KRN7000 Reduces Airway Inflammation via Natural Killer T Cells in Obese Asthmatic Mice.

Although natural killer T cells (NKT cells) are altered in obese asthmatic mice, their function remains completely unclear. To further explore the potential mechanism of NKT cells in airway inflammation of obesity-associated asthma, we examined the effects of α-galactosylceramide (KRN7000) on airway inflammation in obese asthmatic mice. Male C57BL/6J mice were divided into five groups: (1) control; (2) asthma; (3) A + KRN, asthma with KRN7000; (4) obese asthma; and (5) OA + KRN, obese asthma with KRN7000. Cytometric bead array (CBA) was used to detect interleukin-4 (IL-4), IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the serum. Flow cytometry was used to detect NKT cells and CD69+ NKT cells. Airway inflammation was observed in pathological sections, and calmodulin (CaM) expression was observed by immunohistochemistry in lung tissues. Airway inflammation in the obese asthma group was more severe than that of the asthma group. Airway inflammation of the OA + KRN group was reduced more than that of the A + KRN group. CD69+ NKT cells were only significantly reduced in the OA + KRN group. The levels of serum IFN-γ and TNF-α increased more in the OA + KRN group than in the A + KRN group. CaM is widely expressed in the cytoplasm of the lung tissues and was sharply decreased in the OA + KRN group. KRN7000 can significantly reduce airway inflammation in obesity-associated asthma by regulating NKT cell cytokine secretion and intracellular calcium. These results may contribute to the development of novel therapeutic approaches.

Synthesis and biological activities of amino acids functionalized α-GalCer analogues.

Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipid antigens presented by CD1d. In this paper, we wish to report a novel series of α-GalCer analogues which were synthesized by incorporation of l-amino acid methyl esters in the C-6' position of glycolipid. The evaluation of these synthetic analogues for their capacities to stimulate iNKT-cells into producing Th1 and Th2 cytokines both in vitro and in vivo indicated that they were potent CD1d ligands and could stimulate murine spleen cells into a higher release of the Th1 cytokine IFN-γ in vitro. In vivo, Gly-α-GalCer (1) and Lys-α-GalCer (3) showed more Th1-biased responses than α-GalCer, especially analogue 3 showed the highest selectivity for IFN-γ production (IFN-γ/IL-4 = 5.32) compared with α-GalCer (IFN-γ/IL-4 = 2.5) in vivo. These novel α-GalCer analogues might be used as efficient X-ray crystallographic probes to reveal the relationship between glycolipids and CD1d proteins in α-GalCer/CD1d complexes and pave the way for developing new potent immunostimulating agents.

Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain.

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid-protein interactions in KRN7000/CD1d complexes.

AAV-IL-22 modifies liver chemokine activity and ameliorates portal inflammation in murine autoimmune cholangitis.

There remain significant obstacles in developing biologics to treat primary biliary cholangitis (PBC). Although a number of agents have been studied both in murine models and human patients, the results have been relatively disappointing. IL-22 is a member of the IL-10 family and has multiple theoretical reasons for predicting successful usage in PBC. We have taken advantage of an IL-22 expressing adeno-associated virus (AAV-IL-22) to address the potential role of IL-22 in not only protecting mice from autoimmune cholangitis, but also in treating animals with established portal inflammation. Using our established mouse model of 2-OA-OVA immunization, including α-galactosylceramide (α-GalCer) stimulation, we treated mice both before and after the onset of clinical disease with AAV-IL-22. Firstly, AAV-IL-22 treatment given prior to 2-OA-OVA and α-GalCer exposure, i.e. before the onset of disease, significantly reduces the portal inflammatory response, production of Th1 cytokines and appearance of liver fibrosis. It also reduced the liver lymphotropic chemokines CCL5, CCL19, CXCL9, and CXCL10. Secondly, and more importantly, therapeutic use of AAV-IL-22, administered after the onset of disease, achieved a greater hurdle and significantly improved portal pathology. Further the improvements in inflammation were negatively correlated with levels of CCL5 and CXCL10 and positively correlated with levels of IL-22. In conclusion, we submit that the clinical use of IL-22 has a potential role in modulating the inflammatory portal process in patients with PBC.

Intra-peritoneal hyperthermia combining α-galactosylceramide in the treatment of ovarian cancer.

The purpose of this study was to investigate the anti-tumor effect and potential mechanisms of i.p. hyperthermia in combination with α-galactosylceramide (α-GalCer) for the treatment of ovarian cancer. In this study, immuno-competent tumor models were established using murine ovarian cancer cell lines and treated with i.p. hyperthermia combining α-GalCer. Th1/Th2 cytokine expression profiles in the serum, NK cell cytotoxicity and phagocytic activities of dendritic cells (DCs) were assayed. We also analyzed the number of CD8(+)/IFN-γ(+) tumor specific cytotoxic T cells, as well as the tumor growth based on depletion of lymphocyte sub-population. Therapeutic effect on those ovarian tumors was monitored by a non-invasive luminescent imaging system. Intra-peritoneal hyperthermia induced significant pro-inflammatory cytokines expression, and sustained the response of NK and DCs induced by α-GalCer treatment. The combination treatment enhanced the cytotoxic T lymphocyte (CTL) immune response in two mouse ovarian cancer models. This novel treatment modality by combination of hyperthermia and glycolipid provides a pronounced anti-tumor immune response and better survival. In conclusion, intra-peritoneal hyperthermia enhanced the pro-inflammatory cytokine secretion and phagocytic activity of DCs stimulated by α-GalCer. The subsequent CTL immune response induced by α-GalCer was further strengthened by combining with i.p. hyperthermia. Both innate and adaptive immunities were involved and resulted in a superior therapeutic effect in treating the ovarian cancer.

Synthesis of 6"-triazole-substituted α-GalCer analogues as potent iNKT cell stimulating ligands.

We report the synthesis of a small series of 6"-triazol-1-yl-substituted α-GalCer analogues by late-stage conversion of the 6"-OH to an azide group, copper-catalyzed azide-alkyne cycloaddition and final deprotection. When evaluated for their capacity to induce IL-2 secretion in vitro, all compounds proved equally potent or superior to α-GalCer. The S.A.R suggests that the improved antigenic activity is mainly triggered by the triazole functionalization in se. While the introduction of selected substitutuents at C-4 of this heterocyclic ring is tolerated, this generally fails to further improve antigenicity.

A rapid fluorescence-based assay for classification of iNKT cell activating glycolipids.

Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.

Activation of natural killer T cells by α-carba-GalCer (RCAI-56), a novel synthetic glycolipid ligand, suppresses murine collagen-induced arthritis.

Alpha-carba-GalCer (RCAI-56), a novel synthetic analogue of α-galactosylceramide (α-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-γ. IFN-γ exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of α-carba-GalCer on collagen-induced arthritis (CIA) in mice. First, we confirmed that α-carba-GalCer selectively induced IFN-γ in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and α-carba-GalCer. The incidence and clinical score of CIA were significantly lower in α-carba-GalCer-treated mice. Anti-IFN-γ antibodies abolished the beneficial effects of α-carba-GalCer, suggesting that α-carba-GalCer ameliorated CIA in an IFN-γ-dependent manner. Treatment with α-carba-GalCer reduced anti-CII antibody production [immunoglobulin (Ig)G and IgG2a] and CII-reactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-γ-producing T cells to IL-17-producing T cells (Th1/Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with α-carba-GalCer. In addition, we observed higher IFN-γ production by NK T cells in α-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that α-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that α-carba-GalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response.

Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells.

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid α-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-γ) and interleukin-4 (IL-4). However, αGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the αGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-γ secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation.

Stimulation of iNKT cells by α-galactosyl ceramide (α-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells' response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in α-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of α-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation.

Alpha-GalCer ameliorates listeriosis by accelerating infiltration of Gr-1+ cells into the liver.

Alpha-galactosylceramide (alpha-GalCer) activates invariant (i)NKT cells, which in turn stimulate immunocompetent cells. Although activation of iNKT cells appears critical for regulation of immune responses, it remains elusive whether protection against intracellular bacteria can be induced by alpha-GalCer. Here, we show that alpha-GalCer treatment ameliorates murine listeriosis, and inhibits inflammation following Listeria monocytogenes infection. Liver infiltration of Gr-1+ cells and gamma/delta T cells was accelerated by alpha-GalCer treatment. Gr-1+ cell and gamma/delta T-cell depletion exacerbated listeriosis in alpha-GalCer-treated mice, and this effect was more pronounced after depletion of Gr-1+ cells than that of gamma/delta T cells. Although GM-CSF and IL-17 were secreted by NKT cells after alpha-GalCer treatment, liver infiltration of Gr-1+ cells was not prevented by neutralizing mAb. In parallel to the numerical increase of CD11b+Gr-1+ cells in the liver following alpha-GalCer treatment, CD11b-Gr-1+ cells were numerically reduced in the bone marrow. In addition, respiratory burst in Gr-1+ cells was enhanced by alpha-GalCer treatment. Our results indicate that alpha-GalCer-induced antibacterial immunity is caused, in part, by accelerated infiltration of Gr-1+ cells and to a lesser degree of gamma/delta T cells into the liver. We also suggest that the infiltration of Gr-1+ cells is caused by an accelerated supply from the bone marrow.

An alpha-galactosylceramide C20:2 N-acyl variant enhances anti-inflammatory and regulatory T cell-independent responses that prevent type 1 diabetes.

Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

alpha-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

Ovarian cancer is one of the leading causes of death from gynecological cancers in the United States. Conventional therapies are unlikely to control advanced stage ovarian cancers, thus requiring innovative alternative therapies. In the current study, we characterized the therapeutic effect of tumor cell-based vaccines combined with the adjuvant, alpha-galactosylceramide (alpha-GalCer) using two different mouse models. Our data suggests that treatment with alpha-GalCer led to an increase in the IFN-gamma serum levels in the presence or absence of irradiated mouse ovarian surface epithelial tumor cells (MOSEC). Furthermore, administration of irradiated MOSEC tumor cells with adjuvant alpha-GalCer generated significant protective and therapeutic antitumor effects against MOSEC tumors in vaccinated C57BL/6 mice. In addition, immune cells expressing CD4, CD8 or NK1.1 markers were found to be important for the protective antitumor effects generated by irradiated tumor cell-based vaccines combined with adjuvant alpha-GalCer. We also found that treatment of a spontaneous ovarian cancer murine model, the Müllerian inhibiting substance type II receptor T antigen (TgMISIIR-TAg) transgenic mice with ovarian tumor cell-based vaccines combined with adjuvant alpha-GalCer led to prolonged survival as well as increased numbers of tumor-specific CD8+ T cells. Therefore, irradiated tumor cell-based vaccines in combination with alpha-GalCer are capable of breaking immune tolerance and generating significant antitumor effects in two different mouse tumor models. Our study serves as a foundation for future clinical translation.

Polarization of natural killer T cells towards an NKT2 subpopulation occurs after stimulation with alpha-galactosylceramide and rhG-CSF in aplastic anemia.

Natural killer T (NKT) cells play an important role in the regulation of immune responses in a broad range of diseases, including autoimmune disorders, infectious diseases and cancer. So far, few studies have evaluated the roles of NKT cells in the pathogenesis of aplastic anemia (AA), an autoimmune disease. In this study, we investigated the quantitative and qualitative changes in NKT cells in bone marrow (BM) mononuclear cells of AA patients in response to in vitro stimulation with alpha-galactosylceramide. Compared to healthy controls, BM from AA patients had reduced fraction of NKT cells, which possessed a decreased potential to expand in vitro in response to alpha-galactosylceramide stimulation, producing more IFNgamma+ NKT1 cells. In the presence of rhG-CSF, the expansion capacity of NKT cells stimulated by alpha-galactosylceramide was significantly reduced in both AA and control groups, with the majority of the activated NKT cells expressing intracellular IL-4, and the fractions of IFNgamma+ NKT cells were significantly reduced. In summary, our results indicate that polarization of NKT cells towards the NKT2 subpopulation occurs after co-stimulation with alpha-galactosylceramide and rhG-CSF in AA.

Modulation of murine experimental allergic conjunctivitis by treatment with alpha-galactosylceramide.

When mice are treated with alpha-galactosylceramide (alpha-GalCer), NKT cells are activated and suppress the development of experimental airway inflammation. This suppressive effect is believed to be mediated by the upregulation of IFN-gamma. Here, we investigated whether alpha-GalCer treatment can also modulate the development of experimental allergic conjunctivitis (EC). EC was induced in wild-type and IFN-gamma-deficient Balb/c mice by active immunization with ragweed (RW) followed by challenge with RW in eye drops. The mice were intraperitoneally injected with alpha-GalCer or vehicle at the time of immunization or before RW challenge. Twenty-four hours after RW challenge, conjunctivas, spleens and sera were harvested for histological analysis, flow cytometric, proliferation and cytokine assays, and measurement of immunoglobulin levels, respectively. Treatment with alpha-GalCer at the time of the EC-priming immunization significantly increased Th2 responses and markedly upregulated the severity of the EC. However, treatment with alpha-GalCer just before the Ag challenge that triggers EC in primed animals significantly suppressed the disease. This was associated with an increased frequency of CD4(+)CD25(+) cells, which express Foxp3, in the spleen. alpha-GalCer treatment just prior to Ag challenge also suppressed the development of EC in IFN-gamma-deficient mice, and we found apoptosis and anergy are unlikely to play a major role in the mechanism by which pre-challenge alpha-GalCer treatment suppresses EC. These data suggest that NKT cells can play a downregulatory role in the development of EC and that alpha-GalCer may be useful for treating allergic conjunctivitis.

Dendritic catanionic assemblies: in vitro anti-HIV activity of phosphorus-containing dendrimers bearing galbeta1cer analogues.

Two series of water-soluble dendritic catanionic assemblies, acting as multisite analogues of galactosylceramide (Galbeta1cer), have been prepared with the goal of blocking HIV infection prior to the entry of the virus into human cells. Trifunctional and hexafunctional cinnamic acid-terminated dendrimers have been synthesized from phosphorus-containing dendrimers bearing aldehyde end groups. A classical acid-base reaction performed in water between acid-terminated dendrimers and stoichiometric amounts of N-hexadecylamino-1-deoxylactitol provided the expected catanionic assemblies. Antiviral assays on these supramolecular entities confirmed the crucial roles both of multivalency effects and of lipophilicity on the biological activity of Galbeta1cer analogues. Moreover, correlation between in vitro tests and molecular modeling highlights the specific influence of the assembly shape on the anti-HIV efficiency, with the tri- and hexafunctional cored dendrimers, both decorated with 12 sugar moieties, exhibiting IC50 values of 1.1 and 0.12 microM, respectively.

NKT cells promote antibody-induced joint inflammation by suppressing transforming growth factor beta1 production.

Although NKT cells has been known to exert protective roles in the development of autoimmune diseases, the functional roles of NKT cells in the downstream events of antibody-induced joint inflammation remain unknown. Thus, we explored the functional roles of NKT cells in antibody-induced arthritis using the K/BxN serum transfer model. NKT cell-deficient mice were resistant to the development of arthritis, and wild-type mice administrated with alpha-galactosyl ceramide, a potent NKT cell activator, aggravated arthritis. In CD1d-/- mice, transforming growth factor (TGF)-beta1 was found to be elevated in joint tissues, and the blockade of TGF-beta1 using neutralizing monoclonal antibodies restored arthritis. The administration of recombinant TGF-beta1 into C57BL/6 mice reduced joint inflammation. Moreover, the adoptive transfer of NKT cells into CD1d-/- mice restored arthritis and reduced TGF-beta1 production. In vitro assay demonstrated that interleukin (IL)-4 and interferon (IFN)-gamma were involved in suppressing TGF-beta1 production in joint cells. The adoptive transfer of NKT cells from IL-4-/- or IFN-gamma-/- mice did not reverse arthritis and TGF-beta1 production in CD1d-/- mice. In conclusion, NKT cells producing IL-4 and IFN-gamma play a role in immune complex-induced joint inflammation by regulating TGF-beta1.

Survival benefit of KRN7000 immune therapy in combination with TNP470 in hamster liver metastasis model of pancreatic cancer.

Pancreatic cancer is a solid malignancy with the poor prognosis largely due to frequent and lethal liver metastases. The combination of immunotherapy and anti-angiogenesis therapy might be a hopeful strategy for the treatment of distant metastases. The benefits of the combination therapy by an immune stimulator alpha-galactosylceramide (KRN7000) and an angiogenesis inhibitor AGM-1470 (TNP470) were evaluated on the hamster highly aggressive liver metastasis model using the syngeneic pancreatic cancer cell line HPD-NR. KRN7000 immediately activated hepatic mono-nuclear cells to produce IFN-gamma in vitro. Intraportal injection of KRN7000 exhibited a dense accumulation of CD4-CD8- natural killer T cells, around the liver metastases in vivo. KRN7000 treatment significantly inhibited the growth of liver metastases, and importantly, significant survival prolongation was confirmed when TNP470 treatment was added to it. Furthermore, cytotoxic T lymphocytes were induced at the sites of a few residual metastases in the liver of a long-term survivor. Thus, the combination of KRN7000 and TNP470 showed a high effectiveness for the treatment of liver metastases of pancreatic cancer.

Experimental autoimmune encephalomyelitis. Augmentation of demyelination by different myelin lipids.

Alterations in the effect of a known encephalitogenic dose of myelin basic protein (MBP) when inoculated in combination with various myelin lipids have been examined in guinea pigs. A previous study demonstrated that, when MBP was given with galactocerebroside, it produced an acute autoimmune encephalomyelitis similar to that induced by whole white matter in which both inflammation and demyelination were features of the central nervous system lesions. MBP alone, on the other hand, resulted in inflammation only, without demyelination. The present study examined combinations of MBP with the myelin lipids galactocerebroside, sulfatide, ethanolamine phosphoglycerides, and serine phosphoglycerides. The lipids were given with or without MBP, in the same ratio as in intact central nervous system myelin, and were emulsified with complete Freund's adjuvant. An additional group received galactocerebroside and bovine serum albumin in complete Freund's adjuvant. These groups were compared with animals receiving either bovine white matter or MBP in complete Freund's adjuvant. Clinical autoimmune encephalomyelitis was observed in animals receiving bovine white matter, MBP, and all lipid-MBP emulsions; the bovine white matter, galactocerebroside/MBP, sulfatide/MBP, and ethanolamine phosphoglycerides/MBP groups demonstrated central nervous system lesions with a similar picture consisting of inflammation with demyelination, whereas inflammation without demyelination was seen in the MBP and serine phosphoglycerides/MBP groups. Thus, the addition of myelin lipids to MBP leads to the augmentation of demyelination in autoimmune encephalomyelitis lesions in the guinea pig. This might suggest that the immune response against MBP is enhanced by other myelin components. The relevance of these findings to human demyelinating disorders is discussed.