A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model.

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

Serum hepcidin levels are related to the severity of liver histological lesions in chronic hepatitis C.

Hepcidin is synthesized in the liver and has a crucial role in iron homoeostasis. Its synthesis is up-regulated in chronic inflammation and iron excess. We examined the determinants of serum hepcidin and liver hepcidin mRNA levels and their association with histological lesions in patients with chronic hepatitis C (CHC) and healthy controls. We studied 96 patients with CHC and 30 controls. Serum hepcidin levels were measured by an in-house competitive ELISA. Hepcidin mRNA levels were determined by a one-step qRT-PCR in total RNA extracted from liver biopsy specimens of 27 patients with CHC and six disease controls. Histological lesions were evaluated according to Ishak's classification. Serum hepcidin was significantly lower in patients with CHC than healthy controls (14.6 ± 7.3 vs 34.6 ± 17.3 ng/mL, P < 0.001). In patients with CHC, serum hepcidin correlated positively with aspartate aminotransferase (r = 0.334, P = 0.001) and insulin resistance (r = 0.27, P = 0.016) and had a trend for correlation with alanine aminotransferase (r = 0.197, P = 0.057) and serum haemoglobin (r = 0.188, P = 0.067) but not with ferritin. A significant positive correlation was also found between serum hepcidin levels and both necroinflammation (r = 0.259, P = 0.011) and fibrosis (r = 0.214, P = 0.036). Serum hepcidin was among others an independent predictor of cirrhosis (odds ratio: 1.145, P = 0.039). Liver hepcidin mRNA levels did not differ between patients and controls and were relatively lower in patients with than without cirrhosis (19.3 ± 21.7 vs 38.3 ± 26.0, P = 0.067). Patients with CHC have reduced serum hepcidin levels, which correlate with worse necroinflammation and fibrosis. The previously mentioned observations suggest a viral effect on hepatic hepcidin production, but might also support its involvement in the inflammatory process.

Redirecting mouse T hybridoma against human breast and ovarian carcinomas: in vivo activity against HER-2/neu expressing cancer cells.

Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour efficacy of scFv(anti-HER-2/neu)/zeta expressing MD.45 cells in severe combined immunodeficiency disease mice/human and murine tumour models. The adoptively transferred MD.45-HER/zeta cells both slowed significantly the growth of human FM3 melanoma or murine ALC leukaemic cells both transfected to express HER-2/neu. Our data demonstrate the feasibility of redirecting MD.45 CTL with the scFv(anti-HER-2/neu)/zeta chimeric receptor to respond specifically against HER-2/neu expressing tumour cells in vitro and in vivo. Moreover, they make it likely that T cells transduced with the same chimeric gene might be utilised in the treatment of patients with HER-2/neu(+) tumours.

Peptide HER2(776-788) represents a naturally processed broad MHC class II-restricted T cell epitope.

HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) responses, although critical for tumour rejection, are not thoroughly characterized. Here, we report the generation and characterization of CD4+ T cell clones specifically recognizing a HER-2/neu-derived peptide (776-788) [designated HER2(776-788)]. Such clones yielded specific proliferative and cytokine [gamma-interferon(IFN)-gamma] responses when challenged with autologous dendritic cells (DCs) loaded with HER2(776-788). By performing blocking studies with monoclonal antibodies (MAbs) and by using DCs from allogeneic donors sharing certain HLA-DR alleles, we found that HER2(776-788) is a promiscuous peptide presented, at least, by DRB5*0101, DRB1*0701 and DRB1*0405 alleles. One TCRV beta 6.7+ clone recognized the HLA-DRB5*0101+ FM3 melanoma cell line transfected with a full length HER-2/neu cDNA. Moreover, this clone recognized the HER-2/neu+ SKBR3 breast cancer cell line induced to express HLA-DR, thus demonstrating that HER2(776-788) represents a naturally processed and presented epitope. Our data demonstrate that helper peptide HER2(776-788) represents a promiscuous epitope binding to at least three HLA-DR alleles, thus offering a broad population coverage. The use of antigenic peptides presented by major histocompatibility complex (MHC) class II in addition to those presented by class I may improve the therapeutic efficacy of active immunization.

Cloning, expression and localization of human BM88 shows that it maps to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis.

Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88 homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in human brain whereas immunohistochemistry on human brain sections demonstrated that the expression of BM88 is confined to neurons. The initial mapping assignment of human BM88 to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis, was retrieved from the UniGene database maintained at the National Centre for Biotechnology Information (NCBI, Bethesda, MD, U.S.A.). We confirmed this localization by performing fluorescence in situ hybridization on BM88-positive cosmid clones isolated from a human genomic library. These results suggest that BM88 may be a candidate gene for genetic disorders associated with alterations at 11p15.5.

Isolation of a mouse brain cDNA expressed in developing neuroblasts and mature neurons.

A previously uncharacterized 4.5-kb mouse cDNA clone, designated mc7, was isolated and found to be predominantly expressed in brain. This cDNA predicts a 1035-bp open reading frame that encodes for a 345-amino acid polypeptide especially rich in glutamic acid residues located in the region from residues 80 to 174. Computational analysis revealed among other features, putative zinc-finger motifs and coiled-coil regions. The corresponding mc7 gene is detected in mouse, rat, pig and human genomes. In mouse the mc7 mRNA is expressed predominantly in brain and to a much lesser extent in kidney, lung and spleen. In brain it is detectable as early as embryonic day 14 while it is retained in the adult. In situ hybridization studies revealed that mc7 mRNA is widely, albeit unevenly, expressed in neurons throughout the adult brain. Developmental in situ hybridization studies in the cerebellar cortex demonstrated that at postnatal day 5 mc7 mRNA is mainly expressed in neuroblasts of the external granular layer and in developing neurons of the internal granular layer. Some staining is also present in purkinje cells becoming particularly pronounced at postnatal day 10, the time of arborarization of their dendritic tree. In the adult cerebellar cortex expression is mainly confined in purkinje cells and to a lesser extent in granule neurons. The early expression of mc7 in neuroblasts and developing neurons as well as its retention in a wide variety of mature neurons suggest that it may play a role in the process of differentiation and maturation of these cells in the brain.

Reconstitution of conformationally dependent epitopes on the N-terminal extracellular domain of the human muscle acetylcholine receptor alpha subunit expressed in Escherichia coli: implications for myasthenia gravis therapeutic approaches.

Myasthenia gravis (MG) is an autoimmune disease, caused by autoantibodies against the muscle acetylcholine receptor (AChR), an oligomeric transmembrane glycoprotein composed of alpha(2)beta gamma delta subunits. The alpha subunit carries in its N-terminal extracellular domain the main immunogenic region (MIR), a group of conformationally dependent epitopes that seems to be a major target for the anti-AChR antibodies in MG patients. Detailed epitope studies on pathogenic anti-AChR antibodies have been hindered because the binding of most of these antibodies is conformationally dependent, which precludes the use of denatured AChR fragments. The N-terminal extracellular fragment, residues 1-207, of the human AChR alpha subunit was expressed in Escherichia coli in a denatured form, solubilized in a guanidinium hydrochloride-containing buffer, purified, and renatured using a refolding approach which employs a detergent and a cyclodextrin as 'artificial chaperones'. Compared with the non-refolded protein, the refolded molecule exhibited a dramatic improvement in terms of the binding of all anti-MIR mAb tested. Anti-MIR mAb that normally bind weakly to the denatured alpha subunit bound approximately 30-100 times better to the refolded polypeptide and other anti-MIR mAb that bind exclusively to completely conformationally dependent epitopes also bound quite efficiently. These results, in addition to providing a means for the thorough investigation of the antigenic structure of the AChR, show that the conformationally dependent MIR epitopes do not require the participation of the oligosaccharide moiety of the alpha subunit nor the contribution of neighboring subunits for antibody binding. Such AChR fragments may be used in structural studies of the AChR autoantigen, and should prove valuable in the understanding and development of therapeutic approaches for MG.

The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor.

The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (Kd = 0.05 nM) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A2. Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.

The third-dimensional structure of the complex between an Fv antibody fragment and an analogue of the main immunogenic region of the acetylcholine receptor: a combined two-dimensional NMR, homology, and molecular modeling approach.

Binding of autoantibodies to the acetylcholine receptor (AChR) plays a major role in the autoimmune disease Myasthenia gravis (MG). In this paper, we propose a structure model of a putative immunocomplex that gives rise to the reduction of functional AChR molecules during the course of MG. The model complex consists of the [G(70), Nle(76)] decapeptide analogue of the main immunogenic region (MIR), representing the major antigenic epitope of AChR, and the single chain Fv fragment of monoclonal antibody 198, a potent MG autoantibody. The structure of the complexed decapeptide antigen [G(70), Nle(76)]MIR was determined using two-dimensional nmr, whereas the antibody structure was derived by means of homology modeling. The final complex was constructed using calculational docking and molecular dynamics. We termed this approach "directed modeling," since the known peptide structure directs the prestructured antibody binding site to its final conformation. The independently derived structures of the peptide antigen and antibody binding site already showed a high degree of surface complementarity after the initial docking calculation, during which the peptide was conformationally restrained. The docking routine was a soft algorithm, applying a combination of Monte Carlo simulation and energy minimization. The observed shape complementarity in the docking process suggested that the structure assessments already led to anti-idiotypic conformations of peptide antigen and antibody fragment. Refinement of the complex by dynamic simulation yielded improved surface adaptation by small rearrangements within antibody and antigen. The complex presented herein was analyzed in terms of antibody-antigen interactions, properties of contacting surfaces, and segmental mobility. The structural requirements for AChR complexation by autoantibodies were explored and compared with experimental data from alanine scans of the MIR peptides. The analysis revealed that the N-terminal loop of the peptide structure, which is indispensable for antibody recognition, aligns three hydrophobic groups in a favorable arrangement leading to the burial of 40% of the peptide surface in the binding cleft upon complexation. These data should be valuable in the rational design of an Fv mutant with much improved affinity for the MIR and AChR to be used in therapeutic approaches in MG.

Alpha4 but not alpha3 and alpha7 nicotinic acetylcholine receptor subunits are lost from the temporal cortex in Alzheimer's disease.

Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic alpha4-like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p < 0.05). There were no significant changes in alpha3- or alpha7-like immunoreactivity associated with AD or tobacco use. The selective involvement of alpha4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.

Increased generation of autologous tumor-reactive lymphocytes by anti-CD3 monoclonal antibody and prothymosin alpha.

Anti-CD3 monoclonal antibody (mAb) activates in vitro peripheral blood mononuclear cells (PBMC) to lyse a variety of tumor cell lines in a non-major histocompatibility-complex(MHC)-restricted manner [subsequently referred to as anti-CD3-activated killer (AAK) cytotoxicity]. Prothymosin alpha (ProTalpha) is a biological response modifier that exerts its effects primarily on mononuclear cells, especially when these cells' effector functions are impaired. In this study, we report that ProTalpha enhances the AAK cytotoxicity in PBMC from healthy donors. This effect was more profound with cancer patients' PBMC, which were deficient in their ability to respond with enhanced AAK cytotoxicity upon in vitro stimulation with anti-CD3. Thus, cancer patients' PBMC, activated with a combination of anti-CD3 and ProTalpha, exhibited increased AAK activity and efficiently lysed both autologous tumor and Daudi targets. The ProTalpha effect on PBMC was demonstrated to involve stimulation of adhesion molecules (CD2, CD18, CD54, CD49f) and CD25 expression, up-regulation of perforin mRNA transcription, increased numbers of perforin-positive (+) cells and elevated production of interleukin-2 (IL-2), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). Moreover, effector CD8+ and CD56+ cells pretreated with anti-CD3 and ProTalpha contained high cytoplasmic perforin levels and increased expression of IL-1beta- and TNFalpha-specific receptors. The induction of autologous-tumor-reactive CD8+ and CD56+ lymphocytes in anti-CD3-activated PBMC by ProTalpha provides an alternative protocol aimed at the improvement of clinical results in cellular adoptive immunotherapy of cancer.

High affinity single-chain Fv antibody fragments protecting the human nicotinic acetylcholine receptor.

Univalent antibody fragments directed against the main immunogenic region (MIR) of the human acetylcholine receptor (AChR) are capable of protecting the AChR against loss induced by antibodies from myasthenia gravis (MG) patients. Our aim was to construct single-chain Fv (scFv) antibody fragments as a first step towards the production of therapeutic protecting molecules, from two high-affinity anti-MIR monoclonal antibodies (mAb 192 and mAb 195). During the construction of scFv192 fragment, two light chains co-secreted from the hybridoma mAb192 were identified. N-terminal amino acid and cDNA sequence analysis showed that one of the two light chains corresponded to the antigen binding molecule while the other originated from the non-secreting myeloma S194/5.XXO.BU.1 which was used in the production of the hybridoma. Functional scFv 192 and 195 fragments were constructed, expressed in Escherichia coli and affinity purified. The binding affinities of scFv192 and scFv195 (K(D) = 0.6 and 0.8 nM for human AChR) were two orders of magnitude higher than that of the earlier constructed scFv198. The scFv192 almost completely protected human AChR against binding of intact anti-MIR mAbs. Human AChR was also very efficiently protected (74-85%) by the scFv192 against binding of autoantibodies from MG sera with high anti-alpha subunit antibody fractions. These scFvs are good candidates for protection of MG patients after appropriate genetic modifications.

Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor.

The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR (K(D): 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 (K(D): 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-alpha autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.

Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor.

The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti-AChR antibody response is polyclonal. However, a small extracellular region of the AChR alpha-subunit, the main immunogenic region (MIR), seems to be a major target for anti-AChR antibodies. A major loop containing overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) lies within residues alpha 67-76 at the extreme synaptic end of each alpha-subunit: however, anti-MIR mAbs are functionally and structurally quite heterogeneous. Anti-MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplasmic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine-binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti-AChR antibodies cross-reactive with non-AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR; especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in-depth understanding, and for possible specific immunotherapy, of MG.

Overexpression of the neuron-specific molecule BM88 in mouse neuroblastoma cells: altered responsiveness to growth factors.

Previous studies have shown that the BM88 antigen, a novel neuron-specific molecule, promotes the differentiation of mouse neuroblastoma (Neuro 2a) cells. In particular, stably transfected, with the BM88 cDNA, Neuro 2a cells overexpressing the BM88 antigen (Neuro2a-BM88 cells) are morphologically distinct from the nontransfected Neuro 2a cells; they exhibit enhanced process outgrowth and a slower rate of division. In this study we used Neuro2a and the morphologically differentiated Neuro 2a-BM88 cells to compare their responsiveness to growth factors. The growth factors we used were nerve growth factor (NGF), basic-fibroblast growth factor (b-FGF), and glial cell-line derived neurotrophic factor (GDNF). In addition, we used glial conditioned medium derived from either newborn mouse cerebral cortex (NBCC) or aged mouse cerebral hemispheres (MACH), as a source of normal glial factors. Because these cells express the cholinergic phenotype, we used choline acetyltransferase (ChAT) activity as a biochemical marker for comparison. A differential responsiveness to these factors was observed between Neuro 2a and Neuro 2a-BM88. The presence of NGF, 25 ng/ml, in the culture medium did not affect ChAT activity in either cell type. In contrast to NGF, in the presence of b-FGF, 5 ng/ml, the transfected cells, Neuro 2a-BM88, responded with a marked increase in ChAT activity. On the other hand, with GDNF, 1 ng/ml, only Neuro 2a cells showed an increase in ChAT activity. Finally, we found no response to the glial conditioned media, although these media contain several growth factors, including b-FGF. In conclusion, our findings show that overexpression of the neuron-specific antigen BM88 in neuroblastoma cells modifies their properties with respect to growth factor sensitivity, and, hence, the Neuro 2a and Neuro 2a-BM88 are suitable cell models to examine the role of growth factors in neuronal differentiation.

Purification and cDNA cloning of mouse BM89 antigen shows that it is identical with the synaptic vesicle protein synaptophysin.

The BM89 antigen, first identified in porcine brain by means of a monoclonal antibody, is a neuron-specific molecule widely distributed in the mammalian central and peripheral nervous system (Merkouri and Matsas: Neuroscience 50:53-68, 1992). Here we describe the purification of BM89 antigen from porcine and mouse brain by immunoaffinity chromatography using, respectively, the previously described BM89 monoclonal antibody which belongs to the IgM class and a specific polyclonal antibody generated in the present study. This antibody was also used for the cDNA cloning of the BM89 antigen from mouse brain. cDNA sequencing revealed that the mouse BM89 antigen is identical with the synaptic vesicle protein synaptophysin which is implicated in the control of regulated exocytosis and neurotransmitter release. Mouse BM89 antigen/synaptophysin exhibits, except for one extra amino acid, 100% identity with rat synaptophysin and substantial sequence identity with bovine (92.5% identity) and human (94.8% identity) synaptophysin, but only 59.8% identity with Torpedo synaptophysin. Northern and Western blot analyses confirmed that the mouse BM89 antigen/synaptophysin is expressed only in neural tissues.

The BM88 antigen, a novel neuron-specific molecule, enhances the differentiation of mouse neuroblastoma cells.

The BM88 antigen is a neuron-specific molecule widely distributed in the mammalian nervous system. It is a 22-kDa, apparently not glycosylated, integral membrane protein, which appears early during brain development and remains at high levels in the mature animal. Here, we describe the cDNA cloning of the porcine BM88 antigen and present evidence that this protein is involved in neuroblastoma cell differentiation. The deduced protein is a novel molecule consisting of 140 amino acids and bears a putative transmembrane domain at the COOH-terminal region. The mRNA of this protein is expressed only in neural tissues, where it is restricted to neurons. Stably transfected Neuro-2a cells overexpressing the BM88 antigen exhibited a significant change in morphology, reflected by enhanced process outgrowth, and a slower rate of division. Moreover, in the presence of differentiation agents, such as sucrose and retinoic acid, an accelerated differentiation of the transfected Neuro-2a cells was observed. Especially in the presence of sucrose, the consequent overexpression of the BM88 antigen in the transfected cells resulted in their enhanced morphological differentiation accompanied by the induction of neurofilament protein expression. Our results suggest that the BM88 antigen plays a role in the differentiation of neuroblastoma cells.

Expression of endopeptidase-24.11 (common acute lymphoblastic leukaemia antigen CD10) in the sciatic nerve of the adult rat after lesion and during regeneration.

Endopeptidase-24.11, which is identical with the common acute lymphoblastic leukaemia antigen CD10 (CALLA), is a cell surface Zn2+ metalloprotease that regulates peptide-induced responses in different tissues, including the nervous and immune systems. In the peripheral nervous system, high levels of the enzyme are present in all neonatal and early postnatal Schwann cells, while as myelination proceeds it is gradually suppressed in the majority of cells that form myelin but retained in non-myelin-forming cells in the adult animal. In the present study we have investigated the effects of transection, crush and regeneration of the adult rat sciatic nerve on the expression of the endopeptidase by Schwann cells in situ. Endopeptidase-24.11 was monitored by immunocytochemistry using the monoclonal anti-endopeptidase antibody 23B11. For comparison, a parallel study was carried out with a monoclonal antibody directed against the rat nerve growth factor receptor. We found that (i) all Schwann cells of the distal segment re-expressed endopeptidase-24.11 as early as 4 days after axotomy, the level of immunostaining reaching a maximum after 2 weeks, (ii) axonal regeneration repressed Schwann cell expression of endopeptidase-24.11, and (iii) the induction of the nerve growth factor receptor followed a similar pattern to that of endopeptidase-24.11 in the transected and crushed nerve. Enzymatic amplification of endopeptidase-24.11 cDNA from normal and axotomized adult rat sciatic nerve confirmed the expression of endopeptidase-24.11 in these tissues. Our results show that the expression of endopeptidase-24.11 in Schwann cells, as is the case with the nerve growth factor receptor, is induced by the loss of the normal axon-Schwann cell contact. The significant increase in the expression of endopeptidase-24.11 by Schwann cells after axonal damage suggests that the enzyme could play a role in axonal regeneration.