Upregulation of thrombospondin-1(TSP-1) and its binding partners, CD36 and CD47, in sporadic inclusion body myositis

The TSP1/CD36/CD47-complex is involved in T cell expansion and inflammatory responses to beta-amyloid, both relevant to IBM. We report on the mRNA and protein expression of TSP1/ CD36 /CD47-complex in IBM muscles and in human myoblasts after cytokine stimulation. The TSP1/CD36 /CD47 was upregulated in IBM. TSP1 immunolocalized to the connective tissue contiguous to inflammation and CD36/CD47 on the myofibers and CD8+ cells. Further, TNF-alpha upregulated the production of TSP1 and CD47 by myoblasts. The TSP-complex is another inflammatory mediator associated with chronic inflammation in IBM that may perpetuate the immune responses to local antigens in response to TNF-alpha.     

Expression of MUPP1 protein in mouse brain

Localizing cell surface receptors to specific subcellular sites can be crucial for proper functioning. PDZ proteins apparently play central roles in such protein localizations. 5-HT_2C receptors have previously been shown to interact with MUPP1, a multi PDZ domain protein, in heterologous systems and in rat choroid plexus. We now report the generation and characterization of two independent MUPP1 antisera, which recognise distinct areas of the mouse brain in agreement with previous in-situ hybridization studies. Our results indicate that MUPP1 immunoreactivity co-localizes with 5-HT_2A or 5-HT_2C receptor expression in all regions of the mouse brain, including the choroid plexus where 5-HT_2C receptors are highly enriched.     

Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: modulation by FGF-2

CD137 (ILA, 4-1BB), a member of the tumor necrosis factor receptor family, and its ligand CD137-L were assayed by RT-PCR and immunocytochemistry in cultured human brain cells. Results demonstrated that both neurons and astrocytes expressed specific RNA for CD137 and its protein, which was found both on the plasma membrane and in the cytoplasm. Surprisingly, microglia, which also expressed CD137 mRNA, showed negative immunostaining. CD137-L-specific RNA was detected only in astrocytes and neurons. When brain cells were treated with fibroblast growth factor-2 (FGF-2), upregulation of CD137 but not of its ligand was observed in neurons and astrocytes. Protein localization was also affected. In microglia, an inhibition of RNA expression was induced by treatment, whereas CD137-L remained negative. Our data are the first demonstration that human brain cells express a protein found thus far in activated immunocompetent cells and epithelia. Moreover, they suggest not only that CD137 and CD137-L might play a role in interaction among human brain cells, but also that FGF-2 might have an immunoregulatory function in brain, modulating interaction of the central nervous system with peripheral immunocompetent cells.     

P16蛋白在人脑髓母细胞瘤中的表达及意义

目的：研究Ｐ１６蛋白与人脑髓母细胞瘤发病的关系。方法：应用ＳＰ免疫组化技术检测４５例术中切除的肿瘤标本的石蜡切片。结果：Ｐ１６蛋白在４５例髓母细胞瘤中的表达缺失率为８０％。而在１０例正常脑组织中的表达率为１００％。结论：Ｐ１６蛋白的缺失与髓母细胞瘤的发生有密切关系，其中的具体机制有待于在今后的研究中阐明。     

Expression and distribution of trophoblast glycoprotein in the mouse retina

We recently identified the leucine-rich repeat (LRR) adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCα-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal LRR domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled RBC dendrites in the outer plexiform layer and axon terminals in the IPL, as well as a putative amacrine cell with their cell bodies in the inner nuclear layer (INL) and a dense layer in the middle of the inner plexiform layer (IPL). In live transfected HEK293 cells, TPBG was localized to the plasma membrane with the N-terminal LRR domain facing the extracellular space. TPBG immunofluorescence in RBCs was strongly altered by the loss of TRPM1 in the adult retina, with significantly less dendritic and axon terminal labeling in TRPM1 knockout compared to wild type, despite no change in total TPBG detected by immunoblotting. During retinal development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, LRR proteins have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.     

Identification and functional characterization of mouse TPO1 as a myelin membrane protein

TPO1 is a member of the AIGP family, a unique group of proteins that contains 11 putative transmembrane domains. Expression of the rat TPO1 gene is upregulated in cultured oligodendrocytes (OLs) during development from pro-oligodendroblasts to postmitotic OLs. However, the distribution of native TPO1 protein in cultured OLs and in the brain has not been elucidated. We investigated the distribution and cellular function of TPO1 in myelinating cells of the nervous system. In mice, TPO1 gene expression was detected in the central (CNS) and peripheral (PNS) nervous systems and was markedly upregulated at postnatal days 10-20, an early phase of myelination in the mouse brain. To investigate TPO1 localization, we generated affinity-purified antibodies to synthetic peptides derived from mouse TPO1. Immunohistochemical analysis showed that TPO1 was expressed in OLs and Schwann cells but not in neurons and astrocytes. Schwann cells from trembler mice, which lack PNS myelin, had significantly decreased TPO1 expression and an altered localization pattern, suggesting that TPO1 is a functional myelin membrane protein. In OL lineage cell cultures, TPO1 was detected in A2B5+ bipolar early progenitors, A2B5+ multipolar Pro-OLs, GalC+ immature OLs and MBP+ mature OLs. The subcellular localization of TPO1 in OL lineage cells was mapped to the GM130+ Golgi in cell bodies and Fyn+ cell processes and myelin-like sheets. Furthermore, TPO1 selectively colocalized with non-phosphorylated Fyn and promoted Fyn autophosphorylation in COS7 cells, suggesting that TPO1 may play a role in myelin formation via Fyn kinase activation in the PNS and CNS.     

Expression of the EGF-TM7 receptor CD97 and its ligand CD55 (DAF) in multiple sclerosis

CD97 is a recently identified seven-span transmembrane (7-TM) protein that is expressed by leukocytes early after activation. CD97 binds to its cellular ligand CD55 (decay accelerating factor), which protects several cell types from complement-mediated damage. The functional consequences of CD97-CD55 binding are largely unknown, but previous data imply that CD97-CD55 interactions play a role in cellular activation, migration, and adhesion under inflammatory conditions.Here we examined the expression of CD97 and CD55 by immunohistochemistry in multiple sclerosis (MS). On the basis of established criteria for inflammation and demyelination, different lesion stages were distinguished in MS post-mortem brain tissue. In normal white matter, CD97 expression was not found, but CD55 was expressed with weak staining intensity on endothelial cells. In pre-active lesions, defined by abnormalities of the white matter, many infiltrating T cells, macrophages (MPhi) and microglia expressed CD97. CD55 was highly expressed by endothelial cells. In active lesions with myelin degradation, MPhi and microglia expressed both CD55 and CD97. Furthermore, a sandwich ELISA showed significantly (p<0.05) elevated levels of soluble CD97 in serum but not in cerebrospinal fluid of MS patients (37%) compared to healthy controls (8%).Collectively, these data suggest that CD97-CD55 interactions are involved in the inflammatory processes in MS. CD55, which is expressed in lesions by vessels to protect against complement-mediated damage, might bind to CD97 on infiltrating leukocytes. This interaction may facilitate cell activation and migration through the blood-brain barrier. In addition, CD97-CD55 interactions in the parenchyma of the brain may contribute to the inflammation.     

Expression of osteopontin and its ligand, CD44, in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

The expression of osteopontin (OPN) and one of its ligands, CD44, was studied in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that osteopontin significantly increased at the early and peak stage of EAE and slightly declined thereafter. Osteopontin was constitutively expressed in some astrocytes adjacent to pia mater and neurons in normal rats, and was shown to be increased in the same cells and also in some inflammatory cells including macrophages at the early and peak stage of EAE. CD44, a ligand for osteopontin, was constitutively expressed in astrocytes in normal and control spinal cords and was also expressed in inflammatory cells, as well as increased expression in astrocytes in EAE. These findings suggest that inflammatory cells as well as reactive astrocytes are major sources of osteopontin in rat EAE, and osteopontin may interact with its ligand CD44 on astrocytes and inflammatory cells in EAE, possibly mediating autoimmune central nervous system (CNS) diseases in rats.     

Expression of the brain-specific membrane adapter protein p42IP4/centaurin alpha,a Ins(1,3,4,5)P4/PtdIns(3,4,5)P3 binding protein,in developing rat brain

Inositolphosphates and phosphatidylinositides are important second messengers. Previously p42(IP4), a protein with high affinity for both Ins(1,3,4,5)P(4) and PtdIns(3,4,5)P(3) has been characterized in our laboratory. In the present study mRNA levels of p42(IP4) were quantified during development (ages: 7, 14, 21 days and adult) by means of ribonuclease protection assay in various rat brain regions (cerebellum, cortex, striatum, thalamus, hypothalamus, olfactory bulb, hippocampus and tectum (superior and inferior colliculus)). A high level of p42(IP4) mRNA was detected in the cortex (ca. 1 pg specific RNA per microg of total RNA) which stayed highly independent of the age of the animals. In hippocampus and in the thalamus, p42(IP4) mRNA levels were comparable to those in the cortex in the first and second week postnatally, but decreased to lower levels in the adult brain. In striatum, the mRNA increased, albeit less intensely than in hippocampus and thalamus, until day 21 postnatally, and then decreased in the adult rat brain. Cerebellar p42(IP4) mRNA showed a slow increase within the first 3 weeks postnatally, and remained rather high in the adult brain. The protein expression of p42(IP4), tested within the same samples by Western blot staining, was consistent with mRNA values. For comparison, glutamic acid decarboxylase (isoforms GAD65/GAD67), an enzyme, for which some regional brain specific distribution is already known, was also examined. The mRNA levels of GAD and its developmental regulation clearly differed from that of p42(IP4). In summary, p42(IP4) expressed in several neuronal cell types, did not seem to be restricted to specific developmental stages, but the high absolute expression levels at all developmental stages indicated that p42(IP4) is a protein fundamental for neuronal functioning.     

人脑胶质瘤中CD133、SSEA-1、Nestin的表达和意义

目的：研究胶质瘤干细胞标志物CD133、SSEA-1和Nestin在胶质瘤组织中表达及其与病理分级的相关性;探讨三者在人脑胶质瘤的诊断及恶性程度判断中的临床意义。方法应用免疫组织化学方法检测54例脑胶质瘤组织和6例正常脑组织标本中CD133、SSEA-1及Nestin的表达。结果 CD133、SSEA-1和Nestin在胶质瘤组织中的阳性细胞平均表达率分别为25.38%、26.62%和22.39%,而在正常脑组织中均无表达。CD133、SSEA-1和Nestin阳性细胞率在胶质瘤各病理级别间比较,差异均有统计学意义,且三者的表达与胶质瘤病理级别呈正相关（P＜0.05）。SSEA-1与CD133、CD133与Nestin及SSEA-1与Nestin阳性细胞表达均呈正相关（P＜0.05）。结论检测CD133、SSEA-1、Nestin表达,有利于胶质瘤的诊断及恶性程度判断,并在胶质瘤的个性化综合治疗和预后评估发挥作用。     

Expression of the exon 3 skipping form of GLAST, GLAST1a, in brain and retina

GLAST is a glial glutamate transporter; mRNA for a splice variant, GLAST1a, which lacks exon 3, has previously been identified. To detect GLAST1a protein, we generated antibodies against a peptide sequence encompassing the splice site. We demonstrate by Western blotting and immunocytochemistry the expression of GLAST1a in brains and retinae. Robust immunolabelling was present in the cerebellar Bergmann glia, and weaker labelling was evident in the retinal Müller cells. GLAST1a is differentially targeted to some cellular compartments such as the end feet of the Müller cells. As GLAST1a protein may interfere with the transport of glutamate by normally spliced GLAST, differentially targeted expression of GLAST1a may represent a mechanism for selectively regulating GLAST function in the mammalian nervous system.     

Active zone protein CAST is a component of conventional and ribbon synapses in mouse retina

CAST is a novel cytomatrix at the active zone (CAZ)-associated protein. In conventional brain synapses, CAST forms a large molecular complex with other CAZ proteins, including RIM, Munc13-1, Bassoon, and Piccolo. Here we investigated the distribution of CAST and its structurally related protein, ELKS, in mouse retina. Immunofluorescence analyses revealed that CAST and ELKS showed punctate signals in the outer and inner plexiform layers of the retina that were well-colocalized with those of Bassoon and RIM. Both proteins were found presynaptically at glutamatergic ribbon synapses, and at conventional GABAergic and glycinergic synapses. Moreover, immunoelectron microscopy revealed that CAST, like Bassoon and RIM, localized at the base of synaptic ribbons, whereas ELKS localized around the ribbons. Both proteins also localized in the vicinity of the presynaptic plasma membrane of conventional synapses in the retina. These results indicated that CAST and ELKS were novel components of the presynaptic apparatus of mouse retina.     

ARIP1,2 mRNA及其蛋白在小鼠脑组织表达和分布的比较研究

目的比较新发现的激活素受体相互作用蛋白1,2(ARIP1,2)在小鼠脑组织的表达与分布。方法采用Northern杂交检测ARIP1,2 mRNA,Western杂交及免疫组化染色检测ARIP1,2蛋白。结果Northern杂交显示ARIP1,2 mRNA在小鼠组织中的表达形式明显不同,ARIP1主要在脑组织表达,ARIP2组织表达广泛。Western杂交进一步揭示ARIP1在大脑、小脑、海马、下丘脑及垂体表达,而ARIP2在大脑、小脑、海马、下丘脑、垂体及脾脏均有不同程度表达。免疫组化染色显示,ARIP1,2成熟蛋白在大脑、小脑及垂体均有表达,ARIP1在大脑和小脑皮质主要表达在小细胞神经元,而ARIP2主要表达在大细胞神经元。ARIP1在神经垂体和腺垂体表达水平明显高于ARIP2。结论ARIP1,2均表达于脑和垂体,但其表达的细胞类型和强度明显不同,这种差异可能与其在神经细胞中介导激活素生物学活性差异有关。     

Expression and function of the receptor protein tyrosine phosphatase zeta and its ligand pleiotrophin in human astrocytomas

Using subtractive cloning combined with cDNA array analysis, we previously identified the genes encoding for the protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta (PTPzeta/RPTPbeta) and its ligand pleiotrophin (PTN) as overexpressed in human glioblastomas compared to normal brain. Both molecules have been implicated in neuronal migration during central nervous system development, and PTN is known to be involved in tumor growth and angiogenesis. We confirm overexpression of both molecules at the protein level in astrocytic gliomas of different malignancy grades. PTPzeta/RPTPbeta immunoreactivity was associated with increasing malignancy grade and localized predominantly to the tumor cells. PTN immunoreactivity as determined by ELISA and immunohistochemistry analysis was increased in low-grade astrocytomas compared to normal brain. Further increase in malignant gliomas was marginal, and thus no correlation with malignancy grade or microvessel density was present. However, PTN levels were significantly associated with those of fibroblast growth factor-2, suggesting co-regulation of both factors. Functionally, PTN induced weak chemotactic and strong haptotactic migration of glioblastoma and cerebral microvascular endothelial cells. Haptotaxis of glioblastoma cells towards PTN was specifically inhibited by an anti-PTPzeta/RPTPbeta antibody. Our findings suggest that upregulated expression of PTN and PTPzeta/RPTPbeta in human astrocytic tumor cells can create an autocrine loop that is important for glioma cell migration. Although PTN is a secreted growth factor, it appears to exert its mitogenic effects mostly in a matrix-immobilized form, serving as a substrate for migrating tumor cells.