Academic literature on the topic 'RHIgM22'

Background Recombinant human immunoglobulin M22 (rHIgM22) has promoted remyelination in animal models and was well tolerated in people with clinically stable multiple sclerosis. Objective Safety/tolerability of a single rHIgM22 dose was investigated following an acute relapse and to determine whether this enhanced CNS/CSF concentrations. Methods Adults (N = 27) with acute relapse were assigned to rHIgM22 (0.5 or 2.0 mg/kg) or placebo. Study included screening/steroid administration periods and 10 study visits over 6 months. rHIgM22 CSF concentrations were assessed on days 2 and 29. Pharmacokinetic and safety samples were taken for up to 60 days. Assessments included adverse events and other clinical measures. Brain magnetic resonance imaging was performed with/without gadolinium. Results rHIgM22 CSF levels were consistent with dose-dependent concentration on both days 2 and 29. Infusion was generally well tolerated during an acute relapse. Immunogenicity was mild. Most adverse events did not appear to be dose dependent, were mild/moderate, and were events often associated with multiple sclerosis. Conclusion Although limited by high variability and small sample size, the data suggest enhanced CNS uptake associated with a drop in CSF levels. This study demonstrated safety of an antibody directed to myelin and oligodendrocytes in the course of active demyelinating disease. Further research into rHIgM22 is warranted. ClinicalTrials.gov: NCT02398461 https://clinicaltrials.gov/ct2/show/study/NCT02398461?term=M22&draw=2&rank=8

Background Recombinant human immunoglobulin M22 (rHIgM22) has promoted remyelination in animal models and was well tolerated in people with clinically stable multiple sclerosis. Objective Safety/tolerability of a single rHIgM22 dose was investigated following an acute relapse and to determine whether this enhanced CNS/CSF concentrations. Methods Adults (N = 27) with acute relapse were assigned to rHIgM22 (0.5 or 2.0 mg/kg) or placebo. Study included screening/steroid administration periods and 10 study visits over 6 months. rHIgM22 CSF concentrations were assessed on days 2 and 29. Pharmacokinetic and safety samples were taken for up to 60 days. Assessments included adverse events and other clinical measures. Brain magnetic resonance imaging was performed with/without gadolinium. Results rHIgM22 CSF levels were consistent with dose-dependent concentration on both days 2 and 29. Infusion was generally well tolerated during an acute relapse. Immunogenicity was mild. Most adverse events did not appear to be dose dependent, were mild/moderate, and were events often associated with multiple sclerosis. Conclusion Although limited by high variability and small sample size, the data suggest enhanced CNS uptake associated with a drop in CSF levels. This study demonstrated safety of an antibody directed to myelin and oligodendrocytes in the course of active demyelinating disease. Further research into rHIgM22 is warranted. ClinicalTrials.gov: NCT02398461 https://clinicaltrials.gov/ct2/show/study/NCT02398461?term=M22&draw=2&rank=8

Objective The objective of this paper is to assess, in individuals with clinically stable multiple sclerosis (MS), the safety, tolerability, pharmacokinetics (PK) and exploratory pharmacodynamics of the monoclonal recombinant human antibody IgM22 (rHIgM22). Methods Seventy-two adults with stable MS were enrolled in a double-blind, randomized, placebo-controlled, single ascending-dose, Phase 1 trial examining rHIgM22 from 0.025 to 2.0 mg/kg. Assessments included MRI, MR spectroscopy, plasma PK, and changes in clinical status, laboratory values and adverse events for three months. The final cohort had additional clinical, ophthalmologic, CSF collection and exploratory biomarker evaluations. Participants were monitored for six months. Results rHIgM22 was well tolerated with no clinically significant safety signals. Noncompartmental PK modeling demonstrated linear dose-proportionality both of Cmax and AUC0–Last. The steady-state apparent volume of distribution of approximately 58 ml/kg suggested primarily vascular compartmentalization. CSF:plasma rHIgM22 concentration increased from 0.003% on Day 2 for both 1.0 and 2.0 mg/kg to 0.056% and 0.586% for 1.0 and 2.0 mg/kg, respectively, on Day 29. No statistically significant treatment-related changes were observed in exploratory pharmacodynamic outcome measures included for the 21 participants of the extension cohort. Conclusions Single doses of rHIgM22 were well tolerated and exhibited linear PK, and antibody was detected in the CSF.

Understory vegetation cover is an important indicator of forest health, and it can also be used as a proxy in the exploration of soil erosion dynamics. Therefore, quantifying the understory vegetation cover in hilly areas in southern China is crucial for facilitating the development of strategies to address local soil erosion. Nevertheless, a multi-source data synergy has not been fully revealed in the remote sensing data quantifying understory vegetation in this region; this issue can be attributed to an insufficient match between the point cloud 3D data obtained from active and passive remote sensing systems and the UAV orthophotos, culminating in an abundance of understory vegetation information not being represented in two dimensions. In this study, we proposed a method that combines the UAV orthophoto and airborne LiDAR data to detect the understory vegetation. Firstly, to enhance the characterization of understory vegetation, the point CNN model was used to decompose the three-dimensional structure of the pinus massoniana forest. Secondly, the point cloud was projected onto the UAV image using the point cloud back-projection algorithm. Finally, understory vegetation cover was estimated using a synthetic dataset. Canopy closure was divided into two categories: low and high canopy cover. Slopes were divided into three categories: gentle slopes, inclined slopes, and steep slopes. To clearly elucidate the influence of canopy closure and slope on the remote sensing estimation of understory vegetation coverage, the accuracy for each category was compared. The results show that the overall accuracy of the point CNN model to separate the three-dimensional structure of the pinus massoniana forest was 74%, which met the accuracy requirement of enhancing the understory vegetation. This method was able to obtain the understory vegetation cover more accurately at a low canopy closure level (Rlow2 = 0.778, RMSElow = 0.068) than at a high canopy closure level (RHigh2 = 0.682, RMSEHigh = 0.172). The method could also obtain high accuracy in version results with R2 values of 0.875, 0.807, and 0.704, as well as RMSE of 0.065, 0.106, and 0.149 for gentle slopes, inclined slopes, and steep slopes, respectively. The methods proposed in this study could provide technical support for UAV remote sensing surveys of understory vegetation in the southern hilly areas of China.

Abstract The presence of insulin-like growth factor 2 (IGF2) mRNA has been demonstrated in human and mouse spermatozoa. However, the expression of the IGF2 protein in spermatozoa and its function is not known. To accomplish this, we searched for IGF2 protein in spermatozoa of four healthy Caucasian men by Western blot (WB) and immunofluorescence (IM). Semen samples were collected by masturbation into a sterile container after 3-4 days of sexual abstinence. The spermatozoa were separated from leucocytes and epithelial cells by swim-up. The WB demonstrated the presence of the IGF2 protein in each sample examined. IM showed that IGF2 is a cytoplasmic protein with a variable degree of expression in each spermatozoon and localized in the equatorial and post-acrosomal segments. To evaluate the effects of IGF2, porcine Sertoli cells (SCs) were isolated from neonatal Large White pigs (7-15 days) using established methods. SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. At the end of incubation, the following endpoints were evaluated: a) expression of the GDNF, FGF2, and SCF genes, known to be mitogens that promote gonocyte proliferation and differentiation towards their spermatogonial fate, by real-time PCR (RT-PCR); b) gene (RT-PCR) and protein (WB and IM) expression of the follicle-stimulating hormone receptor (FSHR); and c) proliferation of SCs by flow cytometry. Subsequently, the effects of IGF2 were re-evaluated by pre-treating SCs with NVP-AEW541, a not competitive inhibitor of the insulin-like growth factor 1 receptor (IGF1R), known to be activated by IGF2. For this purpose, the NVP-AEW541 was added to the culture medium at a concentration of 1 µg/mL one hour before the IGF2 and was left for the entire duration of the incubation. The results of this study showed that IGF2 significantly downregulates GDNF gene expression in a concentration-dependent manner (-40.3% at 0.33 ng/mL, -48.2% at 3.33 ng/mL, -55.5%, respectively, p<0.01). Gene expression of FGF2 and SCF was significantly downregulated only by IGF2 at the highest concentration used (-23.6% and -39.1%, respectively, p<0.01). Similarly, IGF2 incubation downregulated FSHR mRNA (-27.1% at 0.33 ng/mL, -45.4.2% at 3.33 ng/mL, -21.1%, respectively, p<0.01) and protein (for both WB and IM) expression. Finally, SC proliferation significantly decreased after incubation with IGF2 (2.82±0.1% in untreated controls, -1.87±0.1% at 0.33 ng/mL, -2.19±0.2% at 3.33 ng/mL, and -2.36±0.1% at 10 ng/mL; p<0.01). In the presence of NVP-AEW541, all these effects were counteracted, thus suggesting that they are mediated by the IGF1R. In conclusion, these results suggest the presence of a paracrine regulatory mechanism between germ cells and SCs by which the former can influence the proliferation of SCs, their sensitivity to FSH, and their function by reducing the secretion of germ cell mitogens. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Multiple sclerosis (MS), considered the lead disease featuring demyelination, is the most common cause of non traumatic disability in young people. It is characterized by inflammation, progressive demyelination and gliosis, axonal injury and loss. The pathological hallmarks of all the subtypes of this disease are focal areas, called plaques, of demyelination in the CNS, with surrounding inflammation and neurodegeneration. Despite its high prevalence, multiple sclerosis remains a challenging ailment to study. Currently the most widely accepted hypothesis concerning MS pathogenesis is the autoimmune hypothesis: an autoimmune inflammation is proposed to be the cause of demyelination and auto-reactive leukocytes could be the disease initiators. One of the therapeutic approaches that is currently being developed to improve the regenerative outcome involves the use of CNS reactive antibodies to promote remyelination. One of these antibodies, rHIgM22, is able to bind to oligodendrocytes and myelin in vitro. Moreover, rHIgM22 is able to enter the CNS, accumulate at lesion site and promote remyelination in mouse models of chronical demyelination. As a matter of fact, this antibody has recently passed a phase I clinical trial for treatment of MS. Evidence suggests that the binding target of rHIgM22 could be associated with plasma membrane lipid rafts, and that lipid rafts might be involved in the signaling associated with the biological activity of this antibody. Moreover, it has been demonstrated that, isolated OPCs do not respond to rHIgM22 treatment, instead mixed glial cultures consisting of astrocytes, OPCs and microglial cells demonstrate observable rHIgM22-mediated OPC proliferation. The aim of this study was to analyze the plasma membrane lipid rafts composition in MGC in order to evaluate the effects exerted by rHIgM22 on these cells after single dose treatment of various duration. The analysis of the lipids and proteins distribution in MGC fractions, obtained after discontinuous sucrose gradient centrifugation of cells, and the alteration on lipids and protein composition of MGC due to the rHIgM22 treatment have been tested using TLC immunostaining assays and western blot analysis. The results obtained show that the DRM fraction obtained from MGC was enriched in sphingolipids, in particular sphingomyelin and gangliosides together with Lyn, Caveolin 1 and PrP(SAF32). On the contrary, phospholipids, in particular phosphatididylethanolamine and phosphatidic acid are enriched in the HD fraction, together with integrin αv and Akt. Furthermore we observed that rHIgM22 exerted an effect on the expression of P-Src(Y416) family and Lyn, that show a significant decrease, and PDGFRα that shows a significant increase. Moreover, the rHIgM22 treatment also induces a decrease in the activity of the aSMase. We hypothesize that the treatment with IgM22 could elicit biological responses mediated by alterations of lipid-dependent membrane organization which result in a reorganization of Lyn, integrin αvβ3 and PDGFRα at the cell surface to form a signaling complex. The formation of this complex triggers Lyn activation which in turn promotes oligodendrocyte precursor cells (OPCs) survival and proliferation and an inhibition of the pro apoptotic signaling. Moreover, the increased activation of Lyn could determine a decrease in ASMase activity and consequently in ceramide generation, thus inhibiting pro-apoptotic signaling and/or organization of sphingolipid-dependent signaling platforms. The identification of the binding targets of this antibody, able to promote remyelination in validated mouse models of MS, and the characterization of their membrane microenvironment could significantly contribute to the reveal the signaling mechanisms underlying the biological activity of rHIgM22.

Tutte le cellule eucariotiche sono circondate da una membrana cellulare che funge da barriera tra i compartimenti subcellulari e tra la cellula e il suo ambiente. Oltre alle proteine, le membrane cellulari sono composte da tre diversi gruppi di lipidi: glicerolipidi, sfingolipidi e steroli. I glicerolipidi sono i principali componenti delle membrane cellulari e sono prodotti utilizzando acido fosfatidico (PtdOH) come precursore centrale. Invece, gli sfingolipidi (SL), i componenti cellulari minori, hanno la sfingosina come componente base; l’aggiunta di oligosaccaridi alla sfingosina da origine agli glicosfingolipidi (GSL). SL e GSL non si distribuiscono in modo omogeneo nella membrana plasmatica esterna. Formano domini piccoli, eterogenei, altamente dinamici, arricchiti con steroli e sfingolipidi, chiamati "zattere lipidiche"; in questi microdomini semiordinati, gli SL sono coinvolti i diversi processi, quali quelli di adesione, riconoscimento e trasduzione del segnale. L'organo con il più alto arricchimento di lipidi, come colesterolo e glicosfingolipidi, è il cervello. La mielina, la sostanza isolante che circonda l'assone delle cellule nervose, è caratterizzata da un elevato rapporto lipidi-proteine, in cui i lipidi rappresentano l'80% del suo peso secco. Questa membrana contiene un alto livello di due galattosfingolipidi in particolare, il galattosilceramide (GalCer) e il 3-O-sulfogalattosilceramide (solfatide), che rappresentano rispettivamente circa il 20 e il 5% dei lipidi mielinici. I ruoli specifici di GalCer e solfatide sembrano essere legati alla loro capacità di formare e stabilizzare specifici domini laterali nella membrana mielinica, che regolano il corretto smistamento, traffico, co-clustering e distribuzione laterale delle principali proteine della mielina. Studi recenti hanno suggerito che alcuni lipidi specifici della mielina potrebbero essere i principali responsabili del meccanismo patogeno della sclerosi multipla (SM), la malattia demielinizzante più comune nel sistema nervoso centrale. Gli individui affetti da SM hanno un’alterata composizione lipidica della mielina e elevati livelli di anticorpi anti-solfatide, nei fluidi biologici, rispetto agli individui sani. D'altro canto, gli anticorpi anti-mielina potrebbero rappresentare un importante strumento immunologico per il trattamento di malattie neurologiche che coinvolgono lesioni mieliniche. In particolare, è stato dimostrato che gli anticorpi monoclonali umani che legano la mielina e gli oligodendrociti (OL), come rHIgM22, possono stimolare un meccanismo di rimielinizzazione nei modelli animali di demielinizzazione. Ad oggi, l'esatto meccanismo d'azione di rHIgM22 resta da chiarire, ma alcune prove suggeriscono che il meccanismo è correlato all'organizzazione delle zattere lipidiche presenti sulla superficie della mielina e degli OL. Lo scopo degli esperimenti descritti in questa tesi è quello di individuare i target molecolari dell'anticorpo e caratterizzare il microambiente di membrana in cui si trova, al fine di comprendere meglio l’ attività rimielinizzante di rHIgM22. Il legame di rHIgM22 ai lipidi purificati e agli estratti lipidici da varie fonti è stato testato usando saggi di TLC Immunostaining e saggi SPR. I risultati ottenuti mostrano che rHIgM22 si lega al solfatide e, in misura minore, al lisosolfatide in vitro, mentre non si lega ad altri sfingolipidi della mielina. L'affinità di legame sia per il solfatide che per il suo derivato deacilato è bassa, anche se il legame è specifico. In aggiunta, i nostri dati mostrano che l'affinità di legame di rHIgM22 per solfatide può essere modulata dalla presenza di altri lipidi, suggerendo un possibile ruolo del microambiente di membrana nel riconoscimento dell'antigene da parte di rHIgM22. Inoltre, rHIgM22 reagisce anche con acido fosfatidico, fosfatidilinositolo (PI) e fosfatidilserina (PS). Per verificare la capacità di rHIgM22 di legare sulfatide o altri lipidi, il legame di rHIgM22 è stato testato anche su estratti lipidici purificati da diversi campioni: cervelli di topo WT, ASM (- / -), CST (+/-) e CST (- / -), cellule gliali miste di topo (MGC), astrociti di topo, rHIgM22+ OLs di ratto, microglia di ratto e mielina di topo. Negli esperimenti di TLC Immunostaining per le fasi acquose di questi campioni, abbiamo osservato bande immunoreattive a rHIgM22 che co-migrano con lo standard di solfatide puro e, inaspettatamente, una seconda banda immunoreattiva a rHIgM22 co-migrante con lo standard fosfatidilinositolo e fosfatidilserina puro, confermando i dati di TLC Immunostaining sui lipidi purificati. L'identità delle specie fosfolipidiche riconosciute da rHIgM22 è stata confermata tramite spettrometria di massa; gli spettri mostrano che la specie molecolare del fosfatidilinositolo è 18:0/20:4, mentre per la fosfatidilserina è 18:0/22:6 e 18:0/18:1. Inoltre, l'analisi tramite spettrometria di massa per le frazioni arricchite in solfatide, mostra che rHIgM22 può legare diverse specie solfatate, suggerendo che questo legame non è specifico alle diverse specie degli acidi grassi. Tutti questi dati suggeriscono che non solo il solfatide, ma anche altri lipidi di membrana possono avere un ruolo nel legame di rHIgM22 agli OL o ad altri tipi di cellule. Inoltre, l'antigene riconosciuto da rHIgM22 potrebbe essere associato a zattere lipidiche di membrana plasmatica e incluso in un complesso multimolecolare. L'identificazione dei target riconosciuti da rHIgM22 e la caratterizzazione del microambiente di membrana in cui si trovano, potrebbe contribuire notevolmente a chiarire i meccanismi di segnalazione coinvolti nell'attività di promozione della rimielinizzazione di questo anticorpo, ma anche di quelli coinvolti nell'eziologia della SM, permettendo di identificare nuove potenziali strategie terapeutiche.
All eukaryotic cells are surrounded by a cellular membrane that functions as a barrier between subcellular compartments and between the cell and its environment. In addition to proteins, they are composed by three different set of lipids: glycerolipids, sphingolipids and sterols. Glycerolipids are the major components of cell membranes and they are produced using phosphatidic acid (PtdOH) as a central precursor. Instead, sphingolipids (SLs), the minor cell components, have sphingosine as basic building block; the additions of oligosaccharides to sphingosine giving rise glycosphingolipids (GSLs). SLs and GSLs are not distribute homogeneously in the outer plasma membrane. They form small, heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains, called “lipid rafts”; in these semiordered lipid microdomains, SLs are involved in cell adhesion/recognition processes and signal transduction pathway. The organ with the highest enrichment in lipids such as cholesterol e glycosphingolipids, is the brain. Myelin, the fatty white substance that surrounds the axon of nerve cells, is characterized by a high lipid-to-protein ratio, where lipids representing 80% of its dry weight. The myelin membrane contains a high level of two galactosphingolipids, galactosylceramide (GalCer) and 3-O-sulfogalactosylceramide (sulfatide), account for about 20 and 5 % of myelin lipids respectively. The specific roles of GalCer and sulfatide seem to be linked to their ability to form and to stabilize specific lateral domains in the membrane of myelin forming-cells and in the myelin sheath, that regulate the correct sorting, trafficking, co-clustering and lateral distribution of the major myelin proteins. Recent reports have suggested that some of the myelin-specific lipids may be key contributors to the pathogenic mechanism of multiple sclerosis (MS), the most common demyelinating disease in the CNS. Individuals with MS disease are reported to have different myelin lipid compositions and elevated levels of anti-sulfatide Ab in biological fluids compared with healthy individuals. On the other hand, anti-myelin antibodies might represent an important immunological tool for the treatment of neurological diseases involving myelin lesions. In particular, it has been shown that human monoclonal antibodies that bind myelin and oligodendrocytes (OLs), as rHIgM22, can initiate dramatic increase in remyelination in animal models of demyelination. Nowadays, the exact mechanism of action of rHIgM22 remains to be elucidated, but some evidence suggest that the mechanism is correlated with the organization of lipid rafts on the surface of myelin and OLs. The experiments described in this thesis were aimed at the individuation of the molecular target(s) of the antibody and at the characterization of its membrane microenvironment, in order to better understand the characteristics of rHIgM22 and its remyelinating activity. The binding of rHIgM22 to purified lipids and to lipid extracts from various sources were tested using TLC immunostaining assays and SPR assays. The results obtained show that rHIgM22 binds to sulfatide, and, to a lesser extent, to lysosulfatide in vitro, while it does not bind to other myelin sphingolipids. The binding affinity for both sulfatide and its deacylated derivate is low, even if the binding is specific. On the other hand, our data shows that the binding affinity of rHIgM22 for sulfatide can be modulated by the presence of other lipids suggesting a possible role of the membrane microenvironment in the recognition of the antigen by rHIgM22. In addition, rHIgM22 also reacts with phosphatidic acid, phosphatidylinositol (PI) and phosphatidylserine (PS). To verify whether rHIgM22 can bind sulfatide or other lipids, the binding of rHIgM22 was tested not only to purified lipids, but also to partially purified lipid extracts obtained from wild type, ASM (-/-), CST (+/-) and CST (-/-) mice brains, mouse mixed glial cells (MGC), mouse astrocytes, rat rHIgM22+ OLs, rat microglia, and mouse myelin. In TLC immunostaining experiments for aqueous phases, we observed rHIgM22-immunoreactive bands co-migrating with the pure sulfatide standard and, unexpectedly, a second rHIgM22-immunoreactive band migrating below sulfated, co-migrating with the pure phosphatidylinositol and phosphatidylserine standard, confirming the TLC immunostraining data to purified lipids. The identity of phospholipid species has been confirmed by ESI mass spectrometry experiments; they show that phosphatidylinositol was an 18:0/20:4-PI and phosphatidylserine was 18:0/22:6-PS and 18:0/18:1-PS. In addition, MS analysis for the fractions enriched in sulfatide, shows that rHIgM22 can bind different sulfated species suggesting that this binding is not fatty acid species-specific for the sulfatide. All these data suggest that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to OLs or to other cell types. Moreover, the antigen recognized by rHIgM22 could be associated with plasma membrane lipid rafts in these cells and this target could be including in a multimolecular complex. The identification of the binding target(s) of a rHIgM22, and the characterization of their membrane microenvironment, could greatly contribute to the elucidation of the signaling mechanisms underlying the remyelination promoting activity of this antibody and also of those involved in MS etiology, allowing to define new potential therapeutic strategies.

Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS). They synthesize large amounts of plasma membrane and extends multiple processes that individually wrap around axons generating a multilayered stack of membranes tightly attached at their cytosolic and external surfaces, i.e. myelin. The myelin membrane provides electric insulation of axons and dictates the clustering of the sodium channels at the nodes of Ranvier and the organization of the node itself, allowing saltatory nerve conduction. A number of neurological diseases of the CNS are characterized by destruction of oligodendrocytes with consequent damage or loss of the myelin sheath. In most experimental models, the normal response to this is remyelination, a process mediated by oligodendrocyte precursor cells (OPC) that ultimately leads to functional recovery. However in human diseases, and in specific in multiple sclerosis (MS), this process is inefficient and fails to successfully counteract the accumulation of lasting axonal damage and increasing brain atrophy, thus resulting in motor and neurological deficits. The development of strategies aimed to increase the efficiency of the remyelination process is therefore an important therapeutic goal. One of these strategies involves the use of CNS reactive antibodies to promote remyelination. One of these antibodies, rHIgM22, is able to bind to oligodendrocytes and myelin in vitro. Moreover, rHIgM22 is able to enter the CNS, accumulate at lesion site and promote remyelination in mouse models of chronical demyelination. As a matter of fact, this antibody has recently passed a phase I clinical trial for treatment of MS. rHIgM22 binds to CNS tissues with a pattern very similar to that of the anti-sulfatide antibody O4, and binding of rHIgM22 is abolished in CNS tissue slices from CST (-/-) mice, suggesting that rHIgM22 binding to myelin requires the presence of a product of cerebroside sulfotransferase, possibly sulfatide. However the exact identity of the antigen recognized by this antibody remains to be elucidated. The binding of rHIgM22 to purified lipids and to lipid extracts from various sources, including wild type, ASM (-/-), CST (+/-) and CST (-/-) mice brains, mouse mixed glial cells (MGC), mouse astrocytes, rat rHIgM22+ oligodendrocytes (OL), rat microglia, and mouse myelin, has been tested using TLC immunostaining assays and SPR experiments with lipid monolayers with different composition. The results obtained show that rHIgM22 binds to sulfatide, and, to a lesser extent, to lysosulfatide in vitro, while it does not bind to other myelin sphingolipids, including galactosylceramide and sphingomyelin, suggesting that sulfatide at the oligodendrocyte surface might be important for the binding of rHIgM22 to the surface of these cells and to myelin. The binding affinity for both sulfatide and its deacylated derivate is low, even if the binding is specific. On the other hand, our data shows that the binding affinity of rHIgM22 for sulfatide can be modulated by the presence of other lipids suggesting a possible role of the membrane microenvironment in the recognition of the antigen by rHIgM22. In addition, rHIgM22 also reacts with phosphatidic acid, and with an unknown molecule present in lipid extracts from various sources, including CST knock-out mice brains, MGC, and isolated astrocytes and microglia. The exact identity of this antigen has yet to be confirmed but preliminary data suggests it might be a form of phosphatidylethanolamine with a free amino group and multiple hydroxylation in the fatty acid residues. Remarkably, this antigen is also present in the extracts from mixed glial cultures, which do not contain mature O4-positive oligodendrocytes, and also in isolated astrocytes and microglia suggesting that other glial cells in addition to oligodendrocytes might be important in the response to rHIgM22. All this suggests that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to oligodendrocytes and other cell types. Moreover, binding of rHIgM22 to intact cells might require a complex molecular arrangement, and, in particular, sulfatide might be part of the functional rHIgM22 antigen localized at the cell surface.