liposome-DNA Complexes Research Articles

The adjuvant CLDC increases protection of a herpes simplex type 2 glycoprotein D vaccine in guinea pigs

Herpes simplex virus (HSV) infections are common but there is no vaccine available. We evaluated cationic liposome–DNA complexes (CLDC) as an adjuvant for an HSV gD2 vaccine and compared it to an MPL/Alum adjuvant in a guinea pig model of genital herpes. The addition of CLDC to the gD2 vaccine significantly decreased acute and recurrent disease and most importantly the number of days with recurrent virus shedding compared to gD2 alone. Reductions in these outcomes were also detected when gD2+CLDC was compared to gD2+MPL/Alum. When the vaccine and adjuvants were evaluated as therapeutic vaccines, they were ineffective. CLDC enhanced protection compared to MPL/Alum and is the first vaccine to reduce recurrent virus shedding, a key to decreasing the spread of HSV-2.

Nature as a source of inspiration for cationic lipid synthesis

Synthetic gene delivery systems represent an attractive alternative to viral vectors for DNA transfection. Cationic lipids are one of the most widely used non-viral vectors for the delivery of DNA into cultured cells and are easily synthesized, leading to a large variety of well-characterized molecules. This review discusses strategies for the design of efficient cationic lipids that overcome the critical barriers of in vitro transfection. A particular focus is placed on natural hydrophilic headgroups and lipophilic tails that have been used to synthesize biocompatible and non-toxic cationic lipids. We also present chemical features that have been investigated to enhance the transfection efficiency of cationic lipids by promoting the escape of lipoplexes from the endosomal compartment and DNA release from DNA-liposome complexes. Transfection efficiency studies using these strategies are likely to improve the understanding of the mechanism of cationic lipid-mediated gene delivery and to help the rational design of novel cationic lipids.

Rapid immunity to vaccination with woodchuck hepatitis virus surface antigen using cationic liposome–DNA complexes as adjuvant

Complexes of cationic liposomes and non-coding DNA (CLDC) have shown promise as vaccine adjuvant. Using the woodchuck animal model of hepatitis B virus (HBV) infection, the immunogenic effects of CLDC were evaluated following vaccination with three doses of woodchuck hepatitis virus surface antigen (WHsAg) adjuvanted with either CLDC or conventional alum and administered intramuscularly (im) or subcutaneously (sc). IM vaccination with WHsAg and CLDC elicited antibodies earlier, in more woodchucks, and with higher titers than WHsAg and alum. After two vaccine doses, antibody titers were higher following im than sc administration. Woodchucks administered two vaccine doses sc received the third vaccine dose im, and antibody responses reached titers comparable to those elicited by im administration. Following the second vaccine dose, im vaccination with WHsAg and CLDC induced T cell responses to WHsAg and selected WHs peptides and expression of the leukocyte surface marker CD8 and of the Th1 cytokines interferon-gamma and tumor necrosis factor alpha in woodchucks. T cell responses and CD8/cytokine expression were diminished in woodchucks from the other groups suggesting that this vaccine regimen induced a skew toward Th1 immune responses. The present study in woodchucks demonstrates that CLDC-adjuvanted WHsAg vaccine administered im resulted in a more rapid induction of humoral and cellular immune responses compared to conventional, alum-adjuvanted WHsAg vaccine. While less rapid, the immune responses following sc administration can prime the im immune responses. This adjuvant activity of CLDC over alum may be beneficial for therapeutic vaccination in chronic HBV infection.

Vaccine-Induced CD8+ T Cell-Dependent Suppression of Airway Hyperresponsiveness and Inflammation

Suppressing the abnormalities associated with asthma has been difficult to accomplish using immunotherapy or vaccination once the disease is established. The effector cells necessary for effective immunization/vaccination and immunotherapy of asthma are also not well understood. Therefore, we vaccinated allergen (OVA)-sensitized mice to determine whether therapeutic immunization could suppress airway hyperresponsiveness (AHR) and inflammation and to identify key immune effector cells and cytokines. Mice were immunized with a vaccine comprised of Ag and cationic liposome-DNA complexes (CLDC), a vaccine which has previously been shown to elicit strong CD4(+) and CD8(+) T cell responses and activation of Th1 immunity. We showed that immunization with the OVA-CLDC vaccine significantly suppressed AHR, eosinophilia, goblet cell metaplasia, and Th2 cytokine production. In contrast, immunization with CLDC alone suppressed eosinophilia and Th2 cytokine production, but failed to suppress AHR and goblet cell changes. Using adoptive transfer experiments, we found that suppression of AHR was mediated by Ag-specific CD8(+) T cells and was dependent on IFN-gamma production by the transferred T cells. Thus, we conclude that generation of strong, allergen-specific CD8(+) T cell responses by immunization may be capable of suppressing AHR and allergic airway inflammation, even in previously sensitized and challenged mice.

Mucosal immunotherapy for protection from pneumonic infection with Francisella tularensis

Previous studies have demonstrated that systemically administered immunotherapy can protect mice from systemic challenge with the bacterial pathogen Francisella tularensis. However, for protection from inhalational challenge with this bacterium, we wondered if mucosally administered immunotherapy might be more effective. Therefore, we administered cationic liposome–DNA complexes (CLDC), which are potent activators of innate immunity, intranasally (i.n.) and assessed the effectiveness of protection from lethal inhalational challenge with F. tularensis. We found that pretreatment by i.n. administration of CLDC 24 h prior to bacterial challenge elicited nearly complete protection of BALB/c mice from lethal challenge with F. tularensis LVS strain. We also observed that mucosal CLDC immunotherapy provided a statistically significant increase in survival time in mice challenged with the highly virulent F. tularensis Schu4 strain. Protection was associated with a significant reduction in bacterial burden in the lungs, liver, and spleen. Mucosal administration of CLDC elicited significantly increased expression of IL-12, IFN-γ, TNF-α, IFN-β and IFN-α genes in the lung as detected by real-time quantitative PCR. In vitro treatment of F. tularensis infected macrophages with CLDC-elicited cytokines also significantly suppressed intracellular replication of F. tularensis in infected macrophages. In vivo, depletion of NK cells prior to administration of CLDC completely abolished the protective effects of CLDC immunotherapy. CLDC-elicited protection was also dependent on induction of IFN-γ production in vivo. We conclude therefore that activation of local pulmonary innate immune responses is capable of eliciting significant protection from inhalational exposure to a virulent bacterial pathogen.

Effect of cationic liposomes/DNA charge ratio on gene expression and antibody response of a candidate DNA vaccine against Maedi Visna virus

Maedi Visna virus (MVV) is an ovine lentivirus with high prevalence all over the world. Since conventional vaccines had failed in protecting animals against the infection, the development of a DNA vaccine can be an alternative. The candidate vaccine was constructed by cloning the sequence encoding MVV p25 protein and was tested both in vitro and in vivo experiments associated with cationic liposomes. The lipoplexes (plasmid DNA–liposome complexes) with charge ratios ranging from 0 to 18 were prepared in physiological saline solution and characterized at a physical-chemistry level. Agarose gel electrophoresis was used as a first approach to evaluate qualitatively the amount of unbounded DNA by the liposomes. Dynamic light scattering measurements revealed that under the studied conditions lipoplexes with theoretical charge ratios (+/−) from 3 to 6 are unstable and prone to aggregation displaying sizes higher than 1 μm. At lower and higher charge ratios lipoplex size range from 200 to 500 nm. Using a Foster Resonance Energy Transfer methodology previously reported by us, complexation efficiency of the same complexes was related to in vitro and in vivo results. Higher transfection efficiencies were obtained in vitro with lipoplexes with charge ratio (+/−) = 10, where 97% of the DNA were protected by the liposomes. However, the subcutaneous immunization of mice induced higher antibody titers with lipoplexes at charge ratio (+/−) = 1, in which only 23% DNA is protected by the liposomes. Moreover, use of cationic liposomes has shown an increased antibody response when compared with a naked DNA immunization.

Cationic lipid/DNA complexes (JVRS-100) combined with influenza vaccine (Fluzone®) increases antibody response, cellular immunity, and antigenically drifted protection

Safe and effective adjuvants for influenza vaccines that could increase both the levels of neutralizing antibody, including against drifted viral subtypes, and T-cell immunity would be a major advance in vaccine design. The JVRS-100 adjuvant, consisting of DOTIM/cholesterol cationic liposome–DNA complexes, is particularly promising for vaccines that require induction of high levels of antibody and T-cell immunity, including CD8+ cytotoxic T lymphocytes (CTL). Inclusion of protein antigens with JVRS-100 results in the induction of enhanced humoral and cell-mediated (i.e., CD4+ and CD8+ T cells) immune responses. The JVRS-100 adjuvant combined with a split trivalent influenza vaccine (Fluzone®—sanofi pasteur) elicited increased antibody and T-cell responses in mice and non-human primates compared to vaccination with Fluzone® alone. Mice vaccinated with JVRS-100–Fluzone® and challenged with antigenically drifted strains of H1N1 (PR/8/34) and influenza B (B/Lee/40) viruses had higher grade protection, as measured by attenuation of weight loss and increased survival, compared to recipients of unadjuvanted vaccine. The results indicate that the JVRS-100 adjuvant substantially increases immunogenicity and protection from drifted-strain challenge using an existing influenza vaccine.

A novel vaccine adjuvant for recombinant flu antigens

Many new vaccines under development consist of rationally designed recombinant proteins that are relatively poor immunogens unless combined with potent adjuvants. There is only one adjuvant in common use in the U.S., aluminum phosphate or hydroxide (e.g. alum). This adjuvant, however, has significant limitations, particularly regarding the generation of strong cell-mediated (T-cell) immune responses. A novel adjuvant, JVRS-100, composed of cationic liposome–DNA complexes (CLDC) has been evaluated for immune enhancing activity. The JVRS-100 adjuvant has been shown to elicit robust immune responses compared to CpG oligonucleotides, alum, and MPL adjuvants, and efficiently enhances both humoral and cellular immune responses. Safety has been evaluated in preclinical studies, and the adjuvant is now in early-stage clinical development. One application of this novel adjuvant is to augment the immune responses to recombinant subunit antigens, which are often poorly immunogenic. The JVRS-100 adjuvant, when combined with a recombinant influenza hemagglutinin (H1), elicited increased specific antibody and T-cell responses in mice. Single-dose vaccination and prime/boost vaccinations with JVRS-100-H1 were both shown to be protective (i.e., survival, reduced weight loss) following H1N1 (PR/8/34) virus challenge. Enhanced immunological responses could be critically important for improved efficacy and dose-sparing of a recombinant influenza vaccine.

Potent Adjuvant Activity of Cationic Liposome-DNA Complexes for Genital Herpes Vaccines

Development of a herpes simplex virus (HSV) vaccine is a priority because these infections are common. It appears that potent adjuvants will be required to augment the immune response to subunit HSV vaccines. Therefore, we evaluated cationic liposome-DNA complexes (CLDC) as an adjuvant in a mouse model of genital herpes. Using a whole-virus vaccine (HVAC), we showed that the addition of CLDC improved antibody responses compared to vaccine alone. Most important, CLDC increased survival, reduced symptoms, and decreased vaginal virus replication compared to vaccine alone or vaccine administered with monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) following intravaginal challenge of mice. When CLDC was added to an HSV gD2 vaccine, it increased the amount of gamma interferon that was produced from splenocytes stimulated with gD2 compared to the amount produced with gD2 alone or with MPL-alum. The addition of CLDC to the gD2 vaccine also improved the outcome following vaginal HSV type 2 challenge compared to vaccine alone and was equivalent to vaccination with an MPL-alum adjuvant. CLDC appears to be a potent adjuvant for HSV vaccines and should be evaluated further.

Enhanced in vivo immunogenicity of SIV vaccine candidates with cationic liposome-DNA complexes in a rhesus macaque pilot study

This pilot study tested the immunogenicity of a novel cationic liposome-DNA complex (CLDC) immunomodulatory vaccine adjuvant. Combined with a specific antigen, CLDC enhanced anti-SIV immune responses induced by various SIV vaccine candidates. Rhesus macaques immunized in the presence of CLDC developed stronger SIV-specific T and B cell responses compared to animals immunized without CLDC. These differences persisted and resulted in better memory responses after an in-vivo boost of the animals several months later with whole AT-2 inactivated SIVmac239. Thus, CLDC should be explored further as a potential immunomodulatory adjuvant in HIV vaccine design.

Cationic liposome–DNA complexes as gene delivery vectors: Development and behaviour towards bone-like cells

Modulation of the biological pathways responsible for fracture repair and osteogenisis may accelerate regeneration. Gene therapy is an alternative method for the release of osteogenisis-stimulating proteins into tissues. The development of vectors for gene release is still a problem in terms of ethics and techniques. In this work we evaluated whether cationic liposomes constitute a valuable strategy for the release of genetic material into bone tissue cells as non-viral vectors. Liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)–2-dioleoyl-sn-glycero-3-phosphatidylethanolamine and DOTAP–cholesterol, and characterized according to their size, zeta potential, DNA protection capacity and cytotoxicity. Transfection studies were also carried out using pCMVβ-gal plasmid in two osteoblastic cell lines (MG63 and MC3T3-E1) and in the 294T line, varying the charge ratio and the applied DNA dose. Inclusion of transferrin to increase the expression was also tested. The results suggest that there is great dependency between the transfection activity and the lipid formulation, the charge ratios of the complexes, the applied DNA dose and the cell type. There were even some differences concerning both osteoblastic lines under study. The cells of the MC3T3-E1 line present greater expression levels than the cells of the MG-63 line. The conjugation of the transferrin with the complexes contributes to the increase in transfection levels, possibly due to an increase in internalization of complexes. It is thus a good strategy for inducing the expression of specific genes in osteoblast-like cells.

Protection from Pneumonic Infection withBurkholderiaSpecies by Inhalational Immunotherapy

Burkholderia mallei and B. pseudomallei are important human pathogens and cause the diseases glanders and melioidosis, respectively. Both organisms are highly infectious when inhaled and are inherently resistant to many antimicrobials, thus making it difficult to treat pneumonic Burkholderia infections. We investigated whether it was possible to achieve rapid protection against inhaled Burkholderia infection by using inhaled immunotherapy. For this purpose, cationic liposome DNA complexes (CLDC), which are potent activators of innate immunity, were used to elicit the activation of pulmonary innate immune responses. We found that mucosal CLDC administration before or shortly after bacterial challenge could generate complete or nearly complete protection from inhalational challenge with 100% lethal doses of B. mallei and B. pseudomallei. Protection was found to be dependent on the CLDC-mediated induction of gamma interferon responses in lung tissues and was partially dependent on the activation of NK cells. However, CLDC-mediated protection was not dependent on the induction of inducible nitric oxide synthase, as assessed by depletion studies. We concluded that the potent local activation of innate immune responses in the lung could be used to elicit rapid and nonspecific protection from aerosol exposure to both B. mallei and B. pseudomallei.

Prophylaxis with cationic liposome–DNA complexes protects hamsters from phleboviral disease: Importance of liposomal delivery and CpG motifs

Cationic liposome-DNA complexes (CLDC) are cationic/neutral lipid carriers complexed with plasmid DNA that when administered systemically results in a robust T(H)1 cytokine response. CLDC have been shown to be effective in prophylaxis and therapeutic treatment of animal models of viral disease. To determine the contribution of liposomal delivery and CpG content of the plasmid DNA to the efficacy of CLDC; plasmid, CpG-free plasmid DNA, or CpG-containing oligodeoxynucleotides (ODN) with and without liposomes, as well as poly(I:C(12)U), were evaluated for their ability to elicit protection against lethal Punta Toro virus (PTV, Bunyaviridae, phlebovirus) challenge in hamsters. CLDC-containing plasmid significantly improved survival, decreased systemic and liver viral loads, and reduced liver damage due to progression of viral infection. Mouse-reactive ODNs complexed with liposomes failed to protect hamsters, whereas ODNs known to cross-react with human and mouse (CpG 2006) or non-liposomal poly(I:C(12)U) showed survival benefit but did not limit liver injury. Liposomes complexed with a non-CpG motif-containing plasmid reduced liver viral load and tissue damage, but did not protect hamsters from death. To evaluate the mechanisms of the enhanced activity of CLDC, microarray experiments examined differences in the gene expression profile. The results suggest a broad T(H)1 response elicited by liposomal delivery of a diverse sequence containing CpG and non-CpG elements may be a more effective antiviral treatment than other nucleic acid based immunotherapeutics.

Thermal stability of DNA in DNA-induced DOTAP liposome aggregates

The influence on the melting of calf thymus DNA induced by cationic liposomes, commonly used in gene therapy, was studied by means of ultraviolet spectrophotometry and differential scanning calorimetry. Both the two methods reveal that DNA in DNA-induced liposome complexes undergoes a denaturation process at a much higher temperature than free DNA does. The extent of protection strongly depends on the charge ratio R(+/−) of liposome-DNA complexes. In the case of dioleoyl trimethyl ammonium propane (DOTAP) liposomes, the maximum of the stabilization occurs at R(+/−)=0.7, where the DNA is still native up to temperatures higher than 100°C. This protection against denaturation up to higher temperatures might be of importance for bio-technological applications, such as biomolecular separation, antigene sequencing and for drug design purpose.

Immunotherapy of Acute Leukemia with Cationic Lipid DNA Complexes (JVRS‐100)

Acute leukemias have long been recognized as suitable targets for immunotherapy. Administration of cationic liposome‐DNA complexes (JVRS‐100) elicits cytokines of the TH1 subset, especially IL‐12, IFN‐gamma and IFN‐alpha. These cytokines are particularly important in mediating host defense against cancer. In multiple experiments using the 32Dp210‐GFP model, intravenous dosing of JVRS‐100 prevented or significantly delayed death from leukemia when delivered between 7 and 15 days following leukemic challenge. Specifically, control animals died at ∼19 days of leukemia whereas 80% of similarly leukemia challenged but JVRS‐100 treated animals (1 ug) were alive >40 days with weekly treatment beginning at day 15 (4 days prior to expected mortality). Animals dying of leukemia had splenomegaly and leukemic infiltration of spleen and liver. The GFP expression allowed quantification of leukemia in peripheral blood (PB). At day 12 following challenge PB leukocytes were ∼30% GFP positive with either no treatment, or 0.1 ug JVRS‐100, <0.1% with 1.0 ug or 5.0 ug JVRS‐100 treatment at days 2 and 9. Furthermore there is a similar cell activation profile and cytokine response in vitro following exposure of human PBMCs to JVRS‐100 as is observed following in vivo administration of JVRS‐100 to mice, making this immunostimulation technology a promising candidate for product development and eventual clinical evaluation.

Transferrin-Associated Lipoplexes as Gene Delivery Systems: Relevance of Mode of Preparation and Biophysical Properties

The successful application of gene therapy depends highly on understanding the properties of gene carriers and their correlation with the ability to mediate transfection. An important parameter that has been described to improve transfection mediated by cationic liposomes involves association of ligands to cationic liposome-DNA complexes (lipoplexes). In this study, ternary complexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane:cholesterol, plasmid DNA and transferrin (Tf, selected as a paradigm of a ligand) were prepared under various conditions, namely, in medium with different ionic strengths (HEPES-buffered saline [HBS] or dextrose), at different lipid/DNA (+/-) charge ratios and using different modes for component addition. We investigated the effect of these formulation parameters on transfection (in the absence and presence of serum), size of the complexes, degree of DNA protection and extent of their association with cells (in terms of both lipid and DNA). Our results show that all the tested parameters influenced to some extent the size of the complexes and their capacity to protect the carried genetic material, as well as the levels of cell association and transfection. The best transfection profile was observed for ternary complexes (Tf-complexes) prepared in high ionic strength solution (HBS), at charge ratios close to neutrality and according to the following order of component addition: cationic liposomes-Tf-DNA. Interestingly, in contrast to what was found for dextrose-Tf-complexes, transfection mediated by HBS-Tf-complexes in the presence of serum was highly enhanced.

Liposome-induced DNA compaction and reentrant condensation investigated by dielectric relaxation spectroscopy and dynamic light scattering techniques

Interaction of DNA with oppositely charged objects, such as multivalent ions, cationic surfactants, cationic liposomes, basic proteins, and alcohols, up to nano- or mesoscopic particles, gives rise to a very interesting and fascinating phenomenology, where the shape, size, and stability of the resulting aggregates depend on a delicate balance between different driving forces, mainly of electrostatic origin. We have studied the cationic liposome-DNA complexes during the whole complexation process, below, close to, and above the isoelectric condition, where the number of cationic lipids equals the number of phosphate groups on the DNA chain. We took advantage of the combined use of dynamic light scattering, laser Doppler electrophoretic mobility, and radio-wave dielectric relaxation measurements in order to characterize both the structural parameters (hydrodynamic radius) and the electrical parameters (charge and counterion concentration) of the resulting structures. These structures are fundamentally of two types, clusters of liposomes stuck together by DNA chains (cluster phase in low-density colloidal suspension) and coexisting DNA coils and DNA globules, according to the procedure through which interactions occur (liposomes in excess DNA solution or DNA in excess liposome suspension).

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome–DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.

Systemic and Specific Delivery of Small Interfering RNAs to the Liver Mediated by Apolipoprotein A-I

Tissue-targeted delivery of small interfering RNA (siRNA) must be achieved before RNA interference (RNAi) technology can be used in practical therapeutic approaches. In this study, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of nucleic acids to the liver is demonstrated using real-time in vivo imaging. As a proof of concept, synthetic siRNAs against hepatitis B virus (HBV) were formulated into complexes of apo A-I and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (DTC-Apo) and injected intravenously (i.v.) into a mouse model carrying replicating HBV. We show that administration of these nanoparticles can significantly reduce viral protein expression by receptor-mediated endocytosis. The advantages of the apo A-I-mediated siRNA delivery method are its liver specificity, its effectiveness at low doses (< or = 2 mg/kg) in only a single treatment, and its persistent antiviral effect up to 8 days. The liver-targeted gene silencing was also shown by in vivo images, in which bioluminescent signals emitted from the liver were efficiently reduced after i.v. administration of luciferase-specific siRNA and DTC-Apo lipoplex. Thus, our unique approach to siRNA delivery creates a foundation for the development of a new class of promising therapeutics against hepatitis viruses or hepatocyte genes related to tumor growth.

Cyclosporin A and FK506 Inhibit IL-12p40 Production through the Calmodulin/Calmodulin-dependent Protein Kinase-activated Phosphoinositide 3-Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells

Cyclosporine-A (CyA) and FK506 are potent immunosuppressive agents because of their ability to suppress the production of Th1 cytokines including interleukin (IL)-12. However, the mechanisms underlying the inhibitory effects of CyA and FK506 on the production of IL-12p40, a critical component of IL-12, remain unknown. Both CyA and FK506 are potent inhibitors of calcineurin in the calcium signaling pathway. Interestingly, calcium and phosphoinositide 3-kinase (PI3K) signaling pathways have been shown to negatively regulate lipopolysaccharide (LPS)-induced murine IL-12p40 production. Contrary to these observations, we show that LPS-induced IL-12p40 production in human monocytic cells is positively regulated by the calcium pathway and in particular by calmodulin-(CaM) and CaM-dependent protein kinase-II (CaMK-II)-activated PI3K. Furthermore, LPS-induced IL-12p40 production was regulated by the p110alpha catalytic subunit of PI3K. Moreover, LPS induced IL-12p40 production through the CaM/CaMK-II-activated NFkappaB and AP-1 transcription factors. LPS-induced IL-12p40 production is known to be regulated by the c-Jun N-terminal kinase (JNK) pathway. Importantly, both CyA and FK506 down-regulated LPS-induced IL-12p40 transcription by inhibiting CaM/CaMK-II-activated PI3K and their downstream transcription factors NFkappaB and AP-1 independent of the JNK pathway.