Spatial patterns in markers of contaminant exposure, glucose and glycogen metabolism, and immunological response in juvenile winter flounder (Pseudoplueronectes americanus).

Inshore winter flounder (Pseudoplueronectes americanus) populations in NY, USA have reached record low numbers in recent years, and recruitment into the fishery appears to be limited by survival of post-settlement juvenile fish. In order to identify cellular pathways associated with site-specific variation in condition and mortality, we examined differential mRNA expression in juvenile winter flounder collected from six different bays across a gradient in human population density and sewage inputs. Illumina sequencing of pooled samples of flounder from contrasting degraded sites and less impacted sites was used to guide our choice of targets for qPCR analysis. 253 transcripts of >100bp were differentially expressed, with 60% showing strong homology to mostly teleost sequences within the NCBI database. Based on these data, transcripts representing nine genes of interest associated with contaminant exposure, immune response and glucose and glycogen metabolism were examined by qPCR in individual flounder from each site. Statistically significant site-specific differences were observed in expression of all but one gene, although patterns in expression were complex with only one (vitellogenin), demonstrating a west to east gradient consistent with known loadings of municipal sewage effluent. Principal components analysis (PCA) identified relationships among the genes evaluated. Our data indicate that juvenile winter flounder are responding to estrogenic chemicals in more urbanized coastal bays, and suggests potential mechanistic links between immune response, contaminant exposure and energy metabolism.

Pro-apoptotic and immunomodulatory activity of a mycobacterial cell wall-DNA complex towards LNCaP prostate cancer cells.

BACKGROUND: We have isolated a mycobacterial cell wall-DNA complex (MCC) possessing anti-cancer activity against bladder cancer cells. The anti-cancer activity of MCC appears to be due to two effects: a direct interaction with bladder cancer cells resulting in the induction of apoptosis and an indirect effect via the stimulation of monocytes and macrophages cytokine synthesis. In this study, the direct effect of MCC towards LNCaP cancer cells was evaluated. METHODS: Inhibition of proliferation, cell cycle arrest and induction of apoptosis were evaluated in vitro using LNCaP cells treated with MCC. The synthesis of IL-12, GM-CSF, and TNF-alpha by LNCaP cells in response to MCC was also determined. Experiments were performed to gain insight into the mechanism of action of MCC towards LNCaP cells. RESULTS: MCC caused a dose-dependent inhibition of the proliferation of LNCaP cells that was associated with cell cycle arrest at the G0/G1 phase. MCC-induced apoptosis of LNCaP cells was consistent with a mitochondrial pathway involving mitochondrial disruption, release of cytochrome c, and an increase in Bax protein levels leading to caspase-3 and -7 activation and cleavage of poly (ADP-ribose) polymerase and nuclear mitotic apparatus protein. Surprisingly, MCC also directly induced the synthesis of IL-12 and GM-CSF, but not TNF-alpha, by LNCaP cells. CONCLUSIONS: MCC possesses the ability to directly induce apoptosis of LNCaP cells and to trigger the synthesis of IL-12 and GM-CSF by these cells, suggesting a potential role of MCC for the treatment of prostate cancer.

Pro-inflammatory activity of contaminating DNA in hyaluronic acid preparations.

Hyaluronic acid (HA), an abundant non-sulfated glycosaminoglycan component of the extracellular matrix, has applications in drug delivery, tissue engineering and as an ingredient in cosmetics. HA preparations containing high-molecular-weight polymers are also used in the treatment of inflammatory disorders such as arthritis and interstitial cystitis. Low-molecular-weight fragments derived from HA have been reported to induce pro-inflammatory cytokines such as IL-12 and TNF-alpha, and could therefore potentially exacerbate existing inflammation. We therefore examined the pro-inflammatory activity of HA preparations, since inflammatory reactions are known to occur following administration of HA. We tested low-molecular-weight fragments obtained from seven different HA preparations, either by sonication (approximately equals 3 x 10(5) Da) or by hyaluronidase digestion (approximately equals 1 x 10(4) Da), for the ability to induce the synthesis of IL-12 and TNF-alpha by human monocytic cells. We found that two of the seven HA preparations tested stimulated the synthesis of IL-12 and TNF-alpha by human monocytic cells. We unexpectedly found that the induction of IL-12 and TNF-alpha by these HA preparations was not due to their degradation to low-molecular-weight fragments, since their native high-molecular-weight forms possessed the same ability to stimulate IL-12 and TNF-alpha synthesis, but was due to the presence of contaminating DNA. Treatment of these two HA preparations with deoxyribonuclease I abrogated or reduced the induction of IL-12 and TNF-alpha. It is clear from this study that HA preparations can induce the synthesis of pro-inflammatory cytokines by monocytes. The ability of HA to act as a pro-inflammatory mediator may not, however, be related to the presence of low-molecular-weight HA fragments, but to the presence of DNA. The presence of pro-inflammatory DNA in HA preparations should be evaluated before its use, not only for the treatment of patients with inflammatory disorders, but also before many other applications.

Therapeutic potential of mycobacterial cell wall-DNA complexes.

Cell wall skeletons isolated from many bacteria have been shown to possess anticancer activity. The anticancer activities of such preparations have been attributed to the activation of immune effector cells and not to a direct effect on cancer cell division. A cell wall extract from Mycobacterium phlei, wherein mycobacterial DNA in the form of short oligonulceotides is preserved to the cell wall, has anticancer activity against a wide range of cancer cells. Mycobacterial cell wall-DNA complexes (MCC) exert their anticancer activity by a dual mechanism of action: an indirect effect via the induction of anticancer cytokines and a direct effect on cancer cell division mediated by the induction of apoptosis. In this review, the immunomodulatory and the pro-apoptotic mechanisms of action of MCC will be explored. The identification of the active component in MCC will be discussed, as well as the composition differences with cell wall skeletons and live mycobacteria. Finally, the use of MCC against bladder and prostate cancers will be discussed and compared to standard therapies, particularly therapy using mycobacteria and mycobacteria-derived products.

Anticancer activity of mycobacterial DNA: effect of formulation as chitosan nanoparticles.

Mycobacterium phlei (M. phlei) DNA inhibits cancer cell division but is susceptible to degradation by DNase. Chitosan forms nanoparticulate polyelectrolyte complexes with DNA, and may thus reduce nuclease degradation. We have characterized chitosan-DNA nanoparticle formation, determined DNase susceptibility, and evaluated their antiproliferative activity. Nanoparticle diameter initially decreased with increasing phosphate charge density. However nanoparticle diameter increased above 6 micromol of phosphate. Particle aggregation occurred at 16.2 micromol phosphate and was related to reduced surface charge. Incorporation of DNA within chitosan nanoparticles significantly decreased degradation by DNase. The ability of M. phlei DNA-chitosan nanoparticles to inhibit melanoma cell division was determined relative to M. phlei DNA and a cationic liposomal M. phlei DNA formulation. M. phlei DNA had antiproliferative activity (MTT reduction, IC50 = 0.9 mg/ml) without intrinsic cytotoxicity (LDH release, ED50 > 50 microg/ml). Cationic polyphosphate chitosan nanoparticles were inert (antiproliferative IC50 > 1 mg/ml, ED50 > 1 mg/ml). M. phlei DNA-chitosan nanoparticles were 20-fold more potent than M. phlei DNA. Cationic DOTAP/DOPE liposomes were cytostatic (IC50 = 49 microg/ml) and cytotoxic (ED50 = 87 microg/ml), and complexation of M. phlei DNA resulted in a significant reduction of antiproliferative activity. Chitosan nanoparticles may therefore be appropriate delivery vehicles for M. phlei DNA.

Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and present in a mycobacterial cell wall complex.

A mycobacterial cell wall complex prepared from the non-pathogenic microorganism Mycobacterium phlei, where mycobacterial DNA is preserved and complexed to cell wall fragments, possesses anticancer and immunomodulatory activity. DNA from a number of prokaryotes has been found to modulate the immune system and to induce cytokine synthesis. We have therefore determined whether the DNA associated with this complex has the ability to induce the synthesis of interleukin-12 (IL-12), a potent anticancer cytokine. Mycobacterial DNA complexed with cell wall fragments or DNA purified from M. phlei induced IL-12 synthesis by murine and human monocytes and macrophages in vitro, and was capable of inducing IL-12 synthesis in vivo in mice following i.p. administration. Neutralization of DNA with cationic liposomes or digestion with DNase I significantly decreased the ability of the cell wall complex to induce IL-12. CpG methylation of DNA extracted from these cell walls or from M. phlei did not affect the induction of IL-12 synthesis by monocytes and macrophages. In contrast, CpG methylation of DNA from Escherichia coli abolished its ability to induce IL-12 synthesis. These results demonstrate that unmethylated CpG motifs present in M. phlei DNA are not a prerequisite for the induction of IL-12 synthesis. The size of the mycobacterial DNA, in the range of 5 bp to genomic DNA, did not influence its capacity to induce IL-12. Our results emphasize that M. phlei DNA associated with the cell wall complex makes a significant contribution to the overall immunomodulatory and anticancer activity of this mycobacterial cell wall preparation and that these activities are not correlated with the presence of CpG motifs.

Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells.

Intact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood. Mycobacterium phlei mycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms--a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte-macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12.

Influence of surface properties at biodegradable microsphere surfaces: effects on plasma protein adsorption and phagocytosis.

OBJECTIVE: The objective of this work was to determine plasma protein adsorption and macrophage phagocytosis of biodegradable polyanhydride, polylactic acid and polylactic-co-glycolic acid microspheres prepared by both spray-drying and solvent evaporation techniques. METHODS: Microspheres were characterized by scanning electron microscopy (SEM), confocal laser microscopy, particle size distribution and zeta (zeta) potential determination. Plasma protein adsorption onto the microspheres was determined using a fluoroaldehyde reagent. Phagocytosis was evaluated by incubating microspheres containing the angiotensin II antagonist, L-158,809, with the macrophages in the presence or absence of the phagocytosis inhibitor cythochalasin D. The extent of phagocytosis was established by fluorescence determination of L-158,809 and by optical microscopy. The effect of amphiphilic poly(ethylene glycol) (PEG) derivatives on phagocytosis was determined using PEG-distearate incorporated into the microspheres. RESULTS: The average diameter of the microspheres, which depended on the polymer and the initial formulation, ranged from 0.9 to 3.2 micrometers. Zeta potential studies showed strong negative values irrespective of the polymer used for the spray-dried formulations. The zeta potential was masked by the incorporation of PEG 400- or PEG 1,400-distearate in the formulation. Confocal laser microscopy showed a homogenous dispersion of PEG (measured as PEG-fluorescein) in the microspheres. Protein adsorption was not observed for any of the microsphere formulations following incubation with bovine serum. Incubation of microspheres with murine macrophages showed that PEG-distearate inhibited phagocytosis at appropriate levels (0.1% w/w). Higher levels > 1% w/w of PEG-distearate) resulted in enhanced association with macrophages, despite the presence of the phagocytosis inhibitor cytochalasin D, indicating fusion between the microspheres and the plasma membrane. CONCLUSIONS: These results demonstrate that spray-dried PEG-containing microspheres can be manufactured and that an appropriate concentration of this excipient in microspheres results in decreased phagocytosis.

Drug retention and stability of solid lipid nanoparticles containing azidothymidine palmitate after autoclaving, storage and lyophilization.

Solid lipid nanoparticles (SLNs) were prepared using trilaurine (TL) as the SLN core and phospholipid (PL) as coating. Neutral and negatively charged PLs were used to produce neutral and negatively charged SLNs. An ester prodrug of 3'-azido-3'-deoxythymidine (Zidovudine, AZT), AZT palmitate (AZT-P), was synthesized and incorporated in the SLNs. The stability of SLN formulations containing AZT-P was studied at different temperatures. Drug retention and mean particle diameter of SLNs were determined after autoclaving, during temperature stability testing, and after lyophilization (with or without cryoprotective sugars) and reconstitution. There were no significant changes in the mean diameter and the zeta potential (zeta) of SLNs after autoclaving (121 degrees C for 20 min). The amount of incorporated AZT-P was, however, slightly reduced due to the formation of hydrosoluble AZT. Autoclaved SLNs were stable for a period of 10 weeks at 20 degrees C but an increase in particle size and loss of AZT-P were observed at 4 and 37 degrees C. Trehalose was an effective cryoprotectant for preventing SLN aggregation during lyophilization and subsequent reconstitution. Thermal gravimetric analysis showed that lyophilized preparations contained approximately 1% water. Using appropriate trehalose to lipid ratios, AZT-P retention in the SLNs was 100% after reconstitution. Our results demonstrate that SLNs containing AZT-P can be autoclaved, lyophilized and reconstituted without significant changes in SLN diameter and zeta potential or in the quantity of incorporated prodrug.

Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells.

Liposomal vectors formulated with cationic lipids (cationic liposomes) and fusogenic dioleoylphosphatidylethanolamine (DOPE) have potential for modulating the immune system by delivering gene or antisense oligonucleotide inside immune cells. The toxicity and the immunoadjuvant activity of cationic liposomes containing nucleic acids toward immune effector cells has not been investigated in detail. In this report, we have evaluated the toxicity of liposomes formulated with various cationic lipids towards murine macrophages and T lymphocytes and the human monocyte-like U937 cell line. The effect of these cationic liposomes on the synthesis of two immunomodulators produced by activated macrophages, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), has also been determined. We have found that liposomes formulated from DOPE and cationic lipids based on diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-, dipalmitoyl-, disteroyl-: DOTAP, DMTAP, DPTAP, DSTAP) or dimethyldioctadecylammonium bromide (DDAB) are highly toxic in vitro toward phagocytic cells (macrophages and U937 cells), but not towards non-phagocytic T lymphocytes. The rank order of toxicity was DOPE/DDAB > DOPE/DOTAP > DOPE/DMTAP > DOPE/DPTAP > DOPE/DSTAP. The ED50's for macrophage toxicity were < 10 nmol/ml for DOPE/DDAB, 12 nmol/ml for DOPE/DOTAP, 50 nmol/ml for DOPE/DMTAP, 400 nmol/ml for DOPE/DPTAP and > 1000 nmol/ml for DOPE/DSTAP. The incorporation of DNA (antisense oligonucleotide or plasmid vector) into the cationic liposomes marginally reduced their toxicity towards macrophages. Although toxicity was observed with cationic lipids alone, it was clearly enhanced by the presence of DOPE. The replacement of DOPE by dipalmitoylphosphatidylcholine (DPPC) significantly reduced liposome toxicity towards macrophages, and the presence of dipalmitoylphosphatidylethanolamine-PEG2000 (DPPE-PEG2000: 10 mol%) in the liposomes completely abolished this toxicity. Cationic liposomes, irrespective of their DNA content, downregulated NO and TNF-alpha synthesis by lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-activated macrophages. The replacement of DOPE by DPPC, or the addition of DPPE-PEG2000, restored NO and TNF-alpha synthesis by activated macrophages. Since macrophages constitute the major site of liposome localization after parenteral administration and play an important role in the control of the immune system, cationic liposomes should be used with caution to deliver gene or antisense oligonucleotide to mammalian cells. Cationic lipids show in vitro toxicity toward phagocytic cells and inhibit in vitro and in situ NO and TNF-alpha production by activated macrophages.

Anti-inflammatory activity of cationic lipids.

1. The effect of liposome phospholipid composition has been assumed to be relatively unimportant because of the presumed inert nature of phospholipids. 2. We have previously shown that cationic liposome formulations used for gene therapy inhibit, through their cationic component, the synthesis by activated macrophages of the pro-inflammatory mediators nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha). 3. In this study, we have evaluated the ability of different cationic lipids to reduce footpad inflammation induced by carrageenan and by sheep red blood cell challenge. 4. Parenteral (i.p. or s.c) or local injection of the positively charged lipids dimethyldioctadecylammomium bromide (DDAB), dioleyoltrimethylammonium propane (DOTAP), dimyristoyltrimethylammonium propane (DMTAP) or dimethylaminoethanecarbamoyl cholesterol (DC-Chol) significantly reduced the inflammation observed in both models in a dose-dependent manner (maximum inhibition: 70-95%). 5. Cationic lipids associated with dioleyol- or dipalmitoyl-phosphatidylethanolamine retained their anti-inflammatory activity while cationic lipids associated with dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylglycerol (DMPG) showed no anti-inflammatory activity, indicating that the release of cationic lipids into the macrophage cytoplasm is a necessary step for anti-inflammatory activity. The anti-inflammatory activity of cationic lipids was abrogated by the addition of dipalmitoylphosphatidylethanolamine-poly(ethylene)glycol-2000 (DPPE-PEG2000) which blocks the interaction of cationic lipids with macrophages. 6. Because of the significant role of protein kinase C (PKC) in the inflammatory process we have determined whether the cationic lipids used in this study inhibit PKC activity. The cationic lipids significantly inhibited the activity of PKC but not the activity of a non-related protein kinase, PKA. The synthesis of interleukin-6 (IL-6), which is not dependent on PKC activity for its induction in macrophages, was not modified in vitro or in situ by cationic lipids. The synthesis of NO and TNF-alpha in macrophages, both of which are PKC-dependent, was downregulated by cationic lipids. 7. These results demonstrate that cationic lipids can be considered as novel anti-inflammatory agents. The downregulation of pro-inflammatory mediators through interaction of cationic lipids with the PKC pathway may explain this anti-inflammatory activity. Furthermore, since cationic lipids have intrinsic anti-inflammatory activity, cationic liposomes should be used with caution to deliver nucleic acids for gene therapy in vivo.

Improved activity of a new angiotensin receptor antagonist by an injectable spray-dried polymer microsphere preparation.

PURPOSE: To characterize and evaluate in vitro and in vivo the release mechanisms involved in spray-dried biodegradable microspheres having different Poly(D,L-lactide) blend formulations and containing an antihypertensive drug (L-158,809). METHODS: Microspheres and blended polymers were characterized by DSC, SEM, confocal laser microscopy and size analysis. In vitro release studies were evaluated by using microspheres made from various blends of high and low molecular weight polymer. In vivo studies were evaluated by L-158,809 antagonist AT1 function versus the shift of the normal dose-response curve of blood pressure induced by Angiotensine II. RESULTS: The average yield of L-158,809 microspheres (10% (w/w)) was 95% of the theoretical loading. The average diameter of the microspheres was from 1 to 3 micrometers. In all release experiments, a significant burst effect (< 15%) was observed followed by a near zero-order release kinetics. In vivo studies with two different formulations show a strong shift of angiotensin II dose-response curve. CONCLUSIONS: The release kinetics and photomicrographs suggest that the system is best described as a multi-parameter controlled released system in which the drug is molecularly dispersed. In vivo results demonstrating the controlled release of L-158,809.

Evidence for phospholipid bilayer formation in solid lipid nanoparticles formulated with phospholipid and triglyceride.

PURPOSE: Solid lipid nanoparticles (SLN) are comprised of a high-melting point triglyceride (TG) core with a phospholipid (PL) coating. This study has investigated the possible formation of multiple PL bilayers on the TG core of SLN's as a function of increasing the PL:TG molar ratio. METHODS: Trilaurin (TL) was used as the SLN core. Dipalmitoylphos-phatdylcholine (DPPC) or a mixture of DPPC and dimyristoylphosphatidylglycerol (DMPG) were used to produce neutral and negatively charged SLN's. The volume of aqueous phase associated with the PL was determined using calcein and 6-carboxyfluorescein (6-CF) as hydrophilic markers incorporated during the preparation of the SLN's. RESULTS: The diameter of the SLN's decreased as the molar ratio of PL to TL was increased, until a PL:TL ratio of 0.15 was reached. After this point the diameter was not affected by further increases in the molar ratio. The experimental amount of PL required to prepare SLN's was significantly higher than the theoretical amount required to form a single monolayer on the surface. The aqueous volume associated with the PL was increased with increasing PL:TL molar ratios. CONCLUSIONS: The results obtained suggest that the formation of multiple PL bilayers is probable in SLN's prepared with a high molar ratio of PL to TL. The volume of the aqueous phase between the PL-bilayers, estimated from the amount of the hydrosoluble markers trapped in this phase, provides an indication of the relative number of bilayers at different PL:TL ratios.

Influence of phospholipid composition on antibody responses to liposome encapsulated protein and peptide antigens.

The effect of phospholipid composition on mouse IgG antibody responses to liposomal bovine serum albumin (BSA), murine monoclonal antibody GK1.5 (anti-CD4) or a 21 amino acid peptide from the second conserved domain of HIV gp120 after s.c. administration, and on the IgA, IgE, and IgG antibody response to liposomal Shistosoma mansoni glutathione-S-transferase (Sm28GST) after oral administration, was determined. Antibody responses were compared with alum-adsorbed and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-antigen mixtures. For the s.c. route, dipalmitoylphosphatidylcholine (DPPC)/dimyristoylphosphatidylglycerol (DMPG) liposomes induced 54-60% IgG1 and 35-44% IgG(2a+2b). DPPC/dipalmitoylphosphatidylethanolamine (DPPE) liposomes induced 73-78% IgG1 and 15-25% IgG(2a+2b). DPPC/ phosphatidylserine (PS) liposomes induced 86-89% IgG1 and 8-12% IgG(2a+2b). Alum and MDP induced 79-91% IgG1 and 4-17% IgG(2a+2b). The rank order of adjuvanticity for induction of IgG antibody was DPPC/DMPGDPPC/PE > > alum > > MDPDPPC/PS for all three antigens. DPPC/DMPG liposomes were the only effective adjuvant for the induction of secretory IgA and circulatory IgE and IgG antibodies against Sm28GST after oral administration. The failure of liposome-antigen mixtures to elicit an antibody response showed that liposomal incorporation of the antigens was obligatory for adjuvant activity. These results demonstrate that the correlation between phospholipid composition and adjuvanticity is independent of liposome charge, antigen, or route of administration.

Immunogenicity of immunoliposomes: reactivity against species-specific IgG and liposomal phospholipids.

Immunoliposomes with surface-linked avidin-biotinylated mouse IgG2a were prepared from dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG) and biotinylated dipalmitoylphosphatidylethanolamine (DPPE-biotin) in the molar ratio 10:1:0.1 with or without 5 mol% poly(ethylene glycol) dipalmitate (PEG-(C18)2). The ability of IgG2a immunoliposomes to elicit anti-IgG2a antibodies in mice was compared with alum and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). IgG2a 5 microgram/mouse) did not elicit an IgG1 antibody response after 4 s.c. injections. Alum-adsorbed IgG2a elicited 2.1 +/- microgram IgG1 antibody/ml serum, whereas MDP elicited 24.3 +/- microgram/ml serum. IgG2a immunoliposomes elicited 12.4 +/- 3.7 microgram IgG1 antibody/ml serum, while immunoliposomes containing lipophilic PEG-(C18)2 elicited 21.4 +/- 5.1 microgram IgG1 antibody/ml serum. Elicited antibodies were specific for IgG2a, with no cross-reactivity with IgG2b. Anti-DPPC or anti-DMPG IgG antibody levels did not change during immunization. Anti-DPPE IgG antibody levels were slightly but significantly elevated during immunization, and there was a significant increase in the level of anti-DPPE-biotin antibodies. These results demonstrate that immunoliposomes prepared with species-specific antibody are immunogenic and induce significant levels of isotypespecific antibody upon repeated injection.

Modulation of murine macrophage nitric oxide synthesis by liposomal phospholipids: correlation with liposome immune adjuvant activity.

The influence of alum and liposomal phospholipids on interferon-gamma-(IFN-gamma), IFN-gamma/N-acetylmuramyl-L-alanyl-D-isoglutamine- (MDP) or IFN-gamma/tumor necrosis factor-alpha- (IFN-gamma/TNF-alpha) induced macrophage nitric oxide (NO) synthesis has been investigated. IFN-gamma induced NO synthesis in a dose-dependent manner. TNF-alpha and MDP did not induce NO synthesis, but interacted synergistically with sub-optimal doses of IFN-gamma. Alum strongly inhibited IFN-gamma-induced NO synthesis (ID50 25 microgram/ml). Liposomes composed of dipalmitoylphosphatidylcholine (DPPC) had no effect on IFN-gamma-induced NO synthesis. IFN-gamma-induced NO synthesis was stimulated by DPPC/dimyristoylphosphatidylglycerol (DMPG) liposomes (9:1 mol ratio, ED50 45 nmol phospholipid/ml), and inhibited by DPPC/dipalmitoylphosphatidylethanolamine (DPPE) liposomes (9:1 mol ratio, ID50 > 500 nmol phospholipid/ml), and DPPC/phosphatidylserine (PS) liposomes (7:3 mol ratio, ID50 150 nmol phospholipid/ml). Alum, DPPC/PE and DPPC/PS liposomes also inhibited IFN-gamma/MDP- and IFN-gamma/TNF-alpha-induced NO synthesis. Neither alum or the liposome preparations had significant toxicity towards macrophages in vitro at concentrations that induced maximal inhibition or stimulation of IFN-gamma-induced NO synthesis. Immunization of mice with alum-adsorbed and liposome-incorporated bovine serum albumin (BSA) demonstrated that enhancement or reduction of both IgG antibody and the proportion of IgG2a/IgG2b was correlated with stimulation or inhibition of IFN-gamma-induced NO synthesis.

Tumor cell binding and induction of endothelial cell tumoricidal activity in vitro by muramyl dipeptide is enhanced by liposomal encapsulation.

We investigated the ability of phosphatidylcholine (PC):phosphatidylserine (PS) liposomes containing muramyl dipeptide (MDP) to enhance tumor cell binding and induction of vascular endothelial cell tumoricidal activity in vitro against syngeneic B16-BL6 melanoma tumor cells. Endothelial cell-liposome interaction was influenced by the proportion of PS in the liposome and the length of fatty acid chain of PC. Optimum phagocytosis occurred with multilamellar liposomes (mean diameter 0.8 microns) composed of distearoylphosphatidylcholine (DSPC):PS, 1:1 molar ratio. Hydrosoluble MDP stimulated endothelial cell binding of tumor cells in the dose range 10-100 micrograms/ml. Liposome-incorporated MDP or liposomes containing the lipophilic MDP prodrug MDP-glyceroyl dipalmitate (GDP) significantly enhanced tumor cell binding in the dose range 0.01-1 micrograms/ml. MDP, MDP-containing liposomes, or liposomal MDP-GDP induced endothelial cell tumoricidal activity against B16-BL6 tumor cells with relative potencies of 1.0, 10, and 500, respectively. MDP or liposomal MDP did not induce or enhance endothelial cell nitric oxide (NO) synthase activity (as determined by NO2- production) in the absence or presence of interferon-tau (IFN-tau) or tumor necrosis factor-alpha (TNF-alpha), respectively, indicating the induction of an NO-independent cytotoxic mechanism.

Immunoliposome targeting to murine CD4+ leucocytes is dependent on immune status.

Immunoliposomes were prepared from dipalmitoylphosphatidylcholine, dimyristoylphosphatidylglycerol, biotinylated PE, and surface-linked avidin-biotinylated GK1.5 monoclonal rat (anti-mouse CD4) IgG or F(ab)2. Anti-CD4 immunoliposomes bound quantitatively to CD4+ PBMC's in vitro. Intravenous injection of mice with 1 mumol anti-CD4 immunoliposomes resulted in targeting to a significant proportion (> 75%) of CD4+ PBMCs. Repeated administration of anti-CD4 immunoliposomes induced significant levels of anti-GK 1.5 IgG Ab, with concomitant reductions in immunoliposome plasma t1/2 and targeting efficacy, and increased volumes of distribution. The immunogenicity of the immunoliposome was enhanced if GK1.5 F(ab)2 instead of IgG was used as the targeting ligand. Lipophilic poly(ethylene glycol) derivatives incorporated into the immunoliposomes did not affect targeting efficiency but significantly enhanced immunogenicity. These results demonstrate that IgG or F(ab)2 immunoliposomes are highly immunogenic and that targeting in vivo is conditional on the immune status of the host.

Responsiveness of individual airways to methacholine in adult rat lung explants.

We used a modified adult lung explant technique to directly measure the area of individual airways before and after methacholine (MCh) administration. Lungs were removed from 12-wk-old male Lewis rats under sterile conditions, filled with an agarose-containing solution at 37 degrees C, and cooled to 4 degrees C. Transverse slices (0.5-1.0 mm thick) were cut and cultured overnight. Concentration-response curves to MCh were determined for explant airways from lungs inflated to 25, 50, 75, and 100% total lung capacity (TLC) with a 1.0% agarose solution and to 75% TLC with 0.5 and 2.0% agarose solutions. MCh was added to the medium to achieve final concentrations ranging from 10(-9) to 10(-2) M. Airways were imaged before and 10 min after each increase in MCh concentration with an inverted microscope and video camera, and airway area was determined by computerized image processing. The maximal response (MR) ([1-(minimal area/baseline area)] x 100) and concentration of MCh resulting in 50% MR (EC50) were determined. A total of 217 airways from 3-12 explants per rat constricted in a concentration-dependent manner. Baseline area was larger with both higher lung volumes and agarose concentrations. MR was greatest in the airways from the 25% TLC and 0.5% agarose explants. Although there was considerable heterogeneity toward MCh within rats (EC50 varied up to 5.46 x 10(5)-fold), the median EC50 was similar among all rats (range 1.96 x 10(-6)-5.87 x 10(-4) M). Lung inflation volume and agarose concentration affected baseline area and MR, suggesting that airway-parenchymal interdependence mechanisms are operative in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired stimulation of anti-bovine serum albumin IgG antibodies by vaccine adjuvants in murine acquired immunodeficiency syndrome.

The effect of three adjuvants - alum, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), and liposomes - on the IgG antibody isotype response to bovine serum albumin (BSA), was determined in normal and LP-BM5 retrovirus infected C57BL/6 mice. Alum and MDP induced comparable levels of IgG antibodies in normal mice (predominantly IgG1 (greater than 90%)), whereas liposomes induced IgG1 (60%), IgG2a/b (30%) and IgG3 (10%) antibodies. IgG antibody levels using liposomes as adjuvant were five-fold higher than those observed with alum or MDP. Immunization after LP-BM5 infection significantly reduced the effectiveness of alum and MDP, IgG antibody levels being reduced by 80 and 90% at 3 or 7 weeks respectively. The adjuvant activity of liposomes was reduced by 55 and 65% when immunization was started 3 or 7 weeks post LP-BM5 infection. Boosting of pre-immune mice with BSA and alum, MDP or liposomes 3 weeks after LP-BM5 infection showed that, while the magnitude of the antibody response and isotype distribution was not affected, the persistence of the response was severely diminished compared to control, non-infected mice. The reduced immunoadjuvant activity correlated with a reduction in the frequency of splenic Thy1.2+/CD4+ T cells. These results demonstrated that liposomes were more effective than alum or MDP in inducing IgG antibodies, and that immunoadjuvant activity for prophylactic or therapeutic immunization for all 3 adjuvants was significantly impaired by retroviral infections.