The extracellular matrix affects axonal regeneration in peripheral neuropathies.

OBJECTIVE: Recent evidence in animal models suggests that components of the extracellular matrix (ECM) play a primary role in peripheral nerve degeneration and regeneration. METHODS: We investigated the expression of several ECM molecules in human sural nerves by immunohistochemistry, Western blot, and reverse transcriptase PCR analysis. To unravel the possible role of these molecules in nerve regeneration, we compared results obtained from nerves with abundant signs of regeneration with those with complete absence of axonal regeneration. The role of some ECM components on neurite extension was further tested in dorsal root ganglion cultures. RESULTS: We observed that the ECM composition significantly differs in regenerating compared with nonregenerating nerves, independently from their etiologic background. Fibronectin was abundantly expressed in regenerating nerves, whereas vitronectin and fibrin(ogen) prevailed in nonregenerating nerves. Whereas fibronectin is secreted by endoneurial cells, in vivo and vitro studies showed that the source of vitronectin and fibrin(ogen) is the bloodstream. CONCLUSIONS: These data indicate that nerve regeneration is impaired in the presence of breaches in the blood-nerve barrier or impaired extracellular matrix (ECM) degradation that leads to accumulation of plasma vitronectin and fibrin(ogen). The transformation into mature, fibronectin-enriched ECM is necessary for efficient nerve regeneration in humans.

Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones.

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vbeta chains (Vbeta1, Vbeta2 and Vbeta8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.

Pharmacokinetics of intrathecal transferrin-ricin a chain immunotoxin.

We have studied the pharmacokinetics of an anti-transferrin receptor immunotoxin following intrathecal (i.t.) and intravenous (i.v.) bolus inoculation in healthy rats. After i.t. inoculation of 4.9 microg transferrin-ricin A-chain (Tfn-RTA) we have measured the immunotoxin concentration in the cerebrospinal fluid (CSF), in the brain tissue and in the peripheral blood. After i.v. administration of 4.9 microg Tfn-RTA the concentration of Tfn-RTA immunotoxin was evaluated in the peripheral blood. We found that the clearance of Tfn-RTA from the CSF is rapid (9.1 microLmin(-1)), the immunotoxin then diffuses into the brain tissue and in the peripheral blood where it reaches concentrations below the MTC50 (Minimum Toxin Concentration 50%). The rate of immunotoxin elimination from the peripheral blood following either i.v. or i.t. administration are similar (kel = 0.0021 min(-1) vs. 0.0025 min(-1)). Thus, in the healthy rat the immunotoxin does not accumulate following i.t. inoculation, reaching non toxic concentrations in the brain tissue and in the peripheral blood, whereas in the CSF as well as at the interface CSF/brain tissue the immunotoxin may reach potentially therapeutic concentrations. In conclusion we believe that the i.t. inoculation of an immunotoxin could be considered a potentially useful route of administration in the treatment of leptomeningeal carcinomatosis.

Human IgM anti-GM1 autoantibodies modulate intracellular calcium homeostasis in neuroblastoma cells.

Increased titers of IgM anti-GM1 antibodies are present in some patients with Lower Motor Neuron Disease (LMND) or Motor Neuropathy (MN), but their pathogenic role and the mechanism of action are unclear. Previous studies have shown that the B subunit of Cholera Toxin (CT), which binds and crosslinks ganglioside GM1, modulate intracellular calcium in murine neuroblastoma cells via the activation of L-type voltage-dependent calcium channels (VGCC). Therefore, using a fluorimetric approach, we have examined the hypothesis that the pentameric IgM anti-GM1 antibodies, could similarly alter calcium concentration in N18 neuroblastoma cells. Sera with human IgM anti-GM1 antibodies were obtained from 5 patients with LMND and 2 patients with MN. Human IgG anti-GM1, IgM anti-Myelin Associated Glycoprotein (MAG), IgM anti-sulfatide antibodies and lectin peanut agglutinin (PNA), that recognizes specifically the Gal(betal-3)GalNAc epitope, were used as control sera. Direct application of either human IgM anti-GM1 antibodies or the B subunit of CT to N18 neuroblastoma cells induced a sustained influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. Furthermore, the dihydropyridine L-type channel antagonists completely inhibited the manganese influx, suggesting that it is due to activation of an L-type VGCC. The magnitude of the influx was correlated with antibody titers. None of human IgG anti-GM1, IgM anti-MAG, IgM anti-sulfatide antibodies or PNA induce an ion influx, pointing to the selective participation of the pentameric IgM isotype of anti-GM1 in the modulation of L-type calcium channels opening. Given that L-type calcium channels are present on motor neurons, the modulation of L-type calcium channels by IgM GM1 antisera may have important implications in diseases such as LMND and MN.

Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin-toxin conjugates.

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane-acting peptides: the N-terminus of protein G of the vesicular stomatitis virus (KFT25), the N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane-acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) retained full enzymatic activity in cell-free assays and showed increased ability to induce pH-dependent calcein release from large unilamellar vesicles (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction with LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as compared to wild-type dianthin. Conjugates made by chemically cross-linking KFT25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell-killing efficiency than conjugates of Tfn and wild-type dianthin. As a consequence, by fusion of membrane-acting peptides to the dianthin sequence the specificity factor (i.e., the ratio between non-specific and specific toxicity) of Tfn-KFT25DIA, Tfn-pHA2DIA and Tfn-pJVEDIA was increased with respect to that of Tfn-based conjugates made with wild-type dianthin. Taken together, our results suggest that genetic fusion of membrane-acting peptides to enzymatic cytotoxins results in the acquisition of new physico-chemical properties exploitable for designing new recombinant cytotoxins and to tackle cell-intoxication mechanisms.

Toward a transgenic mouse model of remyelination.

The molecular mechanisms necessary for remyelination by oligodendrocytes remain unexplored. We previously characterized a myelin basic protein promoter-lacZ (MBP-lacZ) transgene whose expression is regulated uniquely during development, and also in pathological situations, suggesting that it may be a useful reporter of molecular mechanisms during remyelination. As a first step toward creating a transgenic mouse model of remyelination, we cultured oligodendrocytes from these transgenic mice and showed that expression of MBP-lacZ appeared in parallel with a marker of oligodendrocyte maturation, galactocerebroside (GC). In addition, basic fibroblast growth factor blocked the expression of both MBP-lacZ and GC in these cells. Therefore, expression of MBP-lacZ reflects not only the developmental stage of oligodendrocytes, but also extrinsic influences on oligodendrocytes. These data suggest that MBP-lacZ may be a useful marker in transgenic mouse models of remyelination.

In vitro production of human anti-sperm antibodies: effect of an oligoclonal antibody (F6) on sperm-egg interaction.

A method has been developed to establish lines of transformed lymphocytes able to produce in vitro the same anti-sperm antibodies as those naturally occurring in immuno-infertile individuals. We utilized lymphocytes from a male donor whose serum contained anti-sperm antibodies of the IgG class up to the dilution 1:10,000, as detected by means of immunobead binding. T lymphocytes were separated from B lymphocytes using magnetic beads coated with anti-T antibody. B lymphocytes were then placed at a concentration of 5 x 10(6)/ml in a 96-well plate, stimulated with phytohaemagglutinin (PHA) and transformed with Epstein-Barr virus. After a few days, only transformed cells continued growing and these were collected. The supernatant was tested for production of anti-sperm antibodies and those transformed lymphocytes shown to be synthesising antibodies directed against the sperm head and the tail were cloned. We obtained a clone of cells producing antibodies of the IgG1 class directed against the head of the spermatozoon. This oligoclonal antibody (F6) recognized a 58-kDa band from a lysate of sperm membranes and was able to reduce the penetration of zona-free hamster oocytes by capacitated spermatozoa.

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction.

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatozoa by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization.

Serum antibodies to Purkinje cells and dorsal root ganglia neurons in sensory neuronopathy without malignancy.

Anti-Purkinje cell antibodies (APCA), believed to be markers of paraneoplastic cerebellar degeneration in females, have been identified in the serum of 3 men with subacute sensory neuronopathies and no evidence of tumors 5 years after the onset of the neurological signs. By indirect immunohistochemistry on sections of rat cerebellum and dorsal root ganglia, the patients' IgG bound to the cytoplasms of both Purkinje cells and dorsal root ganglia neurons. By western blot analysis on whole human cerebellum and whole human dorsal root ganglia homogenates, the IgG from 2 patients bound to a 62-kd protein in both homogenates and the IgG from 1 patient bound to a 110-kd protein in the cerebellum homogenate only. Yo autoantibody test was negative in all patients. Our study provides evidence that non-anti-Yo APCA may be associated with subacute sensory neuronopathies and are not necessarily markers of an underlying tumor. The previously described anti-Yo APCA has only occurred in females with cancer.

Antibodies to sulfatide and to chondroitin sulfate C in patients with chronic sensory neuropathy.

Sera from eight of 25 patients with chronic sensory neuropathy had high titers of antibodies to sulfatide and chondroitin sulfate C or both. Preclearing of patients' sera with either sulfatide or chondroitin sulfate C revealed that in four patients the antisulfatide antibodies crossreacted with chondroitin sulfate C. By indirect immunohistochemistry sera reactive to sulfatide only had a different staining pattern from those reactive to both sulfatide and chondroitin sulfate C. By direct immunohistochemistry we found immunoglobulins bound to nerve fibers only in patients with serum antibodies against both sulfatide and chondroitin sulfate C. Our study provides evidence that antibodies to sulfatide and to chondroitin sulfate C differ in their fine specificity and are present in 30% of patients with chronic sensory neuropathy.

Peripheral neuropathy in scleroderma.

Nervous system involvement is rare in progressive systemic sclerosis (PSS). We present a clinical pathological and immunological study of two patients with peripheral sensory motor neuropathy and PSS. In both, the sural nerve biopsies showed axonal degeneration with increased endoneurial connective tissue. There were also clusters of myelinated fibres indicating axonal regeneration. Only mild microangiopathic changes were evident in the endo, peri and epineurial vessels. By Western immunoblots, patients' sera contained a band of reactivity to a protein from peripheral nerve identified as collagen type I. Primary involvement of the peripheral nerves during PSS is very unusual. Abnormal production of collagen tissue and presence of microvascular disease are considered to be two possible causes of neuropathy. We think that our results suggest the important role of the connective tissue proliferation in the pathogenesis of PSS neuropathy.

Sperm surface proteins after capacitation. Expression of vitronectin on the spermatozoan head and laminin on the sperm tail.

Several integrins recognize as ligands proteins containing the Arg-Gly-Asp (RGD) sequence, such as fibronectin, vitronectin, and laminin. It has been previously demonstrated that oligopeptides containing the RGD sequence competitively inhibit both the adhesion of hamster and human sperm to zona-free hamster eggs and their subsequent penetration. In addition, the appearance of fibronectin on the surface of living human spermatozoa after capacitation has been demonstrated. In this work, it is shown that spermatozoa incubated overnight under capacitating conditions, but not fresh spermatozoa, also display the RGD-containing proteins vitronectin and laminin. Whereas the expression of fibronectin does not appear to be localized to any specific region of the sperm surface, laminin is present solely on the sperm tail, and vitronectin was detected mostly as an equatorial band on the sperm head. The percent of capacitated spermatozoa within each ejaculate reacting with antivitronectin antibodies (51% to 94%) was similar to that observed with antifibronectin antibodies (72% to 100%) in a series of fertile donors, and in a series of infertile men (7% to 98% for vitronectin versus 5% to 100% for fibronectin). In contrast, the percent of spermatozoa displaying laminin was lower, ranging from 2% to 42% for fertile donors and from 5% to 34% for infertile donors, and was unrelated to the expression of fibronectin or vitronectin. The time of appearance of both fibronectin and vitronectin when spermatozoa were incubated under capacitating conditions varied for different sperm donors, suggesting a difference in the process of their expression between different men. The specificity of antivitronectin antibody binding to human spermatozoa was demonstrated by competitive inhibition with purified human vitronectin. That there was no immunologic reaction between the antivitronectin antibodies used and fibronectin was demonstrated both by the failure of free fibronectin to inhibit antivitronectin antibody binding to spermatozoa, and by Dot blot analysis. A partial cross-reaction of the polyclonal antivitronectin antibody with fibronectin was shown by Western blot analysis, but this phenomenon was not present when the monoclonal antivitronectin antibody was used. In addition, both fibronectin and vitronectin could be extracted from capacitated spermatozoa solubilized in Chaps buffer, as shown by Dot blot and Western blot analysis. These observations suggest that vitronectin and fibronectin expressed on the surface of capacitated human spermatozoa could act as a ligand for specific receptors on the egg, and play a role in sperm-oolemmal adhesion.

A clinical, electrophysiological, morphological and immunological study of chronic sensory neuropathy with ataxia and paraesthesia.

We have observed 9 patients (8 men and 1 woman), 58 to 77 years of age with neuropathy with only sensory symptoms and insidious onset. Five of them (4 men and 1 woman) aged 65 to 77 years, had normal serum electrophoretic profiles, while the others (all men), 58 to 74 years, had IgM monoclonal gammopathy of undetermined significance (MGUS). Clinical data were consistent with a sensory neuropathy affecting predominantly the kinesthetic sense (position and vibration sensation). The electrophysiological data indicated predominant sensory axonal neuropathy. Morphological data confirmed the primary axonal damage. Western immunoblot showed that the IgG from a patient without MGUS reacted with a 55 kD protein of dorsal root ganglion homogenate. Three of four patients with IgM MGUS were serum reactive against chondroitin sulfate C (ChS-C) in double immunodiffusion. After absorption with ChS-C the monoclonal peak completely disappeared from two patients and was decreased in the third patient. Our data indicate that immunological abnormalities are part of the pathogenesis for a subgroup of chronic neuropathy with only sensory symptoms.

IgG monoclonal proteins from patients with axonal peripheral neuropathies bind to different epitopes of the 68 kDa neurofilament protein.

We describe three patients with a sensorimotor axonal polyneuropathy and an IgG M-protein that binds to a 68 kDa axonal protein identified as the low molecular weight neurofilament protein (NF-L). The immunological studies revealed that the M-proteins have different target epitopes: one is phosphorylated and the other two are nonphosphorylated. One of the nonphosphorylated epitopes is common to other intermediate filaments, such as desmin and vimentin.

[Autoimmune neuropathy in monoclonal gammopathy]. / Neuropatie disimmuni in corso di gammopatie monoclonali.

The relationship between polyneuropathies and monoclonal gammopathies is well known even though the pathogenetic hypotheses are controversial. The role of autoantibodies against neural antigens has been recently underlined. 45 patients (29 M and 16 F), affected by multiple myeloma (MM) non-Hodgkin lymphoma (NHL) with paraproteinemia and monoclonal gammopathies of undetermined significance (MGUS)4, underwent an EMG study including SCV, MCV and late responses of several nerves, and a search for serum antibodies against neural antigens by immunoblotting assay. 19 out of 45 pts. tested positive to EMG and 15 out of 45 (10 MM and 5 NHL) showed a serological positivity. Among them 11 were positive to EMG too. The results confirm the hypothesis of a possible pathogenetic role of high-titer autoantibodies against neural antigens in cases of polyneuropathy.

Axonal neuropathy in a patient with monoclonal IgM kappa reactive with Schmidt-Lantermann incisures.

We report a patient with a progressive, predominantly sensory neuropathy and a IgM kappa M-protein that binds to Schmidt-Lantermann incisures. A sural nerve biopsy showed primary axonal damage and IgM deposits at Schmidt-Lantermann incisures were seen by direct immunoperoxidase. Serum from the patient injected into rat sciatic nerve reacts with the incisures as with those in the patient's nerve. The IgM kappa M-protein reacts with chondroitin sulfate C and binds to a broad nerve protein band with a mobility of between 170 and 118 kDa. Peripheral neuropathy may be related to the M-protein, which had immunocytochemical reactivity not previously described for patients with polyneuropathy and IgM monoclonal gammopathy.

Polyneuropathy associated with IgA monoclonal gammopathy: a hypothesis of its pathogenesis.

We describe three patients with chronic progressive polyneuropathy associated with IgA monoclonal gammopathy. Two patients had a prominent sensory neuropathy and one had a prominent motor neuropathy. Sural nerve biopsies showed axonal degeneration in all cases. In immunocytochemical studies patients' IgG immunostained axons. By Western immunoblot a band of IgG reactivity with an axonal protein of 66 kDa was found. No band of IgA and IgM were found. We suggest the possibility that the IgA monoclonal protein may act as a stimulating factor of preexisting B cell clones eliciting an immune reaction against nerve antigens.

Axonal neuropathy with monoclonal IgG kappa that binds to a neurofilament protein.

We report a 74-year-old woman with a slowly progressive sensory motor axonal neuropathy and a monoclonal IgG-kappa that bound to a 68-kd axonal protein identified as the low molecular weight neurofilament protein. The sera of control subjects and disease controls did not bind to neurofilament protein.