DIRECT AND INDIRECT MODULATION OF COMPOSITION AND STRUCTURE OF CELL MEMBRANE BY A THERAPEUTIC COMPOUND AND USES THEREOF IN METHODS FOR MONITORING RESPONSIVENESS AND SCREENING METHODS

Field of the Invention

The present invention relates to modulation of structure and composition of cell membranes by a therapeutic compound. More particularly, the invention provides a diagnostic method for predicting response of a subject to treatment with specific therapeutic compound by measuring alterations in the structure and composition of cell membranes of said subject, caused by said compound.

Background of the Invention

The liver provides a unique environment for lymphocytes favoring tolerogenic immune responses. Antigens presented on liver parenchymal cells and resident antigen presenting cells lead preferentially to tolerance rather than immunity. During systemic immune response activated CD8+ T lymphocytes selectively accumulate and undergo apoptosis in the liver. Activated T cell trapping is believed to play a part in liver mediated tolerance induction. NKT cells, a subset of regulatory lymphocytes, are considered to be a part of the innate immune system. Through their T cell receptor these cells recognize glycolipids, anchored by a ceramide tail to CDId, and MHC class I-like molecule. The liver is unique in that it harbors the highest percentage of NK and NKT cells compared to other organs. Classical NKT cells comprise 12% of hepatic lymphocytes in humans as opposed to only 1% in the peripheral blood. The percentage is even higher in the murine liver, where as many as 40% of lymphocytes may be classical NKT cells. Natural killer T (NKT) cells represent a

distinct lineage of T cells that coexpress a conserved αβ T cell receptor (TCR) and natural killer (NK) receptors [Dale I. Godfrey, et al., Nature Reviews Immunology 4: 231 (2004)]. These cells are a heterogeneous population of lymphocytes with different specificities and functions. Prototypical mouse NKT cells are often referred to as invariant NKT (iNKT) cells.

One hallmark of iNKT cells is their capacity to produce, within hours of activation, large amounts of cytokines, including the characteristic THl cytokine interferon-γ (IFN-γ) and the characteristic TH2 cytokine interleukin-4 (IL-4). iNKT cells constitutively express mRNA encoding IFN-γ and IL-4, which allows these cells to rapidly dispatch their effector functions.

Consistent with their capacity to produce copious amounts of cytokines, iNKT cells have been implicated in a myriad of immune responses, including responses to pathogens, tumours, tissue grafts, allergens and autoantigens. In most cases, however, the mechanisms responsible for iNKT-cell activation and the downstream events that result in immune modulation remain elusive. In some systems, the capacity of iNKT cells to produce THl cytokines seems to be important, whereas in others TH2- cytokine production seems to be crucial.

Autoimmune responses are normally kept in check by immune -tolerance mechanisms, which include regulatory T cells. In recent years, research has focused on the role of this subset of NKT cells (iNKT), which are a population of glycolipid-reactive regulatory T cells in controlling autoimmune responses.

A potential link between iNKT cells and autoimmunity was revealed by the finding that, compared with non- autoimmune mouse strains, various

mouse strains that are genetically susceptible to autoimmunity have reduced numbers of iNKT cells, which have defective functions. Defective iNKT-cell function in these mouse strains seems to be genetically determined. In addition, in some experimental models of autoimmunity, depletion of iNKT cells resulted in disease exacerbation. Conversely, restoration of iNKT-cell numbers by adoptive transfer of iNKT cells or by transgenic overexpression of the iNKT-cell TCE. ameliorated disease. Great progress has been recently made with respect to understanding the basic immunobiology of NKT and iNKT cells. In addition, because of their presence in all individuals and their potent cytokine producing ability, these cells might be a promising target for immune-based therapies.

CDl system: Antigen presenting molecules

Recent studies have identified the CDl family of proteins as novel antigen presenting molecules, encoded by genes located outside of the major histocompatibility complex. CDl proteins are conserved in all mammalian species so far examined and are prominently expressed on cells involved in antigen presentation, which suggests a role in activation of cell-mediated immunity. This has now been confirmed by functional studies demonstrating the ability of CDl proteins to restrict the antigen- specific responses of NKT cells in humans and mice.

Identification of naturally occurring antigens presented by GDI has revealed the surprising finding that these are predominantly a variety of foreign lipids and glycoh ^' pids, including several found prominently in the cell walls and membranes of pathogenic mycobacteria. Structural, biochemical, and biophysical studies support the view that GDI proteins bind the hydrophobic alkyl portions of these antigens directly and position the polar or hydrophilic head groups of bound lipids and glycolipids for highly specific interactions with T cell antigen receptors. Presentation of

antigens by CDl proteins requires uptake and intracellular processing by antigen presenting cells, and evidence exists for cellular pathways leading to the presentation of both exogenous and endogenous lipid antigens. NKT cells recognizing antigens presented by CDl have a range of functional activities that suggest they are likely to mediate an important component of antimicrobial immunity and may also contribute to autoimmunity and host responses against neoplastic cells.

The CDl proteins are a family (subdivided to CDla-d), of molecules that have structural homology to major histocompatibility complex (MHC) class I molecules, but are unusual in their ability to present nonpeptide lipid-based antigens to T cells. In mice, there is a single class of CDl molecule (mCDld) which is homologous to the human isoform CDId [Lang, G.A. et al. Immunology 112: 386-396 (2004)].

The CDl isoforms have differing affinities for distinct classes of both foreign and self antigens. CDId and the murine homologue mCDld present galactosylceramides and hydrophobic peptides. Different isoforms of CDl are localized to distinct subcellular locations, and allow sampling of different antigens in different cellular compartments. Recent studies have shown that different glycolipids preferentially target to different organelles. CDl therefore allows APCs to present a variety of glycolipid antigens that enter the cell by different pathways and that are targeted to different subcellular locations [Lang (2004) ibid.]. Murine CDId localizes to the plasma membrane and specialized organelles on the endosomal pathway known as major histocompatibility complex class II-containing compartments (MIIC) [Lang (2004) ibid.].

Natural self and microbial CD Id-presented lipid activators of iNKT cells Because of their role in regulating a range of disease states, the nature of ligands recognized by NKT and iNKT cells, has been the subject of intense research and speculation. Although the TCB, of NKT cells is characteristically autoreactive to CDId, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. Several candidate natural lipid ligands have been investigated. Most recently, a strong candidate for a physiologically relevant natural ligand of iNKT cells has been identified [Zhou D, et al., Science 306(5702): 1786-9 (2004)]. This is a lysosomal glycolipid, isoglobotrihexosylceramide (iGb3), which in either natural or synthetic forms has the ability to activate most human or mouse iNKT cells in vitro.

Impaired generation of lysosomal iGb3 in mice lacking β-hexosaminidase b resulted in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. However, there still is a lack of direct biochemical evidence for the presence of iGb3 in mouse and human. But the combined data suggest that iGb3 or a close structural analog is the principal self antigen of NKT cells. Nevertheless, these results do not rule out the existence of additional endogenous ligands. It is also possible that alternative endogenous ligands are expressed in some disease conditions or in particular cell types.

There has also been some success in identification of foreign microbial glycolipid ligands of CDId that can activate iNKT cells.

Most notably, certain glycosylceramides derived from lipopolysaccharide- negative Sphingomonas bacteria also activate iNKT cells. This suggests a role for iNKT cells in the innate response against pathogens which do not

activate classical pattern-recognition receptors such as Toll-like receptor 4.

Immunomodulatory activities of a-GalCer

There are many glycolipids that affect immune responses, but nothing is quite like the marine sponge-derived, α-galactosyl ceramide (α-GalCer). This compound binds with a very high affinity to CDId, and the α-GalCer- CDId complex is recognized by mouse and human T-cell receptors [Luc Van Kaer, Nature Reviews Immunology 5: 31 (2005)]. α-GalCer was originally discovered by the Pharmaceutical Division of the Kirin Brewery Company during a screen for reagents derived from the marine sponge Agelas mauritianus that prevent tumor metastases in mice. In most studies, a synthetic analogue, KRN7000, of this natural product has been used and is usually still referred to as α-GalCer.

Interestingly, reactivity with α-GalCer is not restricted to iNKT cells from mice but also includes iNKT cells from humans, macaques and rats. α-GalCer is a glycosphingolipid, a chemical category that encompasses abundant glycolipids in the body, including the gangliosides. It is distinguished, however, by the stereochemistry of the bond that joins the asymmetric 1' carbon of the sugar to the lipid. In nearly all natural cases, this bond is in the β-anomeric form, but the sponge-derived glycosphingolipid has an α-linkage.

It is assumed widely that α-GalCer is only a mimic of the natural ligand that the iNKT cells recognize. Given the self-reactivity of these cells, and the limited diversity of the TCRs that they express, it is believed generally that a single autologous glycolipid ligand, or a set of closely related ligands, is present in human cells for possible presentation by CDId and activation of iNKT cells. Several crystal structures of human and murine

CDl molecules with and without α-GalCer have been determined, explaining why α-GalCer binds to CDId with one of the highest affinities of the known ligands.

β-glycolipids are naturally occurring intermediates of complex glycosphingolipids that are found within cell membranes. They were previously suggested to exert a beneficial effect in diverse immune mediated and neoplastic murine models. Lipid rafts are 50 nm structures that contains specialized glycosphingolipids and cholesterol and are enriched in glycosylphosphatidylinositol and play an important role in intracellular trafficking [Helms, J.B. and Zurzolo, C. Traffic. 5(4):247-54 (2004); Manes, S. and Viola, A. MoI. Membr. Biol. 23(l):59-69 (2006)].

Rafls and immune responses

Plasma membranes are composed of different domains maintained by an active cytoskeletal network [Brown, D. A. and London, E. Annu. Rev. Cell Dev. Biol.l4:lll-136 (1998); Edidin, M. Curr. Opin Struct. Biol. 7:528-532 (1997); Hooper, N.M. Curr. Biol. 8:R114-116 (1998); Simons, K, and Toomre, D. Nat. Rev. MoI. Cell Biol. 1:31-39 (2000)]. Glycosphingolipid and cholesterol-rich membrane microdomains, known as lipid rafts, are regulatory components of the immune response. These domains play important roles in intracellular trafficking [Helms, J.B. and Zurzolo, C. Traffic 5:247-254 (2004); Manes, S. and Viola, A. MoI. Membr. Biol. 23:59- 69 (2006)]. Lipid rafts are 50-nm structures that contain specialized glycosphingolipids and cholesterol and are enriched in glycosylphosphatidylinositol (GPI)-linked and palmitoylated membrane proteins. They also contain intracellular signaling molecules, such as Src- family kinases, Lck and Fyn [Rodgers, W. et al. MoI. Cell. Biol.14:5384- 5391 (1994); Shenoy-Scaria, A.M. et al. J. Cell. Biol 126:353-363 (1994)] and transmembrane proteins, such as the adapter protein LAT [Zhang, W.

et al. Immunity 9:239-246 (1998)]. GPI-anchored proteins or proteins that carry hydrophobic modifications divide into rafts owing to preferential packing of their saturated membrane anchors [Simons (2000) ibid.]. The fundamental principle by which lipid rafts exert their functions is via alteration of specific membrane proteins and lipids in membrane microdomains [Brown, D.A. and London, E. J. Biol. Chem. 275:17221- 17224 (2000)], which may be involved in compartmentalizing signal transduction events within different regions of the plasma membrane. Lipid rafts can be purified as low density, detergent-insoluble glycolipid domains (DIGs). It has been suggested that these DIGs are especially efficient at initiating signal transduction pathways [Pike, L.J. Biochem. J. 378:281-292 (2004)]. Clustering small individual rafts together into a larger platform increases the efficiency of the interaction of raft-associated proteins [Janes, P.W. et al. J. Cell. Biol. 147:447-461 (1999)], weak raft affinities of individual proteins are strengthened by oligomerization.

Raft components can be cross-linked with antibodies or toxins in living cells, then clustered together to bind raft and non-raft components separately into micron-sized, quilt-like patches [Harder, T. J. Cell. Biol. 141:929-942 (1998)]. The clustering of separate rafts exposes proteins to a new membrane environment, enriched in specific enzymes. Even a small change of partitioning can initiate signaling cascades [Tuosto, L. et al. Eur. J. Immunol. 31:345-349 (2001)]. This signaling depends on the raft- associated kinases and can be suppressed by reducing membrane cholesterol levels or by changing the membrane lipid composition [Stulnig, T.M. et al. J. Biol Chem. 272:19242-19247 (1997)], indicating that the integrity of raft domains is required. Similarly, the addition of exogenous gangliosides to cells can lead to their incorporation into rafts and, as a result, may cause proteins to dissociate from rafts [Simons, M. et al. MoI. Biol. Cell. 10:3187-3196 (1999)]. Raft microdomains are also enriched

with the ganglioside GMl [Thomas, S. et al. MoI. Immunol. 41:399-409 (2004)] which is recognized by cholera toxin and is used as a raft marker.

In normal T cells, ligation of the TCR induces rapid lipid raft clustering that leads to concentration of signaling proteins at the area of contact between APCs and T cells, known as the immunological synapse [Grakoui, A. et al. Science 285:221-227 (1999)]. Clustering of rafts is an essential feature of this process [Hiltbold, E.M. et al. J. Immunol. 170:1329-1338 (2003)]. Efficient formation of the immunological synapse is critical to amplification of signals downstream of the TCR and to subsequent T cell activation. Conversely, raft integrity is a prerequisite for efficient TCR signal transduction [Xavier, R. et al. Immunity 8:723-732 (1998)], and has been shown to play a major role in the pathogenesis of several diseases, such as infections, allergies, and neoplasms. Several factors can influence the strength of signals controlled by lipid rafts, such as lipid raft pool size, membrane distribution patterns, protein content, and the kinetics of cytoskeletal rearrangements following T cell stimulation [Simons (2000) ibid.].

The distribution of lipid rafts over the cell surface depends on cell type. In lymphocytes and fibroblasts, rafts are distributed over the cell surface without obvious polarity. Typically, sphingolipids make up about 45% of the fibroblast cell surface [Renkonen, O. et al. Virology 46:318-326 (1971)] and roughly 30% of the lymphocyte surface [Levis, G.M. et al. Biochem. J. 156:103-110 (1976)], but these values are upper limits and may also be cell-type dependent.

Rafts also function to concentrate MHC class II molecules on the APC side of the synapse [Hiltbold (2003) ibid.; Anderson, H. A. et al. Nat. Immunol. 1:156-162 (2000)]. Interactions between signaling proteins present within

lipid rafts impose another level of regulation upon TCR signaling. Lipid rafts of naϊve, effector, and memory CD8 T cells display distinct protein profiles with distinct functional outcomes, and ThI and Th2 cells differentially recruit TCE, complexes to lipid rafts and display different functions [Balamuth, F. et al. Immunity 729-738 (2001)]. These membrane-organizing lipids are involved in the formation of distinct MHC-I and MHC-II microdomains at the cell surface and are important in MHC compartmentalization in the processes of antigen presentation [Yaqoob, P. Curr. Opin. Clin. Nutr. Metab. Care 6:133-150 (2003)]. Lipid rafts are also engaged with the MHC class ϊ-like molecule, CDId [Park, Y.K. et al. Biochem. Biophys.Res. Commun 327:1143-1154 (2005)]. CDl molecules are conserved in all mammalian species and are prominently expressed on cells involved in antigen presentation, which suggests a role in the activation of cell-mediated immunity [Porcelli, S.A. and Modlin, R.L. Annu. Rev. Immunol. 17:297-329 (1999)]. CDl receptors have a structural homology to MHC class I molecules, but are unusual in their ability to present glycolipid antigens to natural killer T (NKT) cells [Godfrey, D.I. et al. Nat. Immunol. 6:754-756 (2005)].

CD Id-restricted NKT cells are innate regulatory lymphocytes that express a conserved TCR and play an important role in diverse neoplastic, autoimmune, and infectious processes [Godfrey, D.I. et al. Nat. Rev. Immunol. 4:231-237 (2004); Kronenberg, M. and Gapin, L. Nat. Rev. Immunol. 2:557-568 (2002); Godfrey, D.I. et al. Immunol. Today 21:573- 583 (2000)]. Functional studies have demonstrated the ability of CDl proteins to restrict the antigen-specific responses of NKT cells in both humans and mice [Yu, K.O. et al. Proc. Natl. Acad. Sci. U. S. A. 102:3383- 3388 (2005)]. It was found that murine CDId (mCDld) is raft-localized and disruption of lipid raft structures blocked efficient signaling through mCDld. Partitioning of mCDld into membrane rafts increases the

capacity of APCs to present limiting quantities of glycolipid antigens, perhaps by stabilizing mCD Id/antigen structures on the plasma membrane and optimizing TCR engagement on NKT cells [Lang, G.A. et al. Immunology 112:386-396 (2004)].

The target lipid antigens for NKT regulatory lymphocytes have remained elusive. Lysosomal glycosphingolipid, isoglobotrihexosylcer amide (iGb3), and bacterial ligands were suggested as natural ligands for NKT cells [Zhou, D. et al. Science 306:1786-1789 (2004)]. Synthetic glycolipids, including αGalCer and a number of its derivatives, activate NKT cells [Van Kaer, L. Nat. Rev. Immunol. 5:31-42 (2005)]. Administration of α- GalCer had a protective role in experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis in mice [Furlan, R. et al. Eur. J. Immunol. 33:1830-1838 (2003); Chiba, A. et al. Arthritis Rheum. 50:305-313 (2004)]. β-glucosylceramide (GC), a naturally occurring glycolipid, is a metabolic intermediate in the pathways of complex glycosphingolipids [Hannun, Y.A. et al. Biochemistry 40:4893- 4903 (2001)]. GC can alter both NKT lymphocyte number and function. In vivo, GC administration attenuated immune-mediated liver damage secondary to intravenous administration of concanavalin A (ConA) to mice, and was associated with a decrease in the intrahepatic NKT lymphocyte number [Margalit, M. et al. Am. J. Physiol. Gastrointest. Liver Physiol. 289:G917-925 (2005)]. The mechanism by which β-glycolipids induce an immune modulatory effect remains elusive.

Recently, the present inventors have shown that β-glycolipids, and specifically, β-lactosyl-ceramide (LacC), β-glucosylceramide (GIuC), and β- galactosyl-ceramide (GaIC)] and ceramide may be used for immune- modulation. The inventors further showed a clear synergistic effect of a

particular combination of two β-glycolipids, β-lactosyl-ceramide with β- glucosylceramide (US Application No. 11/287.502).

The inventors have now shown for the first time that administration of glycolipids, especially β-glucosylceramide (GC) ₅ β-lactosylceramide (LC) and mixture thereof (IGL) influences the composition and structure of immune cell membrane and therefore may affect immunological responses.

Therefore, one object of the invention is to provide a method for the treatment of pathologic disorders, preferably, immune-related disorders, by modulating the composition and structure of cell membranes, preferably, of immune cell membranes, using glycolipids.

Another object of the invention is to provide the use of modulation of structure and composition of a cell membrane by a given candidate pharmaceutical compound, as a marker for monitoring and predicting the responsiveness to a treatment of a subject suffering of a pathologic disorder with the candidate therapeutic compound.

More specific object of the invention is to provide a diagnostic method for prediction of response of a subject suffering from an immune-related disorder to treatment with a given therapeutic compound, by monitoring the modulations in structure and composition of the membranes of different cells, preferably, immune cells, obtained from said subject in response to treatment with said therapeutic compound.

Another object of the invention relates to a method for the high- throughput screening of a substance which modulates the structure and composition of the membranes of different cells in response to treatment

with, a given therapeutic compound, preferably, natural glycolipids, and can thus be used for the treatment of pathologic disorders, preferably, immune-related disorders.

These and other objects of the invention will become apparent as the description proceeds.

Summary of the Invention

In a first aspect, the invention relates to a diagnostic method for predicting responsiveness of a subject suffering from a pathologic disorder to treatment with a therapeutic compound. This diagnostic method is based on identifying an alteration in the membrane composition and structure of cells of a subject, in response to treatment with the specific therapeutic compound. The method of the invention comprises the steps of: (a) obtaining cells from the tested subject; (b) exposing these cells to an effective amount of the certain therapeutic compound, in different concentrations or mixtures, under suitable conditions; and (c) monitoring the direct or indirect modulation in structure and composition of cell membrane obtained from the examined subject, as compared to a control. The alteration is measured by a suitable means. It should be noted that an alteration in the membrane composition and structure of said cells as compared to a control is indicative of the potential responsiveness of the diagnosed subject to treatment with the examined therapeutic compound.

According to one preferred embodiment, the therapeutic compound may be a natural or synthetic β-glycolipid.

In a second aspect, the invention relates to a method for high-throughput screening of a substance which modulates the Thl/Th2 cell balance toward

anti-inflammatory cytokine producing cells, in a subject suffering from an immune related disorder. The screening method of the invention, comprises the steps of: (a) providing test substances; (b) selecting from the test substances provided in step (a) a substance which alters, e.g., increase or decrease, the modulation of structure and composition of a cell membrane by a therapeutic compound, as compared to a control; and (c) evaluating the ability of the substance selected in step (b) to modulate the Thl/Th2 cell balance toward anti-inflammatory cytokine producing cells, by a suitable means.

According to a further aspect, the invention relates to a method for the direct or indirect modulation of composition and structure of a cell membrane of a subject suffering from an immune related disorder. This method comprises the step of increasing the intracellular, extra-cellular or serum level of a synthetic or naturally occurring β-glycolipid in said subject, wherein increasing the intracellular, extra-cellular or serum level of a naturally occurring β-glycolipid in said subject is performed by: (I) administering to said subject an effective amount of any one of: (a) a β- glycolipid; (b) a mixture of at least two β-glycolipids; (c) a substance which increases the intracellular, extracellular or serum level of a naturally occurring β-glycolipid; and (d) any combination of the above; or (II) exposing said cells of said subject to an effective amount of any one of: (a) a β-glycolipid; (b) a mixture of at least two β-glycolipids; (c) a substance which increases the intracellular, extracellular or serum level of a naturally occurring β-glycolipid; and (d) any combination of the above; or (III) any combination of (I) and (II).

The invention further provides for a method for the treatment of immune- related disorder in a mammalian subject in need thereof. The method of the invention comprises the step of direct or indirect modulation the

composition and structure of the membrane of immune cells of the subject by in vivo or ex vivo exposing cells of said subject immune-system to an effective amount of any one of: (a) a β-glycolipid; (b) a mixture of at least two β-glycolipids; (c) a substance which increases the intracellular, extracellular or serum level of a naturally occurring β-glycolipid; and (d) any combination of the above.

These and other aspects of the invention will become apparent by the hand of the following figures.

Brief Description of the Figures Figure 1

FACS analysis showing the general effect of GC on liver leukocyte fluorescence as compared to control. The leukocytes were obtained from NASH-model mice. Abbreviations: Liv. (liver), Leuk. (leukocytes), Lymp (lymphocytes), cont. (control), mea. flou. (mean flourocence), gr. (green).

Figure 2

FACS analysis showing the general effect of GC on splenocyte fluorescence as compared to control. The splenocytes were obtained from NASH-model mice. Abbreviations: Sp. (spleen), Lymp (lymphocytes), cont. (control), mea. flou. (mean flourocence), gr. (green), r (red).

Figure 3A-3B

Plasma membrane distribution of raft marker (GMl) in splenocytes obtained from OB/OB mice (NASH-model). Representative cells

(splenocytes) are shown in each panel.

Fig. 3A OB/OB mice treated with PBS served as control.

Fig. 3B OB/OB mice treated with GC.

Magnification used was xlOO oil objective. Abbreviations: cont. (control).

Figure 4A-4B

Plasma membrane distribution of raft marker (GMl) in splenocytes obtained from ConA mice. Representative cells (splenocytes) are shown in each panel.

Fig. 4A ConA mice treated with PBS served as control.

Fig. 4B ConA mice treated with β-lactosylceramide.

Magnification used was xlOO oil objective. Abbreviations: cont. (control).

Figure 5A-5G

Effects of α- and β-glycolipids on lipid rafts and cell membranes in naϊve and ConA-treated animals.

Fig 5A. Immunoblotting of lipid microdomain fractions. Identification of the GMl distribution in the detergent-insoluble fractions (4-2) and in the detergent-soluble (cytosolic) fractions (63—12) was analyzed by immune dot-blot.

Fig. 5B-5C. Densitometric analysis of the immune dot-blot experiments (n

= 3) showing an increase in GMl in both the raft and cytosolic fractions upon β-glycolipid treatment in naϊve (5C) and ConA-treated (5B) animals.

Densitometric quantification was performed using a high-resolution scanner and compared with untreated controls. Data are expressed in arbitrary densitometry units.

Fig. 5D-5E. Surface expression of GMl on NKT lymphocytes is increased after GC treatment. Expression was determined by flow cytometry analysis using the CT-X-FITC conjugate. Plasma membranes were analyzed by three-color FACS using CT-X-FITC, NKl.l-PE, CD3-Cy3, and conjugates. Cells were gated on NKl.1, CD4+, and CD8+ populations, and

GMl expression at a single cell level was based on fluorescence intensity.

Fig. 5D shows GMl analysis in Naϊve animals and Fig. 5E GMl analysis in ConA treated animals.

Fig. 5F- 5G. Densitometric analysis of the immune dot-blot experiments (n

= 3) demonstrating an increase in GMl on whole splenocytes. Freshly derived splenocytes from naϊve and ConA-treated animals were stained using CTX-HRP conjugates. Data are expressed in arbitrary densitometry units. Fig. 5F shows CTX-HRP analysis in Naϊve animals and Fig. 5G

CTX-HRP analysis in ConA treated animals.

Abbreviations: Fr. (fraction), Cyto. (cytosolic), Na. (Naϊve), g. (gated).

Figure 6

Visualization of GMl lipid rafts in splenocyte membranes derived from ConA-treated mice. Representative fluorescent images showing a patchy pattern of GMl distribution in splenocyte cell membranes derived from GC- and LC-treated mice. The right image of each panel shows enlarged green fluorescent patches close to the apical area of the cells, indicating the formation of GMl lipid rafts as identified with the CT-X-FITC conjugate. The left image of each panel is a middle-range cross section of the same microscopic field.

Figure 7

Partitioning of flotillin-2 in splenocyte membranes derived from naϊve and ConA-treated mice. Changes in flotation patterns were observed in GC- treated naϊve mice. Cells were treated with 1% Triton X-IOO lysis buffer for 30 min on ice and subjected to nycodenz density gradient ultracentrifugation. Twelve fractions were collected from the bottom of the gradient and analyzed by SDS-PAGE followed by Western blotting with anti-flotillin-2 antibody. Fraction 1 represents the top of the gradient. Abbreviations: Na. (naϊve).

Figure 8

Effects of α- and β-glycolipids on STATl protein expression. Expression of phosphorylated STAT protein was determined in all groups. Administration of α-GalCer and iGb3 significantly increased the expression of phosphorylated STATl, while treatment with GC and LC did not alter or decreased expression.

Figure 9

Effects of α- and β-glycolipids on serum cytokine levels.

Serum IFNγ levels were measured by ELISA. Levels were significantly reduced in GC-, LC-, and IGL-treated animals.

Abbreviations: Exp. Gr. (experimental group).

Figure 10A-10B

Effects of a- and β-glycolipids on splenic and intrahepatic NKT lymphocytes. FACS analysis of splenic and hepatic NKT lymphocytes on animals from all groups was performed. Administration of ConA was associated with a significant decrease in the number of NKT lymphocytes. Fig. 1OA. Among the ConA-treated groups, the administration of β- and α- glycolipids significantly altered the number of intrahepatic NKT cells, distinct from iGb3. Administration of glycolipids to naϊve mice led to a reduction in intrahepatic NKT cell number. No significant differences were noted between the different glycolipids. Fig. 1OB. In the spleen, a statistically insignificant increase in the number of NKT lymphocytes was observed in all glycolipid-treated groups not associated with ConA administration. Abbreviations: Exp. Gr. (experimental group).

Figure 11

Effects of α- and β-glycolipids on splenic and intrahepatic CD4 and CD8 lymphocytes. FACS analysis of splenic and hepatic CD4 and CD8

lymphocytes on animals from all groups was performed. Administration of ConA was associated with a significant increase in intrahepatic CD8 trapping, signified by an increase in the splenic/intrahepatic CD4/CD8 ratio. Treatment with GC, LC, and IGL in mice that received ConA decreased intrahepatic CD 8 trapping with a significant decrease of the spleen to liver CD4/CD8 lymphocyte ratio. Abbreviations: Exp. Gr. (experimental group).

Figure 12A-12B

Effects of α- and β-glycolipids on serum ALT and AST levels. Fig. 12A. Administration of all β-glycolipids significantly alleviated ConA- induced hepatitis via a decrease in serum AST and ALT levels. In contrast, treatment with α-GalCer led to an increase in transaminase levels. Fig. 12B. In naϊve mice, both α-GalCer and iGb3 induced an elevation of transaminases. Administration of GC, LC, and IGL to naϊve mice did not alter liver enzymes.

Figure 13

Effects of α- and β-glycolipids on liver histology. Histological liver damage was markedly attenuated in GC-, LC-, and IGL-treated groups. Total liver scores were decreased in mice in groups B, D, and H, compared with non- treated controls in group A. Abbreviations: Exp. Gr. (experimental group).

Figure 14A-14B

Protection against intrahepatic apoptosis by β-glycolipids in naϊve mice. The TUNEL assay was performed on liver sections from naϊve and ConA- treated mice. Five standardized microscopic fields (x20 objective) per slide were quantified by fluorescence microscopy. α-GalCer induced large zones of DNA degradation in both naϊve and ConA-treated mice. In contrast,

few TUNEL-positive cells were counted in GC-, LC-, and IGL-treated mice.

Fig. 14A. shows experimental groups A, D, E, F, H.

Fig. 14B. shows experimental groups I, J, L, M, N.

Figure 15A-15B

Plasma membrane distribution of raft marker (GMl) in splenocytes obtained from ConA mice. Representative cells (splenocytes) are shown in each panel.

Fig. 15A HCC mice treated with PBS served as control.

Fig. 15B HCC mice treated with mixture of GC and β-lactosylceramide

(IGL). Magnification used was xlOO oil objective. Abbreviations: cont.

(control).

Figure 16

Histogram of FACS analysis showing the GMl changes among different subsets of splenocytes obtained from HCC mice. Abbreviations: cont. (control).

Figure 17A-17B

Plasma membrane distribution of raft marker (GMl) in cells of TNBS-

Colitis model mice.

Fig. 17A TNBS-Colitis mice treated with PBS served as control.

Fig. 17B TNBS-Colitis mice treated with GC. Magnification used was xlOO oil objective. Abbreviations: cont. (control), col. (Colitis).

Figure 18

Histogram of FACS analysis showing the GMl changes among different subsets of lymphocytes in TNBS-colitis splenocytes as compared to a PBS treated control. Abbreviations: cont. (control), gat. (gated).

Figure 19

Effect of β-glycolpids on lipid rafts and cell membranes by dot blot analysis: The GMl content in the detergent-insoluble and in soluble (cytosolic) fractions was analyzed by dot-blot analysis. Significant changes were noted in raft fractions 1 and 2; and in cytosolic fractions 11-12. In TNBS treated mice the administration of GC, LC, and IGL significantly increased GMl content. Abbreviations: Fr. (fraction), No. (number).

Figure 20

Effect of β-glycolpids on lipid rafts and cell membranes by FACS analysis of GMl expression in lymphocytes subsets. FACS analysis was performed using CT-X-FITC for determination of GMl expression on cell surface. Freshly derived whole splenocytes were stained using CTX-HRP conjugate, as a measure of whole cell GMl content. Administration of TNBS led to a significant decrease of GMl expression in CD4, CD8 and NKT cells. Administration of GC and IGL was associated with a decrease in GMl expression on CD8 and NKT lymphocytes. Abbreviations: g. (gated).

Figure 21

Effect of β-glycolpids on lipid rafts and cell membranes as examined by fluorescent microscopy with cholera toxin. Clustering patterns of GMl using cholera toxin conjugated to FITC (CT-X-FITC) were tested by fluorescent microscopy, to analyze the patching distribution of GMl/cholera toxin. Representative images are shown. Administration of TNBS led to mild cholera toxin induced raft clustering. Mice treated with GC and IGL manifested enlarged GMl patches on cell surfaces.

Figure 22A-22C

Effect of β-glycolipids on splenic and intrahepatic NKT lymphocytes:

Fig. 22A. Effect on intrahepatic NKT lymphocytes: FACS analysis of intrahepatic and intrasplenic NIT lymphoyctes. GC and IGL admisnstration was associated with a significant increase in the number of intrahepatic NKT lymphoyctes (groups B, E and A, p<0.005). In cotrast, administration of LC, GLC and ceramide were associated with a decrease in intrahepatic NKT cell number.

Fig. 22B. Effect on intrasplenic NKT lymphocytes. No significant effect of β-glycolipid administration was noted on the peripheral NKT cell number as evaluated by FACS analysis of intrasplenic NKT lymphoyctes.

Fig. 22C. Effect on intrahepatic to intrasplenic NKT lymphocyte ratio. A singnificant increase in the intrahepatic to intrasplenic NKT lymphocyte ratio ratio for mice treated with GC and IGL (groups B, E and A, p<0.005).

Abbreviations: Exp. Gr. (experimental groups).

Figure 23

Effect of β-glycolipids on intrasplenic to intrahepatic CD4 to CD 8 lymphocyte ratio. FACS analysis for CD4+ and CD8+ T lymphoyctes was perfromed. For each group the CD4 to CD8 lymphocyte ratio was calculated. The ratio between each of the splenic to intrahepatic CD4/CD8 ratios was determined. Induction of experimental colitis was associated with a decrease in intrahepatic CD8 lymphocyte trapping (groups A vs G, p<0.005). Administration of GC and IGL led to a significant increase in this ratio (B, E, and A, p<0.005), suggesting an increased intrahepatic CD8+ T lymphocyte trapping. Abbreviations: Exp. Gr. (experimental groups).

Figure 24A-24B

Effect of β-glycolipids on serum cytokine levels.

Fig. 24A. Effect on IFNγ serum levels. Serum IFNγ levels decreased significantly for animals treated with. GC and IGL as compared with untreated controls (B ₅ E, versus A, p<0.005).

Fig. 24B. Effect on the IFNγ/ILlO cytokine ratio. To assess the effect of

GC and IGL on the Thl/Th2 immune paradigm, the IFNγ/ILlO ratio was calculated. Adminstration of GC and IGL led to a significant reduction in this ratio (B, E, versus A, p<0.005). Abbreviations: Exp. Gr. (experimental groups).

Figure 25A-25B

Effect of β-glycolipids on experimental colitis.

Fig. 25A. Effect on the microscopic score of colitis: Significant improvement in the microscopic colitis score following the administration of GC and IGL in groups B and E (p<0.005).

Fig. 25B. Effect on extent of disease: The extent of bowel affected by the diseae was significantly reduced in animals treated with GC and IGL (B,

E verus A, p<0.005). Abbreviations: Exp. Gr. (experimental groups).

Figure 26

Effect of β-glycolipids on bowel histology. Representative histological slides are shown from mice in treated showed sginifcicant ameliortion of the inflammation and mucose destruction compared with untreated controls (H&E staining, xlO mgnification).

Figure 27

Effect of GC on DN hybridoma fluorescence.

Abbreviations: cont. (control), mea. flou. (mean flourocence), gr. (green), hyb. (hybridoma).

Figure 28

Fluorescence in DN Hybridoma with Rhodamin.

Figure 29

Membrane enhanced fluorescence after treatment with GC.

Detailed Description of the Invention

In a first aspect, the invention relates to a diagnostic monitoring method for predicting responsiveness of a subject suffering from a pathologic disorder to treatment with a given therapeutic compound. This diagnostic method is based on identifying the direct or indirect modulation in structure and composition of a cell membrane obtained from the examined subject in need, in response to treatment with the specific therapeutic compound. The method of the invention comprises the steps of: (a) obtaining cells from the tested subject; (b) exposing these cells to an effective amount of the certain therapeutic compound, in different concentrations or mixtures, under suitable conditions; and (c) monitoring an alteration in the membrane composition and structure of the treated cells, as compared to a control. It should be appreciated that the alteration may be measured by any suitable means. It should be noted that an alteration in the membrane composition and structure of said cells as compared to a control is indicative of the potential responsiveness of the diagnosed subject to treatment with the examined therapeutic compound. Therefore, this diagnostic method may direct the treating physician regarding the appropriate treatment for each specific examined subject. By predicting the responsiveness of the particular tested subject to a certain therapeutic compound, in a certain concentration or mixture thereof, the diagnostic method of the invention provides a powerful tool for

planning a "tailor-made" treatment, personally adjusted and adapted for each specific patient.

It should be further appreciated that the modulation of composition and structure of a cell membrane by said therapeutic compound may be direct or indirect. Direct modulation may be for example, incorporation of said therapeutic compound onto the treated membrane and thereby modulation of its structure and composition. Indirect modulation of the treated membrane, may include as a non-limiting example alteration of down stream elements (signaling pathways or specific molecules) caused for example by modulation of membranal-receptors or other signaling molecules by said therapeutic compound, which eventually may leads to the detectable membranal alteration.

It should be noted that the direct or indirect modulation of composition and structure of the examined cells membrane, may be an alteration of the lipid rafts or of any receptor molecule or any other signaling molecule therein in the membrane of the examined cells. It should be noted that the alteration is measured and evaluated as compared to a suitable control. Such alteration can be optionally detected by a suitable means. Rafts are lateral assemblies of sphingolipids, cholesterol, and glycerophospholipids that contain a specific complement of integral membrane proteins. Detection of alteration in lipid rafts usually involves isolation of these membranal domains. Detergent insolubility can be used to detect microdomains within membranes. So far, TX-100 has been the most popular detergent in the analysis of microdomains, and it has played a central role in delineating the properties of rafts and their molecular composition.

The lipid rafts microdomains extracted from the plasma membrane can be identified by dot blot, Western blot analysis based on several markers. The most common marker used for raft identification, which is constantly associated with the cholesterol-rich domains, is the GMl glycosphingolipid. A variety of signaling molecules are accumulated in raft domains, including the two Src family kinases Lck and Fyn, both implicated in T cell activation, the T cell linker molecule LAT, monomeric and heterotrimeric G proteins, G-coupled protein receptors and molecules involved in Ca ^{2 +} responses Posttranslational addition of lipids (myristylation, palmitoylation and farnesylation) is a critical requirement for the targeting of many of these molecules to the membrane as well as for their functions. Signaling also depends on the raft-associated kinases and can be suppressed by reduction of the membrane cholesterol level or by changing the membrane lipid composition, indicating that integrity of raft domains is required.

According to one embodiment, the alteration in the composition and structure of rafts of the therapeutic compound-treated cell membrane, may be detected by a suitable means such as an assay selected from ELISA, cell fractionations, flow cytometery, microscopy, preferably, confocal microscopy, dot blot, Western blot, immunopercipitation and PCR.

According to another embodiment, the diagnostic method of the invention is based on examining the alteration in the membranal composition and structure of any cell obtained from diagnosed subject. Such cell may be any one of immune-cells, neuronal cells, muscle cells, endocrine cells, hematopoietic cells, epithelial cells, endothelial cells and mesenchimal cells. More particularly, the examined cells may be blood cells, PBMC, brain, heart, kidney and pancreatic cells.

According to a preferred embodiment, and particularly in case the diagnosed subject suffers from an immune-related disorder, the examined cells may be immune cells. Appropriate immune-cells may be for example, a population of lymphocytes, leukocytes or splenocytes. More particularly, such immune cells may be a population of NK T cells. NK T cells can be obtained from bone marrow, liver, spleen, or uterus, but can also be obtained from the peripheral blood, by cytopheresis methods.

According to a specifically preferred embodiment of this aspect, the therapeutic compound may be a natural or synthetic β-glycolipid. More specifically, the β-glycolipid tested for modulation of the cell membrane may be selected from the group consisting of a monosaccharide ceramide, a glucosylcer amide, a galatosylceremide, a lactosyl-ceramide, a gal-gal- glucosyl-ceramide, GM2 ganglioside, GM3 ganglioside, globoside or any other β-glycolipid.

According to one specifically preferred embodiment, said β-glycolipid may be β- lactosyl-ceramide and any analogue or derivative thereof. In yet another specifically preferred embodiment, said β-glycolipid may be glucosylceramide (GC) and any analogue, mixture or derivative thereof.

In yet another preferred embodiment, a mixture of β-glycolipids may be used by the method of the invention. Such mixture may comprise at least two β-glycolipids at a quantitative ratio between 1:1 to 1:1000. It should be appreciated that any quantitative ratio may be used. As a non-limiting example, a quantitative ratio used may be: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1500, 1:750, 1:1000. It should be further noted that where the mixture of the invention comprises more than two glycolipids, the

quantitative ratio used, may be for example, 1:1:1, 1:2:3, 1:10:100, 1:10:100:1000 etc.

According to a specifically preferred embodiment, a mixture of preferred β- glycolipids that may be used by the diagnostic method of the invention may comprise β-lactosyl-ceramide and at least one other β-glycolipid at a quantitative ratio between 1:1 to 1:1000. More preferably, such mixture comprises β-glucosylceramide and β-lactosyl-ceramide at a quantitative ratio between 1:1 to 1:1000.

According to another embodiment, the diagnostic method of the invention is particularly suitable for predicting responsiveness of a certain subject for the treatment of a pathologic disorder, with specific therapeutic compound or any concentration or mixture thereof. Such pathologic disorder may be for example, an immune-related disorder selected from an autoimmune disease, malignant and non-malignant proliferative disorder, graft rejection pathology, inflammatory disease, genetic disease, bacterial infections, viral infections, fungal infections, or parasitic infections, neurodegenerative disorders or vascular diseases.

According to one specific embodiment, a malignant proliferative disorder may be any one of solid and non-solid tumor selected from the group consisting of melanoma, carcinoma, sarcoma, melanoma, leukemia and lymphoma. More particularly, such malignant disorder may be any one of hepaotcellular carcinoma, melanoma, colon cancer, myeloma, acute and chronic leukemia.

In yet another embodiment, the method of the invention is particularly applicable for examining the potential responsiveness to the treatment of a subject with a specific therapeutic compound. According to this preferred

embodiment, the subject suffers from an autoimmune disease such as, rheumatoid arthritis, diabetes, acute and chronic graft versus host disease, systemic lupus erythmatosus, scleroderma, multiple sclerosis, non alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, the metabolic syndrome or any of the diseases comprising the same, obesity, inflammatory bowel disease and immune mediated hepatitis.

According to another embodiment, the diagnosed subject suffers from a viral infection, for example, HBV, HCV or HIV infection.

In a second aspect, the invention relates to a method for high-throughput screening of a substance which modulates the Thl/Th2 cell balance toward anti-inflammatory cytokine producing cells, in a subject suffering from an immune related disorder. The screening method of the invention is based on the surprising finding that β-glycolipids modulate the composition and structure of the treated cells. Therefore, the use of this feature may enable isolation of novel molecules, preferably of small molecules, that further alters the modulation of cell membranes by the certain glycolipids, or by any other therapeutic compound. Therefore, the screening method of the comprises the steps of: (a) providing test substances; (b) selecting from the test substances provided in step (a) a substance which alters e.g., increase or decrease the modulation of structure and composition of a cell membrane by a given therapeutic compound, as compared to a control (treatment only with the therapeutic compound); and (c) evaluating the ability of the substance selected in step (b) to modulate the Thl/Th2 cell balance toward anti-inflammatory cytokine producing cells, by a suitable means. Thereby, such substance may be a Thl/Th2 modulator and therefore may be used for the treatment of immune-related disorders, solely or in combination with the certain therapeutic compound in any concentration or mixture thereof.

According to one embodiment, the selection according to step (b) of the screening method may be performed by the steps of: (a) obtaining cells from the subject and culturing said cells in a first and a second separated solid support. Suitable solid supports may be wells, mixture, containers or even beads, preferably, wells. The solid support of which the cells are cultured on, may be any water-insoluble, water-insuspensible, solid support. Examples of suitable solid support include large beads, e.g., of polystyrene, filter paper, test tubes, and microtiter plates. The solid support mentioned above can include polymers, such as polystyrene, agarose, Sepharose, cellulose, glass beads and magnetizable particles of cellulose or other polymers. The solid-support can be in the form of large or small beads or particles, tubes, plates, or other forms.

In the next step, the cultured cells in both the first and the second solid support are ex vivo exposed to an effective amount of a certain therapeutic compound under suitable conditions; the third step (c) involves contacting the therapeutic compound-exposed cells of the first solid support, obtained in step (b) with a test substance under suitable conditions; (d) determining the effect of the test substance on an end-point indication as compared to a control, for each of the first and second wells (or any other support), whereby difference in said end point between the first and the second wells (or any other solid support) is indicative of alteration (which may be, increase or in the direct or indirect modulation of membrane composition and structure of said cells by the therapeutic compound used) caused by exposure of the cells to the test substance; and (e) selecting a candidate substance which changes (increase or decrease) the modulation of structure and composition of cell membranes by the therapeutic compound.

In one preferred embodiment, the end point indication may be a difference in the alteration of the lipid rafts or of any receptor or signaling molecule therein of these cells as compared to a control, between the first and the second wells (or any other supports), which leads to a visually detectable signal.

The alteration in the composition and structure of rafts or of any receptor or signaling molecule therein, of said cell membrane, preferably, immune cell membrane may be detected by a suitable means. For example, an assay selected from ELISA, flow cytometery, microscopy, preferably, confocal microscopy, cell fractionation, dot blot, Western blot, and PCR.

The third step of the screening method of the invention involves the ability of the selected substances to alter the Thl/Th2 balance. According to another specifically preferred embodiment, where the cells used are immune system cells, the evaluation step (c) according to the method of the invention may be performed by the steps of: (a) providing a test system. Such system may be preferably, a system comprising ThI and Th2 lymphocytes, ThI and Th2 cytokines, or nucleic acids encoding ThI and Th2 cytokines; (b) contacting the test system provided in (a) with a candidate substance obtained and selected by the method of the invention, under conditions suitable for expression of ThI and Th2 cytokines; and (c) determining the effect of the candidate substance on an end-point indication as compared to a control. The effect is indicative of the capability of the tested candidate substance, to modulate expression of said ThI and Th2 cytokines.

According to another preferred embodiment, the test system used may be any one of in-vitro/ex-viυo cell culture, and in-υivo animal model.

According to one preferred embodiment, the test system may be an in vitro / ex-viυo cell culture of any one of lymphocytes, leukocytes and splenocytes, preferably, NK T cells, obtained from the tested subject. According to this particular embodiment, the end point may be therefore the expression of ThI and Th2 cytokines by these cells, which can be detected by a suitable means. ThI cytokines are for example, IFN-γ and IL-2, Th2 cytokines are for example, IL-4, and IL-IO. Suitable means for detection may be for example, ELISA.

Potential effective candidate substances selected and evaluated by the screening method of the invention may be further evaluated using in vivo test systems. Therefore, in yet another preferred embodiment, the test system may be an in vivo test system, preferably, an animal model. Suitable animal models may be for example, ConA induced hepatitis mice, NASH model of lep tin- deficient mice, HCC model of mice (hepatocellular carcinoma) and TNBS-colitis mice model. It should be noted that the use of these animal models was exemplified in the present application.

As shown by the following examples, where the TNBS-colitis model is used as a test system, the end-point indication may be for example, the alleviation in clinical symptoms as reflected by four macroscopic parameters, namely, degree of colonic ulcerations, intestinal and peritoneal adhesions, wall thickness, and degree of mucosal edema. Each parameter should be graded on a scale from 0 (completely normal) to 4 (most severe). For example, Grade 0: normal with no signs of inflammation, Grade 1: very low level of leukocyte infiltration, Grade 2: low level of leukocyte infiltration, and Grade 3: high level of infiltration with high vascular density, and bowel wall thickening, Grade 4: transmural infiltrates with loss of goblet cells, high vascular density, wall thickening, and disruption of normal bowel architecture.

Where the HCC animal model is used, the end point indication for evaluating the test substance may be the examination of tumor size and weight, as well as the examination of intrahepatic and intrasplenic lymphocyte subpopulations, serum cytokine levels and expression of STATl, STAT4 and STAT6 in splenocytes obtained from the tested animals.

To evaluate the effect of the tested substance on the various metabolic and immunologic components of the NASH model, the end point indication may be glucose tolerance tests, as well as determination of hepatic fat content by abdominal MRI (as reflected by SI index IP-OP/IP). Liver size, in area, may also be determined.

It should be noted that other animal models suitable as a test system for different pathologies may be used by the screening method of the invention for evaluating the effect of the tested substance. As a non- limiting example, for diabetes, the diabetic Passamon model or the Cohen rat model may be used. The end point indication in both models may be determined by examination of body and liver weight, glucose tolerance test, fasting serum levels of Insulin and Glucose for HOMA score, Western blot on splenocytes and liver lymphocytes for STAT proteins 1, 3, 4, 5, 6 and NFkB, biopsies from liver & pancreas for pathology, H&E stain, Oil Red O stain, biopsies from liver for RNA (in RNA later), serum for FFA, TG, Cholesterol, AST, ALT, GGT, insulin levels and MRI.

In yet another preferred embodiment, the test substance may be selected from the group consisting of: protein based, carbohydrates based, lipid based, natural organic based, synthetically derived organic based, inorganic based, and peptidomimetics based compounds. More

particularly, the test substance may be a product of any one of positional scanning of combinatorial libraries of peptides, libraries of cyclic peptidomimetics, and random or dedicated phage display libraries.

According to another embodiment, the therapeutic compound used in the screening method of the invention may be a natural or synthetic β- glycolipid. Such β-glycolipid may be selected from the group consisting of a monosaccharide ceramide, a glucosylceramide, a galatosylceremide, a lactosyl-ceramide, a gal-gal-glucosyl-ceramide, GM2 ganglioside, GM3 ganglioside, globoside or any other β-glycolipid. Preferably, the β-glycolipid may be β- lactosyl-ceramide and any analogue or derivative thereof.

In an alternative and preferred embodiment, the β-glycolipid used by the method of the invention may be glucosylceramide (GC) and any analogue or derivative or mixture thereof.

According to one embodiment, the mixture of said β-glycolipids used by the screening method of the invention may comprise at least two β-glycolipids at a quantitative ratio between 1:1 to 1:1000. Preferably, a mixture of the preferred β-glycolipids comprises β-lactosyl-ceramide and at least one other β-glycolipid at a quantitative ratio between 1:1 to 1:1000. More preferably, the mixture comprises β-glucosylceramide and β-lactosyl- ceramide at a quantitative ratio between 1:1 to 1:1000.

In one preferred embodiment, the screening method of the invention is based on the alteration in the modulation of the membrane composition and structure of given cell by a tested substance. According to one preferred embodiment, the cells may be any one of immune-cells, neuronal cells, muscle cells, endocrine cells, hematopoietic cells, epithelial cells, endothelial cells and mesenchimal cells. Preferably, the tested cells may

be immune cells. According to a particular embodiment, the immune cells may preferably be a population of any one of lymphocytes, leukocytes and splenocytes. More preferably, a population of NK T cells.

According to another preferred embodiment, the screening method of the invention is intended for the screening of a substance which modulated the Thl/Th2 cell balance in a subject suffering from a pathologic disorder, or more particularly, an immune-related disorder. Such disorder may be for example any one of an autoimmune disease, malignant and non- malignant proliferative disorder, graft rejection pathology, inflammatory disease, genetic disease, bacterial infections, viral infections, fungal infections, or parasitic infections, neurodegenerative disorders and vascular diseases.

More particularly, malignant proliferative disorder may be any one of solid and non-solid tumor selected from the group consisting of melanoma, carcinoma, sarcoma, melanoma, leukemia and lymphoma. More particularly, the malignant disorder may be any one of hepaotcellular carcinoma, melanoma, colon cancer, myeloma, acute and chronic leukemia.

According to a preferred embodiment, the immune-related disorder may be an autoimmune disease such as rheumatoid arthritis, diabetes, acute and chronic graft versus host disease, systemic lupus erythmatosus, scleroderma, multiple sclerosis, non alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, the metabolic syndrome or any of the diseases comprising the same, obesity, inflammatory bowel disease and immune mediated hepatitis.

In yet another embodiment, the immune-related disorder may be caused by viral infections, for example, by any one of HBV, HCV or HIV. It should be appreciated that the invention further encompasses any pharmaceutical composition comprising as an active ingredient, the substance isolated by the screening method of the invention.

The pharmaceutical compositions of the invention generally comprise a buffering agent, an agent that adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art. Supplementary active ingredients can also be incorporated into the compositions. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

According to a further aspect, the invention relates to a method for the direct or indirect modulation of composition and structure of a cell membrane of a subject suffering from an immune related disorder. This method comprises the step of increasing the intracellular, extracellular or serum level of a synthetic or naturally occurring β-glycolipid in

said subject, wherein increasing the intracellular, extra-cellular or serum level of a β-glycolipid in said subject is performed by: (I) administering to said subject an effective amount of any one of: (a) a β-glycolipid; (b) a mixture of at least two β-glycolipids; (c) a substance which increases the intracellular, extracellular or serum level of a naturally occurring β- glycolipid; and (d) any combination of the above; or

(II) exposing said cells of said subject to an effective amount of any one of: (a) a β-glycolipid; (b) a mixture of at least two β-glycolipids; (c) a substance which increases the intracellular, extracellular or serum level of a naturally occurring β-glycolipid; and (d) any combination of the above; or

(III) any combination of (I) and (II).

According to one embodiment, such substance may be a substance which increases the rate of production of said β-glycolipid in said subject.

For example, such substance may be a substance which decreases the rate of degradation or turnover of said β-glycolipid in said subject.

According to another embodiment, β-glycolipid used by the method of the invention may be selected from the group consisting of a monosaccharide ceramide, a glucosylceramide, a galatosylceremide, a lactosyl-ceramide, a gal-gal-glucosyl-ceramide, GM2 ganglioside, GM3 ganglioside, globoside or any other β-glycolipid. Preferably, such β-glycolipid is β- lactosyl-ceramide and any analogue or derivative thereof. Alternatively, the β-glycolipid may be glucosylceramide (GC) and any analogue or derivative thereof.

In yet another preferred embodiment, the cell modulated by the method of the invention cell may be any one of immune-cells, neuronal cells, muscle cells, endocrine cells, hematopoietic cells, epithelial cells, endothelial cells and mesenchimal cells. According to a specifically preferred embodiment,

the immune cells are a population of any one of lymphocytes, leukocytes and splenocytes. Preferably, the immune cells may be a population of NK T cells.

In yet another embodiment, the modulation of composition and structure of immune cells membrane may be an alteration of the lipid rafts of said cells or of any receptor molecule therein, as compared to a control. Preferably, such alteration may be in the structure of lipid rafts of immune cell membranes. This alteration can be optionally detected by suitable means.

It should be noted that rafts are glycosphingolipid and cholesterol- enriched plasma-membrane domains, which allow association between cell surface receptors and signal transduction molecules.

According to a specific and preferred embodiment, the modulation of membrane rafts composition of the immune cells by said β-glycolipid may results in educated immune cells having the capability of modulating the Thl/Th2 cell balance toward anti-inflammatory cytokine producing cells.

According to one embodiment, administering step comprises oral, intravenous, intramuscular, subcutaneous, intraperitoneal, perenteral, transdermal, intravaginal, intranasal (including inhalation), mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

In yet another embodiment, the immune-related disorder to be treated may be any one of an autoimmune disease, malignant and non-malignant proliferative disorder, graft rejection pathology, inflammatory disease,

genetic disease, bacterial infections, viral infections, fungal infections, or parasitic infections.

More particularly, a malignant proliferative disorder may be any one of solid and non-solid tumor selected from the group consisting of melanoma, carcinoma, sarcoma, melanoma, leukemia and lymphoma.

Particularly, the malignant disorder may be any one of hepaotcellular carcinoma, melanoma, colon cancer, myeloma, acute and chronic leukemia.

Alternatively, the subject suffers from an immune-related disorder, for example, an autoimmune disease such as rheumatoid arthritis, diabetes, acute and chronic graft versus host disease, systemic lupus erythmatosus, scleroderma, multiple sclerosis, non alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, the metabolic syndrome or any of the diseases comprising the same, obesity, inflammatory bowel disease and immune mediated hepatitis.

In yet another embodiment, the immune-related disorder may be a viral infection by HBV, HCV or HW.

The invention further provides a method for the treatment of immune- related disorder in a mammalian subject in need thereof. This method comprises the step of direct or indirect modulation the composition and structure of the membrane of immune cells of the subject by in vivo or ex vivo exposing cells of said subject immune-system to an effective amount of any one of: (a) a β-glycolipid; (b) a mixture of at least two β-glycolipids; (c) a substance which increases the intracellular, extracellular or serum

level of a naturally occurring β-glycolipid; and (d) any combination of the above.

According to a preferred embodiment, modulation of composition and structure of immune cells membrane may be an alteration of the lipid rafts or of any receptor therein, of said immune cells as compared to a control, which alteration can be optionally identified or detected by a suitable means.

According to another preferred embodiment, in vivo exposing cells of the treated subject immune-system to β-glycolipids, comprises administering to said subject an effective amount of any one of β-glycolipids, a mixture of at least two naturally occurring β-glycolipids and a substance which increases the intracellular, extracellular or serum level of a naturally occurring β-glycolipid, and of a composition comprising the same.

In an alternative embodiment, ex vivo exposing cells of the treated subject immune-system to β-glycolipids comprises the steps of: (a) obtaining immune cells from said subject, or from another subject; (b) ex vivo directly or indirectly modulating membrane rafts composition or structure of said immune cells by said β-glycolipid which results in educated immune cells having the capability of modulating the Thl/Th2 cell balance toward anti-inflammatory cytokine producing cells; and (c) re-introducing to said subject the educated cells obtained in step (b) which are capable of modulating the Thl/Th2 cell balance toward anti-inflammatory cytokine producing cells, resulting in an increase in the quantitative ratio between any one of IL4 and ILlO to IFNγ.

It should be noted that the process of modulation of the Thl/Th.2 cell balance toward anti-inflammatory cytokine may be further mediated by at

least one component of the subject immune system and may be changed by the compound isolated by the screening method of the invention. According to a preferred embodiment, such component may be selected from the group consisting of cellular immune reaction elements, humoral immune reaction elements and cytokines. Preferably, such component may be a cellular immune reaction element.

In addition to the treatment with glycolipids and optionally, with the compound isolated by the screening method of the invention, the immune cells may be further treated with any other known condition. For example, cells may be exposed to antigens associated with said immune -related disorder to be treated. These antigens may be for example, any one of allogeneic antigens obtained from a donor subject suffering from said immune-related disorder, xenogeiήc antigens, syngeneic antigens, autologous antigens, non-autologous antigens and recombinantly prepared antigens and any combinations thereof. These antigens can be native or non-native with regards to the subject. They can be natural or synthetic, modified or unmodified, whole or fragments thereof. Fragments can be derived from synthesis as fragments or by digestion or other means of modification to create fragments from larger entities. Such antigen or antigens comprise but are not limited to proteins, glycoproteins, enzymes, antibodies, histocompatibility determinants, ligands, receptors, hormones, cytokines, cell membranes, cell components, viruses, viral components, viral vectors, non-viral vectors, whole cells, tissues or organs. The antigen can consist of single molecules or mixtures of diverse individual molecules. The antigen can present itself within the context of viral surface, cellular surface, membrane, matrix, or complex or conjugated with a receptor, ligand, antibody or any other binding partner.

Polymerization and degradation, fractionation and chemical modification are all capable of altering the properties of a particular antigen in terms of potential immune responses. These small segments, fragments or epitopes can either be isolated or synthesized.

The method of the present invention further encompasses recombinantly prepared antigens. Preparation of recombinant antigens involves the use of general molecular biology techniques that are well known in the art. Such techniques include for example, cloning of a desired antigen to a suitable expression vector.

In another particular embodiment, the ex vivo education of the cells may be further performed by culturing these cells in the presence of liver- associated cells. These cells may be for example Kupffer cells, Stellate cells, liver endothelial cells liver associated stem cells or any other liver- related lymphocytes.

Co-culturing of the cells in the presence of peripheral lymphocytes from tolerized or non-tolerized patients suffering from the same immune- related disorder or from the treated subject, is also contemplated in the present invention. In order to obtain lymphocytes from a subject, particularly human subject, blood is drawn from the patient by cytopheresis, a procedure by which a large number of white cells are obtained, while other blood components are being simultaneously transferred back to the subject.

In another particular embodiment, the ex-vivo education of the cells may be further performed by culturing the cells in the presence of cytokines such as IL4, ILlO, TGFβ, IFNγ, IL12 and IL15, or in the presence of adhesion molecules such as Integrins, Selectin and ICAM. In a specifically

preferred embodiment, the cell that has been ex vivo educated as described above may be re-introduced to the treated subject. This can be carried out by a process that has been termed adoptive transfer. The particular educated cells used for the transfer may preferably originate from the subject (autologous transfer). A syngeneic or non-syngeneic donor (non- autologous transfer) is not excluded. The storage, growth or expansion of the transferred cells may have taken place in vivo, ex vivo or in vitro.

Cell therapy may be by injection, e.g., intravenously, or by any of the means described herein above. Neither the time nor the mode of administration is a limitation on the present invention. Cell therapy regimens may be readily adjusted taking into account such factors as the possible cytotoxicity of the educated cells, the stage of the disease and the condition of the patient, among other considerations known to those of skill in the art.

In one embodiment, β-glycolipid may be selected from the group consisting of: a monosaccharide ceramide, a glucosylceramide, a galatosylceremide, lactosyl-ceramide, gal-gal-glucosyl-ceramide, GM2 ganglioside, GM3 ganglioside, globoside or any other β-glycolipid.

Preferably, the β-glycolipid may be β- lactosyl-ceramide and any analogue or derivative thereof.

Alternatively, the β-glycolipid may beglucosylceramide (GC) and any analogue or derivative thereof.

In yet another embodiment, the administering step comprises oral, intravenous, intramuscular, subcutaneous, intraperitonea, perenteral,

transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

Therapeutic formulations may be administered in any conventional dosage formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof.

According to another embodiment, the method of the invention is intended for the treatment of an immune-related disorder such as an autoimmune disease, malignant and non-malignant proliferative disorder, graft rejection pathology, inflammatory disease, genetic disease, bacterial infections, viral infections, fungal infections, or parasitic infections.

In another specifically preferred embodiment, the method of the invention is intended for the treatment of a malignancy. In cancerous situations, modulation of the Thl/Th2 cell balance may be in the direction of inducing a pro-inflammatory response or in augmenting the anti-tumor associated antigens immunity. As used herein to describe the present invention, "cancer", "tumor" and "malignancy" all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods and compositions of the present invention may be used in the treatment of non-solid and solid tumors.

Malignancy, as contemplated in the present invention may be selected from the group consisting of melanomas, carcinomas, lymphomas and sarcomas. Malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including

leukemia, lymphoma and myeloproliferative disorders), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including lung, liver, breast, colon, prostate GI tract, pancreas and Karposi). More particularly, the malignant disorder may be melanoma, hepaotcellular carcinoma, colon cancer, myeloma, acute or chronic leukemia.

In yet another embodiment, the autoimmune disease may be any one of rheumatoid arthritis, diabetes, acute and chronic graft versus host disease, systemic lupus erythmatosus, scleroderma, multiple sclerosis, non alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, the metabolic syndrome or any of the diseases comprising the same, obesity, inflammatory bowel disease and immune mediated hepatitis.

According to a particular embodiment, the viral infection comprises HBV, HCV or HIV.

According to a specifically preferred embodiment, wherein the treated subject is suffering from any of the diseases indicated above, a preferred result of the treatment by the method of the invention may be for example, an increase in glucose tolerance, reduction in liver fat content or change in cytokine responses.

According to one specific embodiment, the method of the invention is particularly intended for the treatment of a subject suffering from diabetes.

According to another specific embodiment, the method of the invention is particularly intended for the treatment of a subject suffering from non alcoholic fatty liver disease.

According to another specific embodiment, the method of the invention is particularly intended for the treatment of a subject suffering from hyperlipidemia.

According to another specific embodiment, the method of the invention is particularly intended for the treatment of a subject suffering from the metabolic syndrome or any of the diseases comprising the same.

According to another specific embodiment, the method of the invention is particularly intended for the treatment of a subject suffering from obesity.

According to another specific embodiment, the process of the invention is particularly intended for the treatment of a subject suffering from inflammatory bowel disease.

According to another specific embodiment, the method of the invention is particularly intended for the treatment of a subject suffering from immune mediated, viral or chemical mediated hepatitis.

Although the method of the invention is particularly intended for the treatment of immune -related disorders in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions

disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Examples

Experimental procedures

Glycolipids β-Glucosylceramide, β-galactosyloceramide, and β-lactosylcer amide were purchased from Avanti Polar Lipids (Alabaster, AL), dissolved in ethanol and emulsified in PBS. Ceramide was purchased from Alexis Biochemicals (San Diego, USA)

Animals

Twelve-week old male C57B1/6 mice were obtained from Harlan Laboratories (Jerusalem, Israel) and maintained in the Animal Core of the Hadassah-Hebrew University Medical School. Mice were administered standard laboratory chow and water ad libitum, and kept in 12-hour light/dark cycles. Animal experiments were carried out according to the guidelines of the Hebrew University-Hadassah Institutional Committee for Care and Use of Laboratory Animals, and with the committee's approval.

Induction of ConA Hepatitis

ConA (MP Biomedical, USA) was dissolved in pyrogen-free PBS containing 50 mM Tris (pH 7), 150 mM NaCl, and 4 niM CaCl ₂ , and injected into the tail vein at a dose of 500 μg/mouse (15 mg/kg).

Induction of HC:

Mice were subcutaneously implanted with 10xl0 ⁶ Hepa-3B human HCC cells.

Induction of colitis

Trinitrobenzenesulfonic acid (TNBS)-colitis was induced by rectal instillation of TNBS, 1.5 mg/mouse dissolved in 100 ml of 50% ethanol, in the 1 ^st and in the 5^ ¹ day of the experiment.

Isolation of splenocytes and intrahepatic lymphocytes

Splenocytes and intrahepatic lymphocytes were isolated as follows, livers and spleens were kept in RPMI- 1640 + FCS then spleens were crushed through 70Dm nylon cell strainer (Falcon) and centrifuged (1250 rpm for 7 min) for the removal of debris. Red blood cells were lysed with 1 ml of cold 155 mM ammonium chloride lysis buffer and immediately centrifuged (1250 rpm for 3 min). Splenocytes were then washed and resuspended with ImI RPMI + FCS. Remains of connective tissue were removed. The viability by trypan blue staining was above 90%. For intrahepatic lymphocytes, livers were first crushed through a stainless mesh (size 60, Sigma) and the cell suspension was placed in a 50-ml tube for 5 min so cell debris will descend. 10ml of Lymphoprep (Ficoll, Axis-Shield PoC AS, Oslo, Norway) was slowly placed under the same volume of cell suspension in 50-ml tubes. The tubes were then centrifuged at 1800 rpm for 18min. Cells in the interface were collected and moved to new tubes which were centrifuged again at 1800 rpm for lOmin, to obtain a pellet of cells depleted of hepatocytes to a final volume of 250μl. Approximately IxIO ⁶ cells/mouse liver, were recovered.

Cytokine measurement

Serum IFNγ, IL2, IL12, IL4 and ILlO levels were measured in each animal by "sandwich" ELISA, using commercial kits (Genzyme Diagnostics, MA, USA).

Preparation of detergent-resistant membrane fractions Raft micro domains are enriched with glycosphingolipids and the ganglioside GMl, which is recognized by cholera toxin. GMl is currently being used as a raft marker. Lipid raft components were purified using Triton X-IOO insolubility coupled with buoyant density in sucrose or nycodenz gradient. Expression of GMl in murine splenocytes was analyzed by immunoblotting and by FACS analysis.

Flotation study of detergent-insoluble complexes

Flotation study was performed as described [Rouvinski, A. et al. Biochem. Biophys. Res. Commun. 308:750-758 (2003)]. Freshly isolated splenocytes (3xlO ⁷ cells) were washed with ice-cold PBS and lysed with 550μl of TNE buffer (15OmM NaCl ₅ 25mM Tris-HCl, and 5mM EDTA, pH 7.5) containing 1% Triton X-100 for 30 min. Lysates were centrifuged at 3000 rpm, 4 ⁰ C, 5 min. Supernatants were adjusted to 35% Nycodenz (Sigma) by adding an equal volume of ice-cold 70% Nycodenz dissolved in TNE, and loaded at the bottom of TLS-55 tubes (Beckman Instruments). An 8—25% Nycodenz linear step gradient in TNE was overlaid above the lysate (200Dl each of 25, 22.5, 20, 18, 15, 12, and 8% Nycodenz). Tubes were spun at 55,000 rpm for 4h, at 4 ⁰ C in a TLS-55 rotor. Twelve 180μl fractions were sequentially collected from the top of the tubes and analyzed by dot blot.

CTxB-dot blots

To detect the ganglioside GMl on dot blots, 10ml of each flotation fraction was supplemented with lOOμl PBS and blotted on nytran paper using a vacuum dot blotter. The paper was air dried, blocked with 5% BSA in PBS, and reacted with Cholera-Toxin B subunit (CTxB-HRP) (5ng/ml). Blots were developed by chemoluminescence (ECL).

Electrophoresis and immune blotting

For detection of the ganglioside GMl by dot blots, IODl of each flotation fraction was supplemented with 90μl PBS and blotted on Nytran paper (Schleicher & Schuell) using a vacuum dot blotter (Bio-Rad). Papers were blocked with 5% BSA in PBS, and reacted with CTxB-HRP (Sigma, 12.5ng/ml) for 30 min followed by four washes with TBS-T. Membranes were developed using Western Blot-Luminol Reagent (Santa Cruz Biotechnology, USA). Proteins from raft and cytosolic fractions were detected by loading equal volumes of 28μl from each flotation fraction resolved by sodium dodecyl sulphate— polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes and blocked with 5% non-fat dry milk. The following antibodies were used: anti ESA-2 (BD Biosciences, USA), anti Flotillin 1:5000, anti LCK 1:1000, anti Fyn 1:1000 (Lab vision corporation, USA). Horseradish peroxidase (HRP)- coupled Abs (Jackson Immuno Research, USA) were used as secondary Abs, and detection was performed with Western Blot-Luminol Reagent (Santa Cruz Biotechnology, USA).

Detection and quantification of GMl by ELISA.

The analysis of lipid rafts by ELISA appears to be a useful technique to identify and quantify the amount of GMl -lipid rafts when a large number of samples require to be simultaneously analyzed.

To determine the amount of GMl in splenocytes membrane fractions purified by nycodenz gradient centrifugation, a modified ELISA protocol was used. Several dilutions of the nine individual fractions (1:100, 1:5000, and 1:10,000 in PBS) were coated overnight at 4 ⁰ C onto plastic surface with high affinity for glycolipids (96-well Polysorp star well plates, 200 μl/well). The plates were blocked for 30 min at room temperature with 3% BSA/PBS, and then incubated for 1 h at 37 ⁰ C with 5 μg/ml of CTB-HRP

conjugate in 1% BSA/PBS. CTB is a natural ligand that binds strongly and specifically to GMl moieties. The plates were washed with PBS/0.05% Tween 20, incubated for 20 min with 200μl of HRP chromogen substrate and the reaction was stopped by the addition of 25μl of 2M sulphuric acid. The optical density (450 nm) was measured in a Powerwave X ELISA reader.

Flow cytometry for GMl and lymphocyte subsets

Flow cytometry was performed following lymphocyte isolation using IxIO ⁶ lymphocytes in lOOμl PBS. GMl analysis was performed by staining the cells with 0.5μg of CTxB-FITC (Sigma). For determination of the percentage of NKT lymphocytes, PE-Cy5 anti-mouse CD3 and PE anti mouse NKl.1 antibodies were used (eBioscience, USA). CD4 and CD 8 subsets were detected by PE-Cy5 anti-mouse CD3 and by PE-anti-mouse CD4 or CD8. Cells were incubated for 30 min at 4 ⁰ C in the dark, then washed and resuspended in 200μl PBS. Analytical cell sorting was performed on IxIO ⁴ cells from each group with a fluorescence-activated cell sorter (FACSTAR plus, Becton Dickinson). Only live cells were counted and unstained cells served as control background fluorescence. Gates were set on forward- and side-scatters to exclude dead cells and red blood cells. Data was analyzed with the Consort 30 two-color contour plot program (Becton Dickinson, Oxnard, CA), or the CELLQuest 25 program.

Characterization of isolated lipid micro domains

GMl was clustered by incubation of 5xlO ⁶ splenocytes with 20μg/ml CTxB- FITC (Sigma) in 0.1%BSA in PBS at 4 ⁰ C for 30min in the dark. Cells were washed and fixed in 3.7% paraformaldehyde for 15min in room temperature, and attached to polylysine-coated microscope slides and mounted with vecta shield (Vector Laboratories, USA). For splenocyte labeling with CTxB-HRP, 2.5X10 ⁶ splenocytes were stained with 2μg of

CTxB-HRP in 0.1%BSA in ImI PBS for 30min. Cells were lysed with 200Dl splenocyte lysis buffer (0.5% T-XlOO, 0.23% deoxycholate, 1OmM EDTA, 1OmM Tris pH 7.5, 10OmM NaCl) for 15 min on ice. Lysates were centrifuged (2000rpm for 5 min at 4 ⁰ C) and 20μl, equivalent to 2.5X10 ⁵ cells were supplemented with lOOμl PBS and blotted on Nytran paper using a vacuum dot blotter. GMl was detected by developing the membrane with Western Blot-Luminol Reagent.

Cell surface staining using fluorescent microscopy

Freshly isolated splenocytes (5 x 10 ⁶ ) in RPMI/1640, were twice rinsed with ice-cold PBS (800rpm, 4min, 4°C) and then incubated with cholera toxin B-FITC (CT-B-FITC, final concentration 20μg/ml) in phosphate- buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) at 4 ⁰ C for 30 min in dark. The unbound CT-B-FITC was removed by washing twice with ice-cold PBS. The cells were then fixed immediately with 400 μl 3.7% paraformaldehyde (in PBS) and kept on ice for 15 min and then for another 15 min in room temperature. The fixed-stained cells were subsequently washed with PBS and then added to cover slips and mounted to slides.

Assessment of the effect of β-glycolipids on liver damage:

Liver enzymes: Sera from individual mice were obtained. Serum AST and

ALT activities were determined using an automatic analyzer.

Grading of histological lesions

For histological evaluation of inflammation, distal colonic tissue (last 10 cm) was removed and fixed in 10% formaldehyde. Five paraffin sections from each mouse were stained with hematoxylin-eosin by using standard techniques. The degree of inflammation on microscopic cross sections of the colon was graded semi-quantitatively from 0 to 4. Grade 0: Normal

with no signs of inflammation; Grade 1: very low level of leukocyte infiltration; Grade 2: Low level of leukocyte infiltration; Grade 3: High level of infiltration with high vascular density, and bowel wall thickening; Grade 4: Transmural infiltrates with loss of goblet cells, high vascular density, wall thickening, and disruption of normal bowel architecture. Grading was performed by two experienced -blinded examiners. Extent of disease was assessed by evaluation of the bowel taken from each animal.

STAT 1, flotillin-2, and Lck expression

Expression of the transcription factor STAT 1 (signal transducer and activator of transcription 1) and its activated (phosphorylated) form, flotillin-2, and Lck proteins were determined by Western blot analysis of splenocytes harvested from mice in all groups. Splenocytes were lysed in RIPA buffer with a protease inhibitor cocktail (Sigma). Lysates were centrifuged and protein concentrations were determined by Bradford assay (Bio-Rad). β-actin (Abeam, UK) was used as a loading control. Proteins were detected with phospho-specific STATl-Tyr701, flotillin-2, and Lck antibodies that were purchased from Cell Signaling (USA).

TUNEL Staining

The TUNEL assay was performed on paraffin-embedded liver sections using a commercially available kit, . following the manufacturer's instructions (In Situ cell death Apoptosis Detection kit, Fluorescein, Roche Diagnostics, Germany). TUNEL-positive cells were morphologically characterized by DAPI staining. Slides were visualized using a Zeiss (Oberkochen, Germany) Axioplan fluorescence microscope. Images were collected with cooled charge-coupled device camera (Quantix Corp. USA) using Image Pro software.

Cell Counting

For cell counting in tissues processed for TUNEL staining, five standardized microscopic fields (x20 objective) per slide (per animal) were digitally photographed under fluorescence microscopy and then quantified using Total Lab (Phoretix, USA) software.

Statistical analysis

Statistical analysis was performed using the student's t test, p < 0.05 was considered significant. ,

Example 1

Effect of GC on the membrane composition of cells obtained from a murine model of NASH (leptin-deficient ob/ob mice)

Leptin- deficient ob/ob mice, the animal model for non-alcoholic steatohepatitis, exhibit morbid obesity, hyperlipidaemia, glucose intolerance, and steatohepatitis. These mice feature impaired cell mediated immunity, reduced number of hepatic NKT lymphocytes, impaired function of hepatic Kupffer cells, reduced serum levels of interleukin ILlO and IL15, and increased levels of IL12.

As indicated in the background of the invention, the present inventors have previously shown beneficial effect of different glycolipids, and particularly, GC, β-lactosylceramide or mixtures thereof, on murine model of NASH. In order to analyze the mechanism of the CG effect, the inventors first examined the effect of different glycolipids, and particularly, CG on membranes of immune cells obtained from leptin- deficient ob/ob mice treated with glycolipids. Therefore, as a first step, the general effect of GC on liver and spleen leukocytes and lympghocytes was examined by FACS analysis using different fluorescent markers. Briefly,

3

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leptin-deficient ob/ob mice in group A (10 mice per group): 3 mice in each group were administered daily with PBS, as control, for 3 weeks, group B mice were administered daily with GC (2.5 mg/kg) for the same period of time. Mice were sacrificed, and lymphocytes were isolated from spleen and livers of the mice, were stained and subjected to FACS analysis as described in Experimental procedures. As shown by Figure 1, GC treatment clearly increased the total fluorescence of liver leukocytes. However, the effect of GC on lymphocytes obtained from the same mice was less significant. The differential effect of GC on different cell type was also revealed when spleen lymphocytes were next examined. As shown by Figure 2, GC treatment reduced the fluorescence of the splenocytes as compared to PBS treated cells.

A more detailed analysis of lipid rafts was next performed by flow cytometry. FACS technique can provide additional information on the partitioning of lipid rafts in plasma membrane of various immune cells, and therefore CT-B-FITC was used for this purpose. For better identification of splenocytes cell subsets, specific antibodies (anti NKl.1, anti CD45, anti CD3, anti CD4, anti CD8) were also used.

Non-statistical (only 1 mouse from each group) FACS analysis results presented in Table 1, show a GMl content decrease in GC treated mouse. This decline is especially remarkable in the CD4 and CD8 subsets. Table 1

CT- CT- CT- CT-

B/CD3 B/CD8 B/CD4 B/NK #

Al-

36.42 40.73 36.77 5.18 cont

Bl-

32.4 25.1 23.4 5.24 GC

The effect of GC treatment on immune cells obtained from NASH-model mice was further investigated by confocal microscroscopy. The analysis of lipid rafts was performed using Cholera Toxin — B subunit coupled to FITC (CT-B-FITC), which is a familiar marker to monitor the GMl partitioning in plasma membrane.

As shown by Figure 3, analysis of GC treated mouse (group B) in the NASH model showed a small decrease in the CT-B-FITC binding compare to control mice (group A). However, the appearance of the CT-B-FITC binding was similar (a ring like morphology) in both mice groups.

Decreased CT-B-FITC binding to the cell surface of GC treated mouse, shown in the FACS and confocal microscopy analysis, may indicate a decrease in the amount of GMl-lipid rafts on lymphocytes membrane as compared with the control mouse.

Example 2

Effect of β-lactosylceramide on the membrane composition of cells obtained from ConA-induced hepatitis mice

The results obtained from immune cells of NASH-model mice encouraged further investigation of the effect of different glycolipids on immune cells obtained from different mice models representing other immune related disorders. Therefore, the effect of β-lactosylceramide on a murine (C57B1/6) model of ConA-induced hepatitis was next examined. Mice in group A were treated with 1.5 μg of β-lactosylceramide, IP 2h before and 2h following administration of ConA, respectively; group B mice were treated with ConA alone and served as control.

As shown by Figure 4, confocal microscopy analysis performed in splenocytes isolated from β-lactosylceramide (C: 12) treated mice, revealed a special binding pattern of FITC-labeled CT-B, compared to control mice. CT-B-FITC binds in a patchy pattern with several focuses of bound CT-B- FITC. Without being bound by any theory, the enlarged GMl patches on the cell surface may indicate an increase in the amount of GMl-lipid rafts on lymphocytes membrane as compared with the ring-like morphology of the control. Similarly, the addition of exogenous ganglisides to cells can lead to their incorporation into rafts and as a result also cause proteins to dissociate from rafts.

Example 3

Alteration in lymphocyte lipid raft composition and structure via beta glycolipids is associated with amelioration of Concanaυalin-A induced hepatitis

The enlargement of GMl patches caused by the treatment of the ConA hepatitis induced mice with LC, encouraged the inventor to further analyze the effect of other glycolipids and of mixtures thereof on the membrane composition of immune cells obtained from ConA hepatitis induced mice. Hepatitis was induced by ConA administration in the following three tested groups of mice: Groups B and C were treated with glucosylceramide (GC) or lactosylceramide (LC), respectively, control group A received solvent alone. Effect of glycolipids on lipid rafts was determined by Western blot analysis of rafts domains extracted from plasma membrane, FACS analysis for GMl distribution pattern and fluorescence microscopy for raft patching on plasma membrane. Effect of glycolipids on liver damage was assessed by histology, examination of Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) levels, FACS analysis of hepatic and splenic CD4, CD8 and NKT markers, and serum cytokine levels.

Beta glycolipids administration significantly increased GMl content, a key marker of lipid rafts, in NKT, CD4 and CD8 lymphocyte membranes, by 46 and 77%, for groups B and C vs. A, respectively, (p<0.05). Administration of a 1:1 combination of GC and LC increased GMl content in NKT cells by 95%. Dot blot analysis revealed a 4.7 to 9.8 fold increase in detergent-soluble rafts fractions. Tracking protein patterns revealed an increase in detergent-soluble cytosolic fraction, including LCK protein from the Src-family kinases. Modulation of raft size components was suggested by alteration of GMl patching behavior using cholera toxin. Modification of lipid rafts by beta-glycolipids was associated with a significant hepatitis alleviation, noted by a 34% reduction in serum AST and ALT levels in groups B and C vs. A (p<0.05), and marked histological attenuation of hepatocyte necrosis. The beneficial effect was associated with 105% increase in intra-hepatic NKT cells, and with increased liver/spleen NKT ratio (1.46, 1.4 vs. 0.89, for B and C vs. A, respectively, p<0.05). Spleen/liver CD4/CD8 lymphocyte ratio decreased (1.32, 1.36 vs. 1.48, for B, C and A, respectively, p<0.05). A decrease in IFN levels was noted in group B (4457 vs. 8178 pg/ml).

These results show that administration of beta glycolipids alters lymphocyte lipid raft composition and structure, and may affect the intracellular signaling machinery. These effects were associated with alleviation of immune mediated hepatitis, alteration of NKT lymphocyte distribution, and decreased intrahepatic CD 8 lymphocyte trapping.

Example 4

Effects of a- and β-glycolipids on lipid raft composition

The immune microenυironment of the host determines the effect of β- glycolipids on lipid rafts

The clear effect of GC and LC as demonstrated by the ConA model, encouraged the inventors to further examine the effect of other glycolipids. Therefore, in a next step sixteen experimental and control groups, 10 mice per group, were studied (Table 2). Mice in experimental groups A-H were injected with ConA. Group A mice were administered a single intraperitoneal injection of 100 DL PBS two hours prior to IV administration of ConA. Mice in groups B, C, D, E, F, and G were administered a single intraperitoneal injection of β-glucosylceramide (GC), β-galactosylceramide (β-Gal), β-lactosylcer amide (LC), α- galactosylceramide (α-GalCer), iGb3, and a 1:1 ratio of β-glucosylceramide and β-lactosylceramide (IGL), respectively (1.5 μg in 100 μL PBS) 2 hours prior to IV administration of ConA. Mice in groups I— P were similarly injected with the different glycolipids without ConA administration. Animals were sacrificed ten hours following the glycolipid injections, 8 hours after the injection of ConA. Membranal fractions were prepared as described in Experimental procedures. GMl content in both the detergent- insoluble [Vratsanos, G.S. et al. J. Exp. Med. 193:329-337 (2001)] and detergent-soluble (cytosolic) fractions was analyzed by dot-blot analysis. As shown by Figure 5A, the most significant changes were noted in raft fractions 1 and 2 and in cytosolic fractions 11 and 12. Quantitative analysis is shown in Figures 5B, 5C.

In naϊve mice, administration of β-glycolipids, including GC, LC and IGL, but not β-Gal and iGb3, increased splenic GMl content. This effect was not observed with α-GalCer. In ConA-treated mice, only the administration of GC or LC significantly increased both raft and cytosolic

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fractions, while IGL only increased raft fractions. No effect was seen after iGb3 administration in either fraction. While α-GalCer increased both raft and cytosolic fractions, the raft to cytosolic fraction ratio was significantly increased compared with administration of either GC or LC. To further determine the effects of distinct α- and β-glycolipids on lipid rafts in NKT lymphocytes, mice were treated with various glycolipids. FACS analysis was performed using CT-X-FITC for determination of GMl expression on the cell surface. Administration of ConA caused a significant decrease in GMl expression in CD4, CD8, and NKT cells (60- 70% decrease). Administration of α-GalCer, β-GalCer, and iGb3 led to a further decrease in GMl expression in these cells. In contrast, treatment of mice with GC, LC, and IGL prevented or attenuated this decrease in NKT lymphocytes (Figures 5D and 5E). A lesser effect was noted on CD4 and CD 8 lymphocyte subsets (data not shown).

Freshly derived whole splenocytes from naϊve and ConA-treated animals were stained using the CTX-HRP conjugate, as a measure of whole-cell GMl content. As shown by Figures 5F and 5G, ConA-induced hepatitis led to a 50% increase in whole-cell GMl expression. Administration of GC, LC, and IGL further enhanced this effect. In contrast, α-GalCer and iGb3 reduced GMl content in ConA-treated mice. It should be noted that these effects were not observed in naϊve animals.

β-glycolipids affect lipid raft structure.

The structure of raft domains in plasma membrane of splenocytes derived from untreated and glycolipid-treated mice were analyzed in order to determine whether administration of various glycolipids could affect lipid raft disruption on the plasma membrane. Clustering patterns of GMl using CT-X-FITC were compared. Fluorescent microscopy was used to analyze the patching distribution of GMl/cholera toxin. Representative

images are presented in Figure 6. Administration of ConA led to mild cholera toxin-induced raft clustering. Mice treated with GC and LC showed enlarged GMl patches on the cell surface, suggesting an increase in the GMl content on the splenocyte membrane. In contrast, a decrease in clustering was noted in mice treated with α-GalCer and iGb3 (data not shown).

Table 2 Experimental groups

Example 5

Effects of a and β-glycolipids on expression of flotϊllin-2 and Lck in detergent-insoluble and soluble fractions in lipid rafts

The effect of different glycolipids on lipid rafts composition led the inventors to further examine the involvement of molecules which participate in signal transduction pathways know to be involved. Therefore, flotation assays were performed to determine the expression of flotillin-2 and Lck in detergent-insoluble and soluble fractions (#1-12)

derived from splenocytes of ConA- treated and naϊve mice (Figure 7). ConA-induced hepatitis was associated with the disappearance of flotillin- 2 from detergent-insoluble membrane fractions 1-5. Lck distribution was similar to that of flotilin-2 (data not shown).

Effect of β-glycolipids on phosphorylation of STATl

The inventors next studied the expression of STATl and its phosphorylated state, which is associated with activation of the IFN-γ receptor. Administration of α-GalCer and iGb3 significantly increased the expression of phosphorylated STATl, while treatment with GC and LC either did not alter or decreased this expression (Figure 8).

Effects of β-glycolipids on serum cytokine levels

Serum IFNγ levels were significantly reduced in GC-, LC-, and IGL- treated animals (86.1, 126.8, and 188.8 pg/mL for mice in groups B, D, and H, respectively, compared with 1264, 1902, 1343, and 1051 for groups C, E ₅ F, and G, respectively, (p < 0.005, for B, D, and H compared with C, E, F, and G, Figure 9). Serum IL-10 levels decreased in animals treated with β-glycolipids compared with those treated with ConA alone (p NS). Serum IL- 12 levels did not change significantly between treated and untreated groups (data not shown).

Effects of β-glycolipids on the immune response

Effects of β-glycolipids on splenic and intrahepatic NKT lymphocytes Administration of ConA was associated with a significant decrease in the number of NKT lymphocytes (1.66 vs. 22.55, in groups I and A, respectively, p < 0.005). Among the ConA-treated groups, administration of β- and α-glycolipids significantly altered the number of intrahepatic NKT cells, distinct from iGb3 (Figure 10A). Administration of glycolipids to naϊve mice (groups J through P) led to a reduction of the intrahepatic

NKT cell number (22.5 to 1.3; p < 0.005, for group I compared with J through P). No significant differences were noted between the different glycolipids. In the spleen, a statistically insignificant increase in the number of NKT lymphocytes was observed in all glycolipid-treated groups with and without the administration of ConA (Figure 10B).

Effects of β-glycolipids on splenic and intrahepatic CD4 and CD8 lymphocytes.

As shown above, administration of ConA was associated with a significant increase in intrahepatic CD8 trapping (signified by an increase in the splenic/intrahepatic CD4/CD8 ratio). Treatment with GC, LC, and IGL in mice that received ConA decreased intrahepatic CD 8 trapping with a significant decrease of the spleen to liver CD4/CD8 lymphocyte ratio (1.23, 1.38, and 1.24, for mice in groups B, D, and H, respectively, compared with 1.53, 1.51, 1.61, and 1.53 for groups C, E, F, and G, respectively, p < 0.005, for B, D, and H compared with C, E, F, and G) (Figure 11).

Effect of β-glycolipids on liver damage in ConA-induced hepatitis

Effect of β-glycolipids on serum ALT and AST levels

Administration of all β-glycolipids significantly alleviated ConA-induced hepatitis via a decrease of serum AST and ALT levels (Figure 12A). In contrast, treatment with α-GalCer led to a 2.5-fold increase in transaminase levels. In naϊve mice, both α-GalCer and iGb3 induced an elevation of transaminases. Administration of GC, LC, and IGL to naϊve mice did not alter liver enzymes (Figure 12B).

Effect of β-glycolipids on liver histology

Histological liver damage was markedly attenuated in GC-, LC- and IGL- treated groups. Total liver scores were decreased to 1.5, 1.75, and 1.16 for mice in groups B, D, and H, respectively, compared with 6.0, in non-

treated controls in group A, and 2.66, 3.16, 3.0, and 2.33 in mice in groups C, E, F, and G, respectively (p < 0.005 for B, D, and H, compared with C, E, F, and G, Figure 13).

Effect of β-glycolipids on intrahepatic apoptosis.

The TUNEL assay was performed on liver sections. Figure 14 shows a representative densitomemtric quantification that emphasizes the significant decrease in TUNEL-positive cells noted in mice treated with GC, LC, and IGL compard with animals treated with ConA alone. In contrast, treatment with α-GalCer and iGb3 induced massive apoptosis, showing a 5-fold increase in the number of TUNEL-positive cells. In naϊve mice, only treatment with α-GalCer, but not iGb3, led to an increase in TUNEL-positive cells.

Example 6

Effect of IGL on the membrane composition of cells obtained from

Hepatocellular carcinoma (HCC) model mice

The effect of IGL, which is a mixture of GC and β- lactosylceramide, on immune cells obtained from a murine model of hepatocellular carcinoma, was next examined.

HCC (mice injected with Hepa-3B human hepatocellular carcinoma cells) mice in group A were administered with PBS, HCC group B mice were administered with IGL (2.5 mg/kg of GC and of β- lactosylceramide).

The treated immune cells were then analyzed by confocal microscopy as shown by Figure 15. Analysis of IGL treated mice in the HCC model did not show any remarkable change pattern in CT-B-FITC binding compare

to control mice. It should be noted that GC or β-lactosyceramide treated mice were not available for this analysis.

These cells were further analyzed by FACS analysis. Non-statistical (only 2 mice from each group) results presented by Table 3 and the graph in Figure 16, show a GMl content decrease in IGL treated mice, though not seen in microscopy. It should be noted that the decline is more remarkable in the CD4 subset, than in CD8 subset.

Table 3

CT- CT- CT- CT-

B/CD3 B/CD8 B/CD4 B/NK #

2.54 2.63 4.34 1 58 Al-cont

2 99 8.16 11.53 1.89 A2-cont

1.55 2 4 2 41 1.14 Bl-IGL

3.06 2.91 4.05 1.33 B2-IGL

Example 7

Suppression of hepatocellular carcinoma by β-glycolipids is associated with alteration of lipid raft composition in splenocytes

The effect of IGL on reducing GMl content in HCC model was further analyzed by the inventors. Athymic Balb/c mice (n=8/group) were sublethally irradiated and transplanted with human Hep3B HCC, followed by daily intraperitoneal injections of PBS, GC, LC or IGL (1.5μg in lOOμl PBS, groups A, B, C and D, respectively) for 25 days. Animals were followed for serum α-fetoprotein (AFP) and for intrahepatic and

intrasplenic lymphocyte subpopulations. Lipid rafts in splenocytes were studied by FACS analysis for GMl and by dot blot analysis for GMl in raft fractions (1-4) following nycodenz gradient centrifugation of detergent- insoluble membrane complexes in 4-6 mice from each group. Administration of GC, LC and IGL resulted in reduced serum AFP, reflecting suppression of HCC, that was most prominent in GC-treated animals (34560 vs. 169600 ng/ml in groups B and A, respectively). The beneficial effect of β-glycolipids was associated with increased intrahepatic NKT lymphocytes (hepatic/splenic NKT lymphocyte ratio 0.38, 6.13, 1.94, and 3.41 in groups A, B, C and D, respectively, p<0.05). Membrane GMl content, as assessed by FACS analysis, was significantly decreased in NKl.1 positive cells in all β-glyeolipid treated groups (1.63% vs. 0.79%, 1.06% and 0.82% gated in groups A, B C and D ₅ respectively, p<0.05), and in CD4 and CD8 T cells in IGL-treated animals (5.2% vs. 2.6% for CD4 and 4.2% vs. 2.2% for CD8 in group A vs. group D, respectively, p≤0.05).

Dot blot analysis revealed a significant increase (1.55-2.03 fold, p<0.05) in raft fraction GMl content in the IGL-treated group, but not in the GC or LC-treated groups; in cytosolic fractions, quantitative analysis of LCK, a Src-family kinase, did not differ between the tested groups.

Alteration of lipid rafts in splenocytes by different β-glycolipids may mediate the immune-modulatory anti tumor effect associated with suppression of HCC by these compounds. Without being bound by any theory, the decreased membrane GMl content demonstrated herein may suggest that lipid raft disruption, which results in inhibition of T cell receptor activation-related lipid raft assembly, is the mechanism underlying this anti tumor effect.

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Example 8

Effect of GC on the membrane composition of cells obtained from

TNBS-colitis model mice

The effect of GC on membrane composition and structure of immune cells obtained from TNBS-colitis model mice was next investigated. TNBS colitis was induced in groups A and B as indicated in Experimental procedures. Group A served as control whereas group B was administered with 15μg GC/day, starting at the first day post-colitis induction up to day 10. All animal groups were sacrificed following isoflurane anesthesia at day 10 post-colitis induction.

As shown by Figure 17, confocal microscopy analysis of GC treated mice, revealed an altered binding of FITC-labeled CT-B, compared to control mice. CT-B-FITC binds in a patchy pattern with several focuses of bound CT-B- FITC. It should be noted that this pattern is less remarkable than in the ConA model (Figure 4), but still noticeable.

These cells were further analyzed by FACS. Non-statistical (only 2 mice from each group) results presented in Table 4 and Figure 18, show a significant GMl content increase in GC treated mice. As shown by the table, the increase is especially remarkable in the NKl.1 subset. In the CD4 and CD8 subsets, a small decrease in GMl content is seen.

Table 4

CT- CT- CT- CT-

B/CD3 B/CD8 B/CD4 B/NK #

26.03 17.65 15.28 4.61 Al-cont

24.89 22.86 22.43 4.19 A2-cont

MUM mmm mean

22.2 11.96 12.5 7.65 B1-GC 33.99 16.21 19.89 7.22 B2-GC

Example 9

Redistribution of intrahepatic NKT regulatory lymphocytes and increased intrahepatic T lymphocyte trapping: a possible mechanism for the beneficial effect of beta glycolipids in immune mediated collitis

To further investigate the mechanism of the immune-modulatory effect of

GC on the TNBS induced colitis mice model, the effect of different beta- glycolipids on intra hepatic NKT regulatory lymphocytes and lymphocyte trapping was further examined by the inventors.

Four groups of mice were studied. Immune mediated colitis was induced by intracolonic instillation of trinitrobenzenesulfonic-acid (TNBS) in all groups. Groups B-D were treated by daily administration of glucosylceramide (GC), lactosylceramide (LC), and a combination of both GC and LC (1:1 ratio), respectively. Mice in control group A received solvent alone. Mice were evaluated for macroscopic and microscopic colitis scores. The immune modulatory effect of beta-glycolipids was determined by FACS analysis of intrahepatic and intrasplenic lymphocytes for NKT, CD 4 and CD 8 markers, and by measurement of serum cytokine levels. Lipid rafts were investigated by detergent-insoluble splenic membrane fractions, FACS analysis of raft lipid ganglioside-GMl and fluorescent microscopy of lipid raft structure.

Administration of β-glycolipids resulted in an increased intrahepatic/peripheral NKT ratio (7, 15, 4 vs. 2, for groups B-D compared

with A, respectively, p<0.05), and in a decreased peripheral/intrahepatic CD4+/CD8+ ratio (0.3, 0.86, 0.4 vs. 0.87, for groups B-D compared with A, respectively). Beta -glycolipids led to a 43% increase in survival and significant alleviation of colitis with improvement in the macroscopic and microscopic scores (82, 56, 92% for groups B-D compared with A, respectively, p<0.05), and to a decrease in serum IFNγ levels and IFNγ/IL- 10 ratio in groups B-D compared with group A (83, 58, 184 vs. 437 pg/ml; 4, 4, 3 vs. 14; respectively, p<0.01).

These beneficial effects were associated with a significant increase in the raft lipid, GMl, by dot blot in detergent- soluble raft fractions (1.25, 2.51, and 4.55 fold for groups B-D, respectively, p<0.05). FACS analysis of GMl cell surface expression showed a 20% decrease in group D. Fluorescent microscopy demonstrated structural changes in lipid raft patching.

Administration of beta -glycolipids increased NKT regulatory lymphocyte redistribution, intrahepatic T lymphocyte trapping, and alleviated immune mediated colitis.

Example 10

Effect of β-glycolpids on lipid rafts and cell membranes

The significant effect of GC demonstrated in the TNBS colitis experimental model, encouraged the inventors to further broaden the examination to other glycolipids. Particularly, analyzing their effect on membrane rafts composition and structure as well as lymphocyte trapping. Therefore, twelve experimental and control groups, 12 mice per group, were studied (Table 5). Mice in experimental groups A-F were injected with TNBS. Group A mice were administered daily for 12 days with intra-peritoneal injection of lOOμl PBS. Mice in groups B, C, D, E,

and F, were administered with daily intra-peritoneal injections of β- glucosylceramide (GC), β-lactosylceramide (LC), β-galactosyloceramide (GLC), IGL, or ceramide, the 1:1 combination of β-glucosylcer amide and β- lactosylceramide, respectively (1 μg in 100 μl PBS). Naϊve mice in groups G-L were similarly injected with the different glycolipids without TNBS administration. Animals were sacrificed on day 12.

Dot blot analysis

The GMl content in the detergent-insoluble [Vratsanos (2001) ibid.] fractions and in the detergent- soluble (cytosolic) fractions was analyzed by dot-blot analysis (Figure 19). The most significant changes were noted in raft fractions 1 and 2; and in cytosolic fractions 11-12. In naϊve mice administration of β -Glycolipids was not associated with a significant change in GMl content. In TNBS treated mice only the administration of GC, LC, and IGL significantly increased both raft and cytosolic fractions.

Effect of β-glycolpids on lipid rafts and cell membranes: FACS analysis of GMl expression in lymphocytes subsets

To further determine the effect of distinct α and β-glycolipids on lipid rafts in NKT lymphocytes, FACS analysis was performed using CT-X-FITC for determination of GMl expression on cell surface. Freshly derived whole splenocytes from naϊve and from TNBS-treated animals were stained using CTX-HRP conjugate, as a measure of whole cell GMl content. Administration of TNBS led to a significant decrease of GMl expression in CD4, CD8 and NKT cells. Administration of GC and IGL was associated with a decrease in GMl expression on CD8 and NKT lymphocytes (Figure 20, p<0.005). No major effects were noted on CD4 cells, and for the other tested glycolipids. These effects were not observed in naϊve animals.

Effect of β-glycolpids on lipid rafts and cell membranes: Fluorescent microscopy with cholera toxin: Lipid rafts are highly dynamic, submicroscopic assemblies that float freely within the liquid disordered bilayer in cell membranes and can coalesce upon clustering of their components. By clustering small individual rafts together into larger visible units, efficient interaction of raft-associated proteins can be noted. The structure of raft domains in plasma membrane of splenocytes derived from untreated and glycolipid-treated mice were analyzed in order to determine whether administration of various glycolipids could affect lipid raft disruption on the splenic plasma membrane. Clustering patterns of GMl using cholera toxin conjugated to FITC (CT-X-FITC) were compared. Fluorescent microscopy was used to analyze the patching distribution of GMl/cholera toxin. Representative images presented in Figure 21. Administration of TNBS led to mild cholera toxin induced raft clustering. Mice treated with GC and IGL showed enlarged GMl patches on cell surface, suggesting an increase in the GMl content on splenocytes membrane. No major changes were noted in naϊve animals.

Effect of β-glycolipids on splenic and intrahepatic NKT lymphocyte distribution

Administration of β-glycolipids was assoicated with alteration of intrahepatic NKT lymphocte number. This effect was dependent on the immune microenvronment and most profound in experimental colitis harboring mice. Both GC and IGL admisnstration was associated with a significant increase in the number of intrahepatic NKT lymphoyctes (22 and 19 vs. 13% for groups B and E vs. A, respectively, p<0.005, Figure 22A). In cotrast, administration of LC, GLC and ceramide were associated with a decrease in intrahepatic NKT cell number (4, 7, and 9%, for groups C, D, and F, respectively). No significant effect was noted on the peripheral NKT cell number as evaluated by FACS analysis of

intrasplenic NKT lymphoyctes (Figure 22B). Calculation of the intrahepatic to intrasplenic NKT ratio, revelaed a singnificant increase in the ratio for mice treated with GC and IGL (12.22 and 10.00 vs. 5.00, for mice in groups B, E and A respectively, p<0.005, Figure 22C). β-glyolipids did not led to a significant change in the NKT ratio in naϊve animals.

Effect of β-glycolipids on intrasplenic to intrahepatic CD4 to CD8 lymphocyte ratio

To determine the role of the liver on CD 8+ T lymphocte trapping, FACS analysis for CD4+ and CD8+ T lymphocytes was perfromed for animals from all treated and control groups. For each group the CD4 to CD8 lymphocyte ratio was calculated. The ratio between each of the splenic to intrahepatic CD4/CD8 ratios was determined (Figure 23). Induction of experimental colitis was associated with a decrease in intrahepatic CD8 lymphocyte trapping (ratio of 0.55 vs. 0.61, for groups A vs G, respectivley, p<0.005). Administration of GC and IGL led to a significant increase in this ratio (0.95, and 0.83 vs. 0.55, for group B, E, and A, respectively, p<0.005, Figure 23), suggesting an increased intrahepatic CD8+ T lymphocyte trapping. A lesser effect was noted in mice treated with GLC, and cer amide.

Effect of β-glycolipids on serum cytokine levels

Treatment with β-glycolipids was associated with a profound alteration of the ThI to Th2 immune balance in colitis harboring animals. Serum IFNγ levels decreased significantly for animals treated with GC and IGL as compared with untreated controls (135 and 110 vs. 455 pg/ml, for groups B, E, and A, respectively, p<0.005, Figure 24A). To further assess the effect of GC and IGL on the Thl/Th2 immune paradigm, the IFNγ/ILlO ratio was calculated. Adminstration of GC and IGL led to a significant reduction in this ratio (5.0, 4.0 vs 13.3, for groups B, E, and A,

respectively, p<0.005, Figure 24B). This data suggests that the β- glycolipids alteration in NKT regulatory lymphoycte distribution and intrahepatic CD 8 lymphoycte trapping, is associated with a Th2 to ThI immune shift in the TNBS model.

Effect of β-glycolipids on microscopic score of colitis and on extent of disease

Administration of GC and IGL were associated with a significant improvement in the microscopic colitis score. The total microscopic score decrease from 5.6 to 3.9 and 3.5 for mice in groups A, B and E, respectively (p<0.005, Figure 25A). Similarly, the extent of bowel affected by the diseae was significantly reduced in animals treated with GC and IGL (0.85 and 0.75 vs. 1.9 for mice in groups B, E, and A, respectively, p<0.005, Figure 25B). Representative histological slides are shown in Figure 26, manifesting siginifcicant ameliortion of the inflammation and mucose destruction in treated versus untreated control mice.

Table 5: experimental groups

In summary, the results indicate that the effect of GC on different lymphocyte subsets may be variable.

The patches on the cell surface of cells obtained from the GC treated mice may indicate an alteration in the normal distribution of GMl, not necessarily an increase in the glycolipid marker. The control mice exhibit the familiar ring-like morphology. Similarly, the addition of exogenous ganglisides to cells can lead to their incorporation into rafts and as a result, also cause proteins to dissociate from rafts.

Example 11

Effect of GC on the membrane composition of NKT hybridoma cells

The effect of GC on NKT cells was further examined by using three different hybridoma cell lines, TCBlI, TBA, and DN. FACS analysis performed in DN hybridoma shown in Figure 27, clearly revealed increase in the general fluorescence of the GC treated cells. This enhanced fluorescence after treatment with GC is also shown by the confocal microscopy analysis in Figures 28 and 29.