Keratinocyte Growth Factor Gene Delivery via Mesenchymal Stem Cells Protects against Lipopolysaccharide-Induced Acute Lung Injury in Mice

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality, and have no specific therapy. Keratinocyte growth factor (KGF) is a critical factor for pulmonary epithelial repair and acts via the stimulation of epithelial cell proliferation. Mesenchymal stem cells (MSCs) have been proved as good therapeutic vectors. Thus, we hypothesized that MSC-based KGF gene therapy would have beneficial effects on lipopolysaccharide(LPS)-induced lung injury. After two hours of intratracheal LPS administration to induce lung injury, mice received saline, MSCs alone, empty vector-engineered MSCs (MSCs-vec) or KGF-engineered MSCs (MSCs-kgf) via the tail vein. The MSCs-kgf could be detected in the recipient lungs and the level of KGF expression significantly increased in the MSCs-kgf mice. The MSC-mediated administration of KGF not only improved pulmonary microvascular permeability but also mediated a down-regulation of proinflammatory responses (reducing IL-1β and TNF-α) and an up-regulation of anti-inflammatory responses (increasing cytokine IL-10). Furthermore, the total severity scores of lung injury were significantly reduced in the MSCs-kgf group compared with the other three groups. The underlying mechanism of the protective effect of KGF on ALI may be attributed to the promotion of type II lung epithelial cell proliferation and the enhancement of surfactant synthesis. These findings suggest that MSCs-based KGF gene therapy may be a promising strategy for ALI treatment.     

Porous Silk Scaffolds for Delivery of Growth Factors and Stem Cells to Enhance Bone Regeneration

Stem cell-based tissue engineering shows promise for bone regeneration and requires artificial microenvironments to enhance the survival, proliferation and differentiation of the seeded cells. Silk fibroin, as a natural protein polymer, has unique properties for tissue regeneration. The present study aimed to evaluate the influence of porous silk scaffolds on rat bone marrow stem cells (BMSCs) by lenti-GFP tracking both in vitro and in vivo in cranial bone defects. The number of cells seeded within silk scaffolds in rat cranial bone defects increased from 2 days to 2 weeks after implantation, followed by a decrease at eight weeks. Importantly, the implanted cells survived for 8 weeks in vivo and some of the cells might differentiate into endothelial cells and osteoblasts induced by the presence of VEGF and BMP-2 in the scaffolds to promote angiogenesis and osteogenesis. The results demonstrate that porous silk scaffolds provide a suitable niche to maintain long survival and function of the implanted cells for bone regeneration.     

Exposure to Factor VIII Protein in the Presence of Phosphatidylserine Induces Hypo-responsiveness toward Factor VIII Challenge in Hemophilia A Mice

Administration of recombinant factor VIII (FVIII), an important co-factor in blood clotting cascade, elicits unwanted anti-FVIII antibodies in hemophilia A (HA) patients. Previously, FVIII associated with phosphatidylserine (PS) showed significant reduction in the anti-FVIII antibody response in HA mice. The reduction in the immune response to FVIII-PS could be due either to a failure of the immune system to recognize the antigen (i.e. immunological ignorance) or to an active induction of an antigen-specific nonresponsiveness (i.e. immunological tolerance). If it were a result of tolerance, one would predict that pre-exposure to FVIII-PS would render the mice hypo-responsive to a subsequent FVIII challenge. Here, we have demonstrated that naive HA mice that were pretreated with FVIII-PS showed a significantly reduced FVIII immune response to further challenge with native FVIII and that this decreased responsiveness could be adoptively transferred to other mice. An increase in number of FoxP3-expressing CD4⁺ regulatory T-cells (Treg) was observed for the FVIII-PS-immunized group as compared with animals that received FVIII alone, suggesting the involvement of Treg in PS-mediated hypo-responsiveness. The PS-mediated reduction in antibody response was reversed by the co-administration of function-blocking anti-TGF-β antibody with FVIII-PS. The decreased response to FVIII induced by FVIII-PS was determined to be antigen-specific because the immune response to another non-cross-reactive antigen (ovalbumin) was not altered. These results are consistent with the notion that FVIII-PS is tolerogenic and suggest that immunization with this tolerogenic form of the protein could be a useful treatment option to minimize immunogenicity of FVIII and other protein-based therapeutics.     

小鼠颌下腺表皮生长因子的分离、纯化与鉴定

雄性成年小鼠颌下腺醋酸提取物经Bio-Gel p-10凝胶层析可得七个色谱峰,经放射受体结合及放射免疫分析后,证实第七峰的EGF比活性最高。收集该组分,再经Sephadex G-10层析及DEAE-52离子交换层析,可得纯化的EGF样品。SDS-PAGE证明样品的分子量为6 000。RRA,RIA结果表明,样品与标准mEGF具有相同的受体结合活性及抗原性。生物学研究证明样品可以促进新生鼠睁眼和萌牙,刺激成纤维细胞DNA的生物合成。     

Placental and Embryonic Growth Restriction in Mice With Reduced Function Epidermal Growth Factor Receptor Alleles

Embryos lacking an epidermal growth factor receptor (EGFR) exhibit strain-specific defects in placental development that can result in mid-gestational embryonic lethality. To determine the level of EGFR signaling required for normal placental development, we characterized congenic strains homozygous for the hypomorphic Egfr^wa2 allele or heterozygous for the antimorphic Egfr^Wa5 allele. Egfr^wa2 homozygous embryos and placentas exhibit strain-dependent growth restriction at 15.5 days post-coitus while Egfr^Wa5 heterozygous placentas are only slightly reduced in size with no effect on embryonic growth. Egfr^wa2 homozygous placentas have a reduced spongiotrophoblast layer in some strains, while spongiotrophoblasts and glycogen cells are almost completely absent in others. Our results demonstrate that more EGFR signaling occurs in Egfr^Wa5 heterozygotes than in Egfr^wa2 homozygotes and suggest that Egfr^wa2 homozygous embryos model EGFR-mediated intrauterine growth restriction in humans. We also consistently observed differences between strains in wild-type placenta and embryo size as well as in the cellular composition and expression of trophoblast cell subtype markers and propose that differential expression in the placenta of Glut3, a glucose transporter essential for normal embryonic growth, may contribute to strain-dependent differences in intrauterine growth restriction caused by reduced EGFR activity.EPIDERMAL growth factor receptor (EGFR) is the prototypical member of the ERBB family of receptor tyrosine kinases and is known to regulate many aspects of cellular biology including cell proliferation, survival, differentiation, and migration (reviewed in Yarden and Sliwkowski 2001). Eleven known ligands bind the extracellular region of ERBB-family receptors, and activation of the tyrosine kinase domain occurs following receptor homo- or heterodimerization. The resulting biological responses are dependent upon specific signaling cascades initiated by ERBBs and can be influenced by the particular ligand–ERBB combination (Yarden and Sliwkowski 2001). Studies using cultured cells have underscored the importance of EGFR in modulating various cellular processes, while animal models have been able to demonstrate that EGFR is required for numerous developmental and physiological processes (Casalini et al. 2004). In vivo studies have shown that EGFR is particularly important for normal placental development in mice; placentas from Egfr nullizygous (Egfr^{tm1Mag/tm1Mag}) embryos exhibit strain-specific defects that result in differential embryonic lethality (Sibilia and Wagner 1995; Threadgill et al. 1995). Two additional Egfr alleles result in reduced EGFR signaling in mice: the recessive hypomorphic Egfr^wa2 and dominant antimorphic Egfr^Wa5 alleles (Luetteke et al. 1994; Fowler et al. 1995; Du et al. 2004; Lee et al. 2004). These alleles can provide insight into the level of EGFR signaling required for normal placental development.Egfr^wa2 is a classical spontaneous mutation that arose in 1935 that causes a distinct wavy coat phenotype in the homozygote (Figure 1; Keeler 1935). This recessive mutation was subsequently found to be a single nucleotide transversion resulting in a valine → glycine substitution in the highly conserved kinase domain of EGFR (Luetteke et al. 1994; Fowler et al. 1995). Since mice homozygous for the Egfr^tm1Mag null allele die before or shortly after birth depending on genetic background, the hypomorphic Egfr^wa2 allele has been the primary model used to study the effect of attenuated EGFR signaling in a variety of adult physiological and disease states. In addition to eye and hair phenotypes, the adult Egfr^wa2 homozygous mouse exhibits delayed onset of puberty, abnormal ovulation, enlarged aortic valves and cardiac hypertrophy, decreased body size, defects in mammary gland development and lactation, increased susceptibility to colitis, and impaired intestinal adaptation following small bowel resection (Fowler et al. 1995; Helmrath et al. 1997; Chen et al. 2000; Egger et al. 2000; O''Brien et al. 2002; Prevot et al. 2005; Hsieh et al. 2007). Despite the widespread use of the Egfr^wa2 allele, there are limitations in using Egfr^wa2 homozygous mice to clearly define the physiological roles of EGFR. Egfr^wa2 has traditionally been maintained in cis, tightly linked with a hypomorphic Wnt3a allele, Wnt3a^vt (vestigal tail), making phenotypic analysis of reduced EGFR signaling by itself difficult. Furthermore, Egfr^wa2 has also typically been maintained on a mixed genetic background and since the Egfr nullizygous phenotype is similarly influenced by genetic modifiers, a mixed background could mask phenotypes that become evident when Egfr^wa2 mice are inbred.Open in a separate windowFigure 1.—Congenic 129 Egfr allelic series. Wild-type (left), Egfr^wa2 homozygote (middle), and Egfr^wa5 heterozygote (right) mice. As weanlings and adults, the Egfr^wa2 homozygotes and Egfr^wa5 heterozygotes are grossly indistinguishable.The Egfr^Wa5 allele arose in a large, genomewide N-ethyl-N-nitrosourea mutagenesis screen for dominant visible mutations in the mouse. Egfr^Wa5 heterozygous mice were first identified by their open eyelids at birth and by development of a wavy coat, similar to the phenotype of Egfr^wa2 homozygous mice (Figure 1). Egfr^Wa5 failed to complement the Egfr^tm1Mag null allele and was shown to function as an antimorph since Egfr^Wa5, but not Egfr^tm1Mag, heterozygotes exhibit eyelid and coat phenotypes (Lee et al. 2004). A single nucleotide missense mutation was found in the Egfr^Wa5 allele that results in an Asp → Gly substitution in the highly conserved DFG domain of the EGFR kinase catalytic loop (Du et al. 2004; Lee et al. 2004). Although Egfr^Wa5 heterozygotes are viable, Egfr^Wa5 homozygotes die prenatally and exhibit placental defects identical to those from Egfr^tm1Mag homozygous null embryos. Placentas from Egfr^Wa5 heterozygotes on a mixed background show variable reduction in the spongiotrophoblast layer and minor abnormalities in the labyrinth region, but no effects on embryo survival have been reported.In vitro studies with Egfr^Wa5 suggest that it encodes a kinase-dead EGFR since no phosphorylation of EGFR^Wa5 is detected following stimulation with ligands. In agreement with the genetic data showing that Egfr^Wa5 is an antimorph, in vitro studies have demonstrated that the EGFR^Wa5 receptor can inhibit phosphorylation of EGFR and MAPK in a dose-dependent manner (Lee et al. 2004). In Chinese hamster ovary cells expressing an equimolar ratio of EGFR and EGFR^Wa5 receptors, <10% of wild-type phosphorylation levels were observed by Western blot analysis.The Egfr allelic series available in the mouse has high utility for studying gene function since EGFR is involved in a multitude of developmental processes and human diseases. Although both Egfr^wa2 and Egfr^Wa5 alleles result in reduced EGFR signaling, the activity and phenotypic consequences of Egfr^wa2 homozygosity has not been compared to that of Egfr^Wa5 heterozygosity when both are on the same genetic backgrounds. Adult Egfr^Wa5 heterozygous mice appear highly similar to Egfr^wa2 homozygotes, but crosses with the Apc^Min intestinal tumor model have shown that a more substantial reduction in tumor number occurs when the Apc^Min mutation is bred onto the Egfr^wa2 homozygous vs. Egfr^Wa5 heterozygous background (Roberts et al. 2002; Lee et al. 2004). These results suggest that Egfr^Wa5 heterozygous mice retain higher levels of EGFR activity than Egfr^wa2 homozygous mice; however, the data are confounded by the fact that the crosses were performed using different mixed genetic backgrounds.This study reports a comprehensive genetic analysis of reduced EGFR signaling in Egfr^wa2 homozygotes and Egfr^Wa5 heterozygotes in placental development and embryonic growth for three congenic backgrounds, C57BL/6J (B6), 129S1/SvImJ (129), and BTBR/J-T+, tf/tf (BTBR). Wild-type placenta weight, embryo weight, and mRNA levels of genes selected for their trophoblast-specific expression were found to be highly strain dependent. Egfr^wa2 homozygous placentas are reduced in size in all three strains, and a proportion of 129-Egfr^wa2 homozygotes die before 15.5 days post-coitus (dpc). Egfr^wa2 homozygous embryos also display background-dependent intrauterine growth restriction (IUGR) in late gestation, which is most severe on 129 and BTBR backgrounds and models EGFR-associated IUGR in humans. Egfr^Wa5 heterozygous placentas exhibit a minor reduction in size on all three backgrounds with no impact on embryonic growth. These results suggest that reduced levels of EGFR signaling can interfere with normal placental development and that embryo development is affected only after placental size is sufficiently reduced. In addition, our data show that the level of EGFR signaling in Egfr^Wa5 heterozygous mice is higher than in Egfr^wa2 homozygotes and suggests that different Egfr allele combinations can be generated to “genetically titer” total EGFR activity in vivo.     

Generation and Characterization of Conditional Heparin-Binding EGF-Like Growth Factor Knockout Mice

Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c) decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e) decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II) signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.     

Effects of Ovariectomy and Prolactin on Mammary Gland Expressions of Transforming Growth Factor alpha and Epidermal Growth Factor Receptor mRNAs in Mice

Beginning 15 days after ovariectomy (OVX), a high mammary tumor strain of SHN virgin mice at 3 months of age received subcutaneous injections of danazol (0.5 mug / 0.1 ml olive oil, once a day), perphenazine (0.05 mg / 0.1 ml saline, twice a day) or ovine prolactin (oPRL: 0.25 mg / 0.05 ml buffer, twice a day) for 3 days to modulate their circulating PRL levels. The serum PRL level was significantly decreased by danazol and increased by perphenazine compared to the intact and OVX-control groups. The expression of both transforming growth factor alpha (TGFalpha) mRNA and epidermal growth factor receptor (EGFR) mRNA in the mammary gland was increased by danazol. However, TGFalpha mRNA expression was decreased by perphenazine. Meanwhile, mammary end-bud formation was inhibited in danazol-treated group. All findings suggest that the manifestation of the effect of TGFalpha on mammary gland is rather suppressed by PRL, while mammary gland growth needs the participation of PRL; in other words, PRL is dominant to TGFalpha on the mammary gland growth. OVX resulted in a significant decrease of TGFalpha mRNA expression in the mammary gland despite of little alteration in serum PRL, confirming the previous observations. The similar trend was observed in ICR mice; however, the response to hormonal modulation is generally less susceptible than SHN mice.     

Antibodies to Gangliosides and Myelin Autoantigens Are Produced in Mice Following Sciatic Nerve Injury

Abstract: Autoimmune response following peripheral nerve injury in mice was investigated in the present work. Antibodies specific to gangliosides and myelin proteinaceous constituents are produced in mice following sciatic nerve cut. Results are discussed with regard to the possible relevance of the immunological response to nerve regeneration.     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

Cell-Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice

Total polyribosomes were isolated from the brains of 16-20 day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shil/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 +/- 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and less than 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an "assembly" or "synthesis" defect.     

鼠神经生长因子对电烧伤患者神经修复的作用及其机制研究

目的:探讨鼠神经生长因子对电烧伤患者神经修复的作用及其机制。方法:选取2013年2月至2014年11月期间我院确诊治疗的四肢电烧伤患者128例,依据随机分配原则分为对照组和神经组,对照组患者给予常规皮瓣修复术治疗,神经组患者在此基础上给予鼠神经生长因子治疗,且依据给药方式分为全身亚组和局部亚组。统计分析所有患者创面愈合时间、感染和出血发生情况,采用BMRC感觉、运动功能评级法评估患者感觉、运动功能恢复情况,应用Spearman分析法分析二者之间的关系。结果:神经组患者创面愈合时间、感染和出血发生率明显低于对照组,差异有统计学意义(P0.05);局部亚组患者感觉功能优良率为90.63%,全身亚组为84.38%,对照组为71.88%,局部亚组全身亚组对照组,差异有统计学意义(P0.05);局部亚组患者运动功能优良率为93.75%,全身亚组为84.38%,对照组为76.56%,局部亚组全身亚组对照组,差异有统计学意义(P0.05);Spearman分析法结果显示,感觉功能与运动功能呈正相关(r=0.812,P0.05)。结论:鼠神经生长因子可有效提高电烧伤患者神经修复的作用,有利于改善患者术后创面愈合、感染、出血情况,促进患者感觉、运动功能恢复,且通过局部给药方式具有更为良好的神经修复作用,值得临床作进一步推广。     

Immunochemical Investigation of Thylakoid Coupling Factor Protein during Photosynthetic Acclimation to Irradiance

Davies, E. C, Jordan, B. R., Partis, M. D. and Chow, W. S. 1987.Immunochemical investigation of thylakoid coupling factor proteinduring photosynthetic acclimation to irradiance.—J. exp.Bot. 38: 1517—1527 A double antibody sandwich enzyme-linked immunosorbent assay(ELISA) has been used to determine the amount of coupling factorprotein (CF₁ present on thylakoid membranes isolated from lettuceleaves. Thylakoid CF₁ was purified from lettuce chloroplastsand used to raise antibodies. After purification of the IgGits specificity to CF₁ was examined by Ouchterlony double diffusion,Western blotting and control ELISA experiments. The specificityof the response for CF₁ was confirmed and the ELISA found togive a log-linear measurement of purified CF₁ concentrationswithin a range of 10 µg cm³ to 25 ng cm ³. The ELISA wasthen used to determine the level of CF₁ protein associated withthe thylakoid membrane. Comparable values were obtained forCF₁ levels, either assayed directly on the thylakoid or afterrelease from the membrane by chloroform treatment. Determinationof CF₁ protein content on thylakoids isolated from lettuce grownat different irradiances showed that the amount of CF₁ proteinper chlorophyll and chloroplast increased with increased lrradiance.In addition, the changes in CF₁ protein as determined by ELISAare reflected in changes that have been found in enzymic activityunder these same light environments. The implications for thephotoregulation of CF₁ are discussed.     

The Tissue Fibrinolytic System Contributes to the Induction of Macrophage Function and CCL3 during Bone Repair in Mice

Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg ^–/–), urokinase-type plasminogen activator (uPA ^–/–) or tissue-type plasminogen activator (tPA ^–/–) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA ^–/– mice until day 6, compared with wild-type (uPA ^+/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA ^–/– mice at the bone injury site on day 4, compared with those in uPA ^+/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA ^–/– and Plg ^–/–, but not in tPA ^–/– mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1β, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA ^–/– and Plg ^–/–, but not in tPA ^–/– mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA ^+/+, but not in uPA ^–/– mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.     

Demyelinating Antibodies to Myelin Oligodendrocyte Glycoprotein and Galactocerebroside Induce Degradation of Myelin Basic Protein in Isolated Human Myelin

Abstract: Although the specificity of multiple sclerosis (MS) brain immunoglobulins (lgs) remains unknown, the incubation of these lgs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS-specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin-associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti-MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody-induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8-18C5), the neutral protease involved in this antibody-induced degradation of MBP could be calcium-activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.