified on the initial or DSHCT scan. DICT scanning, as described, is useful in defining and detecting hepatic metastases, especially where there is questionable hepatic involvement or better quantification of size is necessary.     

Lung lining fluid modification of asbestos bioactivity for the alveolar macrophage.

It is likely that chrysotile fibers deposited in the lower respiratory tract become rapidly coated by components of lung lining fluid. Therefore, we have used lung lining fluid and its components as part of an in vitro model to study chrysotile stimulation of superoxide anion production by the alveolar macrophage. In terms of superoxide anion production, lung lining fluid-treated chrysotile was 50% as effective as the untreated fibers. Fractionated lung lining fluid components and pure phospholipids were tested individually for their effects on chrysotile bioactivity. Pretreatment of chrysotile with lung surfactant isolated from a 30,000g pellet of lung lining fluid decreased chrysotile-stimulated superoxide anion production by 90%. The inhibitory activity of lung surfactant was found to reside in a chloroform extract containing hydrophobic proteins and lipids. Total proteolysis of the proteins did not affect the inhibitory activity of the chloroform extract, but treatment with phospholipase C significantly decreased its inhibitory activity. The inhibitory effects of lung surfactant could be simulated with phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol at concentrations equivalent to those found in lung lining fluid. These results strongly suggest that phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol in lung lining fluid can modify chrysotile bioactivity for the alveolar macrophage. Together with previous results indicating that IgG enhances asbestos bioactivity, it would appear that lung lining fluid contains components that can either inhibit or enhance the bioactivity of asbestos and that it is the relative amounts of these components that determines the overall bioactivity of the fiber.     

Mechanistic aspects of cromolyn sodium action on the alveolar macrophage: inhibition of stimulation by soluble agonists.

Despite wide use of the drug cromolyn sodium, its mechanism of action remains unknown. The alveolar macrophage plays a major role in the regulation of the inflammatory responses of the lung which may contribute to asthma. Since the biochemical mechanism by which agonists stimulate the alveolar macrophage to produce superoxide anion has been described, the effects of cromolyn sodium on this process were examined. Cromolyn sodium (0.5-4 mM) reversibly blocked macrophage stimulation by formyl peptide and leukotriene B4, but not by phorbol diester and concanavalin A. Cromolyn sodium inhibition was not calcium dependent and could be reversed by increasing the dose of agonist. Cromolyn sodium did not elevate intracellular cAMP, nor did the characteristics of inhibition resemble those observed using cAMP to inhibit agonist stimulation. However, cromolyn sodium did block agonist-mediated stimulation of the phosphatidylinositol (PI) pathway. Taken together, the present results suggest that one site of action of cromolyn sodium may be at the GTP-binding protein of the PI pathway.     

Timing of therapeutic intervention determines functional and survival outcomes in a mouse model of late infantile batten disease.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice. Intracranial injection of AAV1-hCLN2 resulted in widespread human TPP1 (hTPP1) activity in the brain that was 10-100-fold above wild-type levels. Injections before disease onset prevented storage and spared neurons from axonal degeneration, reflected by the preservation of motor function. Furthermore, the majority of CLN2 mutant mice treated pre-symptomatically lived for at least 330 days, compared with a median survival of 151 days in untreated CLN2 mutant controls. In contrast, although injection after disease onset ameliorated lysosomal storage, there was evidence of axonal degeneration, motor function showed limited recovery, and the animals had a median lifespan of 216 days. These data illustrate the importance of early intervention for enhanced therapeutic benefit, which may provide guidance in designing novel treatment strategies for cLINCL patients.