Mario C. Filion

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 ED50s 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.

Major limitations in the use of cationic liposomes for DNA delivery

Liposomal vectors formulated with cationic lipids and the fusogenic phospholipid dioleyolphosphatidylethanolamine (DOPE) are usually used to target DNA inside mammalian cells. Since macrophages constitute the major site of liposome localisation after parenteral administration we felt it prudent to examine the effect of cationic liposomes on the production of several important immuno-inflammatory modulators secreted by activated macrophages. In addition, we have evaluated the toxicity of different cationic liposome formulations towards phagocytic macrophages and non-phagocytic T-lymphocytes. Our results indicate that cationic liposomes are able to down-regulate the synthesis of the protein kinase C (PKC)-dependent mediators nitric oxide (NO), tumour necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) by activated macrophages after in vitro incubation under non-toxic conditions or after in vivo treatment, while the production of PKC-independent IL-6 is not modified. We have shown that cationic lipids possess potent anti-inflammatory activity in vivo. Prolonged incubation (>3 h) of macrophages with cationic liposomes induced a high level of toxicity (ED50 1000 nmol/ml). The rank order of toxicity was DOPE/dimethyldioctacylammonium bromide (DDAB)>DOPE/dioleyoltrimethylammonium propane (DOTAP)=DOPE/dimethylaminoethanecarbamoyl cholesterol (DC-Chol)>DOPE/dimyristoyltrimethylammonium propane. The replacement of DOPE by dipalmitoylphosphatidylcholine (DPPC) or the incorporation of dipamitoylphosphatidylethanolamine-PEG2000 (DPPE-PEG2000) in DOPE/cationic lipids reduced the toxicity toward macrophages and restored the synthesis of PKC-dependent modulators. The incorporation of DNA, either as an antisense oligonucleotide (15-mers) or as the plasmid vector pBR322 (4363 bp), in cationic liposomes did not reduce these adverse effects. These results, in addition to the observation that cationic liposomes are extremely toxic following oral administration, indicate that DOPE/cationic lipid liposomes are not appropriate for DNA (or drug) delivery.

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‐α (TNF‐α). 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‐α 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.

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‐α, 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 (≅ 3 times 105 Da) or by hyaluronidase digestion (≅ 1 times 104 Da), for the ability to induce the synthesis of IL‐12 and TNF‐α by human monocytic cells. We found that two of the seven HA preparations tested stimulated the synthesis of IL‐12 and TNF‐α by human monocytic cells. We unexpectedly found that the induction of IL‐12 and TNF‐α 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‐α 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‐α. 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.

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

Abstract 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.

The mouse keratin 19-encoding gene: sequence, structure and chromosomal assignment

Keratin 19 (K19) is synthesized mainly in embryonic and adult simple epithelia, but has also been found in stratified epithelia as well. K19 is the smallest known keratin and is remarkable in that, contrary to all other keratins, it does not have a designated partner for the formation of filaments, implying that regulation of its expression is different from other keratin-encoding genes. As a first step in elucidating the mechanisms by which the K19 gene is regulated in relatively undifferentiated embryonic and in terminally differentiated adult tissues, a series of overlapping clones containing the complete mouse K19 gene was isolated from a mouse genomic library and characterized. The nucleotide (nt) sequence extends over 5119 nt and includes six exons. A region of 303 nt upstream from the transcription start point (tsp) was also sequenced. Comparison with the human and bovine K19 genes revealed the existence of homologies in both the coding and noncoding regions. The putative promoter region of the mouse K19 gene is highly homologous to the corresponding sequences of the human and bovine K19 genes. It contains an ATA box, a CAAT box and two potential Sp1-binding sites. Significant homologies were also found between the sequences of the introns of the mouse, human and bovine genes: this was particularly evident in introns 2, 3, 4 and 5. Intron 1, which showed the greatest degree of divergence, was found to contain many repetitive elements. Finally, it is shown that the mouse K19 gene cosegregates with the type-I keratin-encoding gene locus (Krt-1) on chromosome 11.

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Mycobacterial cell wall-DNA complex induces apoptosis in cancer cells

pathogenic organism Mycobacterium phlei (M. phlei) has been shown to have anti-tumour activity in animal tumour models (Chm et al, 1996) and in clinical studies in patients with carcinoma in situ of the bladder (Morales and Chin, 1997). We have demonstrated that MCC is a powerful inducer of a number of macrophagederived cytolunes such as IL12 that have indirect anti-cancer activity, and that it can directly induce apoptosis in human bladder cancer cells (Filion et al, 1998).

Cloning and identification of genes differentially expressed in metastatic and non-metastatic rat rhabdomyosarcoma cell lines.

The objective of this study was to identify genes involved in invasion and metastasis using a rat rhabdomyosarcoma model (SMF-A and RMS-B cell lines). The SMF-A cell line was established from a metastatic nodule of an induced rhabdomyosarcoma in syngeneic F344 rats. Two cell lines with defined metastatic potentials, SMF-Ai and SMF-Da, were cloned from the SMF-A line. The cell line SMF-Ai is tumorigenic, highly invasive and highly metastatic. On the other hand, the revertant line SMF-Da is less tumorigenic, non-invasive and non-metastatic. We have isolated from a SMF-Ai cDNA library eight cDNA clones which are differentially expressed by the metastatic SMF-Ai and the non-metastatic SMF-Da cell line using Northern blot analysis. Five of these clones, smf-4, smf-6, smf-41, smf-42 and smf-44, are overexpressed in the SMF-Da cell line and have homology with beta-2-microglobulin, lactate dehydrogenase, ribosomal protein L38, ribosomal protein S4 and acidic ribosomal phosphoprotein P1, respectively. The three other clones, smf-7, smf-40 and smf-61, are overexpressed in SMF-Ai. Clones smf-40 and smf-61 show significant homology with the human TB3-1 gene and the human fus gene respectively. The clone smf-7 has no significant homology with known sequences. We also analyzed the expression of these clones in other rat rhabdomyosarcoma cell lines (RMS-B and their clones) and in tumors obtained by injection of these cell lines into rats or nude mice. Smf-61 and smf-7 were the only clones with a differential expression pattern associated with the invasive or metastatic potential of all cell lines examined. A preliminary study of the expression of smf-7 and smf-61 in other cancer cell lines also showed mRNA expression in two human rhabdomyosarcomas and a human epidermoid carcinoma suggesting the existence of genes homologous to smf-7 and smf-61 clones in human cancers. Our findings suggest an association between the expression of smf-7 and smf-61 and invasive or metastatic potential of rhabdomyosarcoma cells.