Presence Of Myelin Basic Protein Research Articles

A scaffold containing zinc oxide for Schwann cell-mediated axon growth

Objective. Schwann cells (SCs) transplanted in damaged nervous tissue promote axon growth, which may support the recovery of function lost after injury. However, SC transplant-mediated axon growth is often limited and lacks direction. Approach. We have developed a zinc oxide (ZnO) containing fibrous scaffold consisting of aligned fibers of polycaprolactone (PCL) with embedded ZnO nanoparticles as a biodegradable, bifunctional scaffold for promoting and guiding axon growth. This scaffold has bifunctional properties wherein zinc is released providing bioactivity and ZnO has well-known piezoelectric properties where piezoelectric materials generate electrical activity in response to minute deformations. In this study, SC growth, SC-mediated axon extension, and the presence of myelin basic protein (MBP), as an indicator of myelination, were evaluated on the scaffolds containing varying concentrations of ZnO in vitro. SCs and dorsal root ganglion (DRG) neurons were cultured, either alone or in co-culture, on the scaffolds. Main results. Findings demonstrated that scaffolds with 1 wt.% ZnO promoted the greatest SC growth and SC-mediated axon extension. The presence of brain-derived neurotrophic factor (BDNF) was also determined. BDNF increased in co-cultures for all scaffolds as compared to SCs or DRGs cultured alone on all scaffolds. For co-cultures, cells on scaffolds with low levels of ZnO (0.5 wt.% ZnO) had the highest amount of BDNF as compared to cells on higher ZnO-containing scaffolds (1 and 2 wt.%). MBP immunostaining was only detected in co-cultures on PCL control scaffolds (without ZnO). Significance. The results of this study demonstrate the potential of the ZnO-containing scaffolds for SC-mediated axon growth and its potential for use in nervous tissue repair.

Niche-dependent inhibition of neural stem cell proliferation and oligodendrogenesis is mediated by the presence of myelin basic protein.

Neural stem and progenitor cells (collectively termed neural precursor cells [NPCs]) are found along the ventricular neuraxis extending from the spinal cord to the forebrain in regionally distinct niches comprised of different cell types, architecture, and cell‐cell interactions. An understanding of the factors that regulate NPC behavior is critical for developing therapeutics to repair the injured central nervous system. Herein, we demonstrate that myelin basic protein (MBP), the major cytoplasmic protein constituent of the myelin sheath in oligodendrocytes, can regulate NPC behavior. Under physiological conditions, NPCs are not in contact with intracellular MBP; however, upon injury, MBP is released into the neural parenchyma. We reveal that MBP presented in a spinal cord niche is inhibitory to NPC proliferation. This inhibitory effect is regionally distinct as spinal cord NPCs, but not forebrain‐derived NPCs, are inhibited by MBP. We performed coculture and conditioned media experiments that reveal the stem cell niche is a key regulator of MBP's inhibitory actions on NPCs. The inhibition is mediated by a heat‐labile protein released by spinal cord niche cells, but not forebrain niche cells. However, forebrain NPCs are also inhibited by the spinal cord derived factor as revealed following in vivo infusion of the spinal cord niche‐derived conditioned media. Moreover, we show that MBP inhibits oligodendrogenesis from NPCs. Together, these findings highlight the role of MBP and the regionally distinct microenvironment in regulating NPC behavior which has important implications for stem cell‐based regenerative strategies.

Tissue Transglutaminase contributes to myelin phagocytosis in interleukin-4-treated human monocyte-derived macrophages

Macrophages exert either a detrimental or beneficial role in Multiple Sclerosis (MS) pathology, depending on their inflammatory environment. Tissue Transglutaminase (TG2), a calcium-dependent cross-linking enzyme, has been described as a novel marker for anti-inflammatory, interleukin-4 (IL-4) polarized macrophages (M(IL-4)), which represent a subpopulation of macrophages with phagocytic abilities. Since TG2 is expressed in macrophages in active human MS lesions, we questioned whether TG2 drives the differentiation of M(IL-4) into an anti-inflammatory phenotype and whether it plays a role in the phagocytosis of myelin by these cells. In macrophage-differentiated THP-1 monocytes, TG2 was increased upon IL-4 treatment. Reducing TG2 expression impairs the differentiation of M(IL-4) macrophages into an anti-inflammatory phenotype and drives them into a pro-inflammatory state. In addition, reduced TG2 expression resulted in increased presence of myelin basic protein in macrophages upon myelin exposure of M(IL-4) macrophages. Moreover, the elevated presence of an early endosome marker and equal expression of a lysosome marker compared to control macrophages, suggest that TG2 plays a role in phagosome maturation in M(IL-4) macrophages These data suggest that tuning macrophages into TG2 producing anti-inflammatory cells by IL-4 treatment may benefit effective myelin phagocytosis in e.g. demyelinating MS lesions and open avenues for successful regeneration.

Molecular Mechanism of Central Nervous System Myelinogenesis: In Vitro Self-Assembly of Myelin Membrane Lipid and Protein Structures

The myelin sheath is an insulating, compacted, multilamellar biological membrane that facilitates efficient propagation of action potentials down neuronal axons, and is critical for proper physiological function. Recently, EM imaging has provided compelling in vivo data that puts to question the long-established mechanism for the formation of central nervous system (CNS) myelin. The new data have led to the proposal of a new model involving (1) the preformation of myelin membrane tubules that are trafficked to the neuronal axon, where they (2) undergo a transition from tubular to lamellar form and thus form the final compact myelin sheath [Szuchet et al. J. Struct. Biol. 190, 56-72 (2015)]. To investigate this mechanism, we designed in vitro experiments to probe the interactions of myelin lipids as they (1) self-assemble into tubules and (2) transition into lamellar form. Using fluid-cell AFM, TEM, and DLS, we have investigated the self-assembly of lipidic tubules, their transition into the multilamellar structure of mature myelin, and how this process is modulated by lipid composition and the presence of myelin basic protein. Our data support a galactosylceramide (GalCer) concentration-dependent response in lipid morphology that drives a transition from stable tubules to nonspecific aggregates with decreasing GalCer concentration. Our in vitro findings at high GalCer concentrations align with the in vivo tubules observed in ovine embryonic oligodendrocytes, suggesting that these structures of major myelin lipids can be stable precursors for myelination.

Association and Risk of Canine distemper with Respect to Age, Sex and Breed of Dogs Suffering from Demyelinating Neuropathies

A total of 3934 canine patients were screened clinically and dogs showing neurological signs were further examined for presence of Myelin basic protein and CD specific immunoglobulin (IgG and IgM) both in plasma and cerebrospinal fluid. The overall incidence of DMN in dogs was 3.53%, that of CD was 2.39% and DMN due to other causes was 1.14%. Age wise incidence of DMN was higher in age group >9 years (32.37%). Among the CD origin DMN cases incidence was higher in age group of 0-1 years (32.98%) and lowest in 1-3 years (9.57%). Sex wise incidence of CD was higher in male (53.33%) than female (46.67%). Further analysis of risk factor for CD in dog suffering from DMN and its association with age, sex and breed revealed that the dogs aged between 0 -1 years was 4.051, 1.384, 1.292 and 1.292 times more at risk when compared with dogs aged > 9 years (0.247), 3-6 years (0.723), 6-9 years (0.774) and 1-3 years (0.774) of age groups respectively. Breed wise Mongrel was 3.00, 2.250, 1.875, 1.263, 1.105 and 1.500 times higher at risk when compared with Dalmatian, Doberman, German shepherd, Labrador, Pomeranian and Spitz breed respectively. Dogs were 2.288 times more prone to CD than bitches.

Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer**Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central Universities of China (Grant No. GK201604004), and the National

Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure–area (π–A) and pressure–time (π–T) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value.

Axonal Loss and Neurofilament Phosphorylation Changes Accompany Lesion Development and Clinical Progression in Multiple Sclerosis

Neuroaxonal damage and loss are increasingly recognized as disability determining features in multiple sclerosis (MS) pathology. However, little is known about the long-term sequelae of inflammatory demyelination on neurons and axons. Spinal cord tissue of 31 MS patients was compared to three amyotrophic lateral sclerosis (ALS) and 10 control subjects. MS lesions were staged according to the density of KiM-1P positive macrophages and microglia and the presence of myelin basic protein (MBP) positive phagocytes. T cells were quantified in the parenchyma and meninges. Neuroaxonal changes were studied by immunoreactivity (IR) for amyloid precursor protein (APP) and variably phosphorylated neurofilaments (SMI312, SMI31, SMI32). Little T cell infiltration was still evident in chronic inactive lesions. The loss of SMI32 IR in ventral horn neurons correlated with MS lesion development and disease progression. Similarly, axonal loss in white matter (WM) lesions correlated with disease duration. A selective reduction of axonal phosphorylated neurofilaments (SMI31) was observed in WM lesions. In ALS, the loss of neuronal SMI32 IR was even more pronounced, whereas the relative axonal reduction resembled that found in MS. Progressive neuroaxonal neurofilament alterations in the context of chronic inflammatory demyelination may reflect changes in neuroaxonal metabolism and result in chronic neuroaxonal dysfunction as a putative substrate of clinical progression.

Lymphocyte transformation in the presence of myelin basic protein in multiple sclerosis and control subjects.

The transformation of lymphocytes in vitro in the presence of human myelin basic protein has been investigated in normal people, patients with multiple sclerosis (MS) and controls with other neurological diseases. There was little or no response at low concentrations (1--10 microgram/ml) but significant transformation at higher concentrations (100--1000 microgram/ml) in all three groups. There was no significant difference among the groups as a whole, but those MS patients who had had disease for more than 10 years did show greater responses than normal subjects (P less than 0.05). Increased responses could not be correlated with any other aspect of disease activity: in particular they were not increased in patients with acute relapses. The use of autologous serum instead of homologous AB Rhesus positive serum did not significantly alter lymphocyte responsiveness. The absence of any response in the presence of purified calf thymus histone suggests that the response to myelin basic protein indicates a low level of lymphocyte sensitization to this antigen even in normal subjects. The present evidence does not support a primary pathogenetic role for such a reaction in MS. The increased response in patients with a long duration of disease might merely be an effect of white matter damage or might represent an amplification of the normal immune response contributing to myelin breakdown and leading to the emergence of the progressive stage of the disease. The study of lymphocyte responsiveness over a wide range of concentrations of myelin basic protein is considered to resolve some of the controversy surrounding this subject in the literature.

The effect of myelin basic protein on the protease inhibitors alpha 1 antitrypsin and alpha 2 macroglobulin.

The effect of myelin basic protein (MBP) of central nervous tissue on the protease inhibitors, alpha 1 antitrypsin (a1AT) and alpha 2 macroglobulin (a2M) was studied in vitro. For this purpose, 2 characteristics of the protease inhibitors were used, viz. their pattern after isoelectric focusing and their trypsin-binding capacity. Both features of purified a2AT disappeared in the presence of MBP. The tests with a2M revealed that the formation and stability of the complex between a2M and protease were reduced. The results of this study suggest that MBP causes an increase in proteolytic activity by inactivating protease inhibitors. The potential relevance of these results for demyelinating processes in man and animal is discussed.

Myelin basic protein is temporospatially expressed in developing rat molars

Tooth development occurs through a complex and intricate set of gene-expression cascades. Although its early events have been examined extensively, there are fewer reports on the late events, such as dental hard-tissue formation. This study searched for genes involved in the late stages of tooth development. Differential display-polymerase chain reaction revealed myelin basic protein (MBP) mRNA to be expressed differentially in the second molar root stage germs compared with the third molar cap/early bell stage germs. The MBP expressed during hard tissue formation was confirmed to be a 21.5 kDa molecule by Western blotting. Immunoreactivity of MBP in the third molar (cap/early bell stage) germs was barely detectable in the dental papilla and inner enamel epithelia, whereas strong reactivity was noted in the differentiating and differentiated ameloblasts and odontoblasts in a temporospatial pattern. However, after complete formation of the full-thickness enamel, no reactivity was observed in the maturation-stage and protection stage ameloblasts. Myelin basic protein immunoreactive nerve fibers were also observed near the developing molar germs. This is the first report showing the presence of MBP in dental hard tissue cells, and its functional implications should be studied further.

Immunohistochemical Evidence of Inducible Nitric Oxide Synthase and Nitrotyrosine in a Case of Clinically Isolated Optic Neuritis

Optic neuritis (ON) is a demyelinating inflammation of the optic nerve that may occur as an isolated disease or related to multiple sclerosis (MS). There is little evidence of whether the immunohistochemistry of ON resembles that of typical cerebral MS lesions. Pathologic optic nerves were obtained from a patient who died of causes unrelated to ON after clinical recovery from clinically isolated ON. Normal control optic nerves were obtained from an eye bank. Normal and pathologic tissues were probed with antibodies to pathologic proteins including myelin basic protein (MBP) fragment, the inducible form of nitric oxide synthase (iNOS), macrophage markers CD14 and CD64, nitrotyrosine, and cyclooxygenase (COX-2). We also examined MBP, the oligodendrocyte marker cyclic nucleotide phosphodiesterase (CNPase), and glial fibrillary acidic protein. In the affected pathologic nerve, iNOS-positive macrophages/microglia, iNOS-positive astrocytes, COX-2, and nitrotyrosine were observed. iNOS and COX-2 were occasionally observed in the unaffected nerve. Decreased expression of MBP and CNPase was seen in the pathologic optic nerves, along with evidence of gliosis and ongoing myelin degradation indicated by the presence of MBP fragment. The immunohistochemistry of clinically isolated optic neuritis, as judged by this single case, resembles that of cerebral lesions of MS in showing abnormally high levels of iNOS and nitrotyrosine as well as other mediators of immune damage.

SLAM-associated Protein as a Potential Negative Regulator in Trk Signaling

Neurotrophin signaling plays important roles in regulating the survival, differentiation, and maintenance of neurons in the nervous system. Binding of neurotrophins to their cognate receptors Trks induces transactivation and phosphorylation of the receptor at several tyrosine residues. These phosphorylated tyrosine residues then serve as crucial docking sites for adaptor proteins containing a Src homology 2 or phosphotyrosine binding domain, which upon association with the receptor initiates multiple signaling events to mediate the action of neurotrophins. Here we report the identification of a Src homology 2 domain-containing molecule, SLAM-associated protein (SAP), as an interacting protein of TrkB in a yeast two-hybrid screen. SAP was initially identified as an adaptor molecule in SLAM family receptor signaling for regulating interferon-gamma secretion. In the current study, we found that SAP interacted with TrkA, TrkB, and TrkC receptors in vitro and in vivo. Binding of SAP required Trk receptor activation and phosphorylation at the tyrosine 674 residue, which is located in the activation loop of the kinase domain. Overexpression of SAP with Trk attenuated tyrosine phosphorylation of the receptors and reduced the binding of SH2B and Shc to TrkB. Moreover, overexpression of SAP in PC12 cells suppressed the nerve growth factor-dependent activation of extracellular signal-regulated kinases 1/2 and phospholipase Cgamma, in addition to inhibiting neurite outgrowth. In summary, our findings demonstrated that SAP may serve as a negative regulator of Trk receptor activation and downstream signaling.

Antibodies to myelin basic protein, myelin oligodendrocytes peptides, alpha-beta-crystallin, lymphocyte activation and cytokine production in patients with multiple sclerosis.

To measure neurone-specific humoral and cellular immune parameters in MRI-positive patients with multiple sclerosis (MS). It has been postulated from animal models for MS and in situ evidence in MS patients that antibodies, activated T cells and proinflammatory cytokines are involved in the destruction of myelin sheaths and loss of oligodendrocytes in active areas. Blood samples were obtained from 20 healthy control subjects and 20 patients with abnormal MRI and clinical diagnosis of MS. Sera were tested for levels of IgG, IgM and IgA against myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) peptides, and a small heat-shock protein, alpha-beta-crystallin. Lymphocytes were isolated and cultured in the presence or absence of MBP, MOG peptides and alpha-beta-crystallin, measured for stimulated T cells, cytokine production and compared with controls. Patients with MS showed the highest levels of IgG, IgM or IgA antibodies against one or all three tested antigens. Moreover, in the presence of MBP, MOG peptides or alpha-beta-crystallin, a significant percent- age of lymphocytes from MS patients underwent blast transformation, which resulted in high levels of interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and tumour necrosis factor beta (TNF-beta) production. Sensitivity of these assays was 60-80% and specificity, 65-70%. Detection of antibodies against MBP, MOG peptides, alpha-beta-crystallin, lymphocyte stimulation and production of proinflammatory cytokines in response to these antigens could be used as surrogate markers for the confirmation of MS diagnosis. A combination of antibodies, lymphocyte activation or cytokine production with abnormal MRI may significantly increase the sensitivity and specificity of MS diagnosis.

Changes in the anisotropy of oriented membrane dynamics induced by myelin basic protein

We report recent results showing the evidence of the effect induced by physiological amounts of myelin basic protein (MBP) on the dynamics of dimyristoyl L-a-phosphatidic acid (DMPA) membranes. Incoherent elastic neutron scattering scans, performed over a wide temperature range, have shown that the anisotropy of motions in oriented membranes is significantly enhanced by the presence of MBP.

The mechanism of action of anthrax toxin lethal factor

The mechanism of action of anthrax toxin lethal factor

The zinc environment in Langmuir-Blodgett phospholipid multi-layers.

The Zinc environment of a system formed by Langmuir-Blodgett phospholipid multi-layers is investigated by employing X-ray absorption spectroscopy. A comparative analysis of the Extended X-ray Absorption Fine Structure and Near Edge regions of the X-ray absorption spectra at the Zinc K-edge, in presence and in absence of the Myelin Basic Protein, clearly indicates that Zinc ions are bound to the heads of the phospholipidic molecules, while the presence of Myelin Basic Protein induces a visible distorsion of the geometry of the Zinc environment. These findings represent a first important step in understanding the interplay among the lipids of the myelin sheath, Myelin Basic Protein and Zinc, as Langmuir-Blodgett phospholipid multi-layers represent a valuable model for the multilamellar structure of the membrane surrounding the nerve axon.

Microglia induce myelin basic protein-specific T cell anergy or T cell activation, according to their state of activation.

Microglial cells are non-professional antigen-presenting cells (APC) the function of which is still controversial. Here, we studied the function of microglia derived from H-2(u) mice. We show that these microglia express a low level of B7.2 and CD40 and, interestingly, lack surface expression of B7.1. Resting and IFN-gamma-activated microglia were unable to activate naive and primed myelin basic protein (MBP)-specific CD4(+) T cells in the presence of MBP and encephalomyelitic MBP Ac1-11 peptide. Furthermore, in the presence of Ac1-11 peptide, CD4(+) TCR-transgenic T cells became anergized. Microglia became professional APC only after a multistep activation process involving both stimulation through cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-gamma] and cognate signaling (B7-CD28 and CD40-CD40 ligand interactions). As such they were able to present MBP to both unprimed and primed T cells. Co-culture of microglia with GM-CSF up-regulated co-stimulatory molecules, in particular B7.1. Additional activation with IFN-gamma induced MHC class II and CD40 up-regulation. CD40-CD40 ligand interaction significantly enhanced microglial ability to prime TCR-transgenic T cells and was essential for presentation of MBP to in vivo primed non-transgenic T cells. We propose that microglia may serve different functions under different inflammatory conditions, depending on the cytokine milieu and the type of cognate interaction they are involved in.

Reduced Levels of a Specific Myelin-Associated Oligodendrocytic Basic Protein Isoform in shiverer Myelin

Myelin-associated oligodendrocytic basic protein (MOBP) and myelin basic protein (MBP) share many structural similarities. MOBP is synthesised by mature oligodendrocytes and localised at the major dense line (MDL), suggesting a role in the myelin compaction process. The shiverer mouse, a deletion mutant of the myelin basic protein (Mbp) gene, has poorly compacted myelin with essentially no MDL. In this study we compare the developmental expression of the Mobp gene in wild-type and shiverer mice. The significant finding is that one of the two abundant MOBP isoforms, the ∼20-kD species, is poorly incorporated into shiverer myelin. The absence is specific to shiverer and is not a feature of dysmyelinating mutants with an abnormal intraperiod line. Our data suggest that incorporation of this MOBP isoform into shiverer myelin may be influenced by the presence of MBP or be a consequence of a disrupted MDL.

Force Measurements on Myelin Basic Protein Adsorbed to Mica and Lipid Bilayer Surfaces Done with the Atomic Force Microscope

The mechanical and adhesion properties of myelin basic protein (MBP) are important for its function, namely the compaction of the myelin sheath. To get more information about these properties we used atomic force microscopy to study tip-sample interaction of mica and mixed dioleoylphosphatidylserine (DOPS) (20%)/egg phosphatidylcholine (EPC) (80%) lipid bilayer surfaces in the absence and presence of bovine MBP. On mica or DOPS/EPC bilayers a short-range repulsive force (decay length 1.0–1.3 nm) was observed during the approach. The presence of MBP always led to an attractive force between tip and sample. When retracting the tip again, force curves on mica and on lipid layers were different. While attached to the mica surface, the MBP molecules exhibited elastic stretching behavior that agreed with the worm-like chain model, yielding a persistence length of 0.5 ± 0.25 nm and an average contour length of 53 ± 19 nm. MBP attached to a lipid bilayer did not show elastic stretching behavior. This shows that the protein adopts a different conformation when in contact with lipids. The lipid bilayer is strongly modified by MBP attachment, indicating formation of MBP-lipid complexes and possibly disruption of the original bilayer structure.

IL-4 is a differentiation factor for transforming growth factor-beta secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis.

We have previously shown that following oral administration of myelin basic protein (MBP), regulatory T cells are generated from gut-associated lymphoid tissue and that these cells suppress experimental allergic encephalomyelitis (EAE). These regulatory T cells produce transforming growth factor-beta (TGF-beta) with various amounts of IL-4 and IL-10 and these TGF-beta-secreting T cells have been termed Th3 cells. T cells in lymphoid organs drained by mucosal sites secrete IL-4 as a primary T cell growth factor. In the present study, we examined the role of IL-4 on oral tolerance and in the generation of TGF-beta secreting cells. Treatment of (PLJ x SJL)F1 mice with intraperitoneal (i. p.) IL-4 and low-dose oral MBP (0.5 mg) given three times reduced the severity of EAE, whereas i.p. injection of IL-4 alone or oral MBP alone given in these suboptimal doses, showed no protection. Spleen cells from protected mice produced increased amounts of TGF-beta and reduced IFN-gamma upon stimulation with MBP in vitro. Mucosal MBP-specific IgA production was significantly increased in IL-4 plus MBP fed animals. Moreover, oral administration of IL-4 (1 microg per feeding) also enhanced the suppression of EAE by oral MBP and this protective effect was reversed by administration of anti-TGF-beta antibody in vivo. Reverse transcription-PCR showed enhanced suppression of IFN-gamma in Peyer's patch in animals fed MBP and IL-4 versus those fed MBP alone. We then investigated the role of IL-4 in the generation of TGF-beta-secreting cells using MBP Ac1-11 TCR transgenic animals. Cells were cultured with IL-2, IL-4, or IFN-gamma in the presence of MBP and limiting dilution analysis for cytokine-secreting cells performed. We found that IL-4, but not IL-2 or IFN-gamma, generated TGF-beta-secreting T cells from naive splenic T cells and that these cells provided help for IgA production. These findings demonstrate that IL-4 is a differentiation factor for TGF-beta-secreting Th3 cells and oral IL-4 has a synergistic effect on low-dose oral tolerance that is associated with increased TGF-beta secretion.