Early and long-lasting protection from arthritis in tumour necrosis factor alpha (TNFalpha) transgenic mice vaccinated against TNFalpha.

OBJECTIVE: To evaluate the effect in mice with arthritis of active anti-tumour necrosis factor (TNF)alpha immunotherapy based on a keyhole limpet haemocyanin-human TNFalpha heterocomplex (hTNFalpha kinoid or TNFK) adjuvanted in incomplete Freund adjuvant. Immunotherapy was evaluated also with methotrexate. METHODS: Human TNFalpha-transgenic mice received TNFK with or without methotrexate. Follow-up ranged from 6 weeks (short term) to 17 weeks (long term). Arthritis was evaluated clinically and histologically. Monitoring included titration of anti-hTNFalpha antibodies by ELISA and neutralisation assay. RESULTS: Vaccination with TNFK was associated with rapid-onset, long-lasting protection. Long-term results showed significantly milder arthritis in vaccinated animals than in control animals at the peak of the disease. Vaccination was followed by resolution of the clinical evidence of arthritis, contrasting with severe progressive arthritis in the control group. Histological improvements with decreased inflammation and destruction were noted in all immunised groups, even after the shortest follow-up (6 weeks). High titres of neutralising anti-hTNFalpha antibodies were detected as early as the fifth week post immunisation and persisted over time. Methotrexate given concomitantly with the vaccine did not influence either the effect on arthritis or the anti-hTNFalpha antibody titres. CONCLUSION: Anti-cytokine induction of autoimmune protection against chronic hTNFalpha overproduction is an efficient alternative to TNFalpha blockade in experimental arthritis and can be achieved using a TNFK vaccine.

Active versus passive anti-cytokine antibody therapy against cytokine-associated chronic diseases.

Current therapeutic vaccine trials in major chronic diseases including AIDS, cancer, allergy and autoimmunity, target antigenic pathogens but not the pathogenic stromal cytokines which can be major sources of histopathologic processes. Considering that the limited efficacy of these vaccines has been ascribed to local pathogen-induced cytokine dysfunction, we propose to antagonize pathogenic cytokine(s) by high affinity neutralizing auto-Abs triggered by specific anti-cytokine vaccines. As anticipated by theoretical considerations, animal experiments and initial clinical trials showed that anti-cytokine immunization was safe, well tolerated and triggered transient high titers Abs neutralizing pathogenic cytokines but, in contrast to conventional vaccines, no relevant cellular response was observed. Advantages of active versus passive anti-cytokine Ab therapy, particularly for long-term treatments, as those required in AIDS, cancer, allergy and autoimmunity include greater ease of maintaining high Ab titers, lack of anti-antibody responses and low cost.

Are CD4 and Fas peptide identities of gp120 relevant to the molecular basis of AIDS pathogenesis?

The release of virions from HIV-1-infected CD4 cells, although occurring readily as a result of immune activation, does not appear to be the only mechanism mediating T-cell loss in AIDS. Three other interacting HIV-1-induced immune disorders in association with viral release (the source of gp120 molecules) may also account for the constitutive T-cell depletion and functional immune suppression: 1. gp120-induced CD4(+) cell anergy, which can be reproduced in cultures of immune activated normal T-cells in the presence of gp120 or gp120 peptide containing the SLWDQ sequence identity to the CD4 molecule; 2. overproduction of IFNalpha and gamma, 3. activation-driven apoptosis of non infected T-cells. Apoptosis of T-cells could also be: 1. induced by effector components - particularly CTL and lymphotoxins produced by helper T-cells of the anti-Fas autoimmune reaction triggered by gp120 epitopes shared with the Fas/APO-1 molecule; 2. enhanced by IFN overproduction. These molecular mechanisms stress the importance in the progression to AIDS of both the viral load and HIV-induced cytokine dysregulation, including overproduction of IFNalpha, which should be considered as targets in the development of strategies for AIDS prophylaxis and immunotherapy.

Neurotropism of rabies virus. An in vitro study.

The relative susceptibility of neurons and glia, grown as monolayers in vitro, to rabies virus infection was explored. Established cell lines of neuronal or glial phenotype and primary cultures of cells derived from mouse dorsal root ganglia (DRC) or brain were used as homologues of the targets of rabies virus in the nervous system. Fixed rabies virus (CVS) strain was used in most experiments; other fixed rabies strains (PV, HEP, ERA) and a street rabies virus isolate were used in some. Virus-cell tropism was determined by immunofluorescence assay for rabies nucleocapsid antigen and cell permissivity was assessed by titration of virus yields. Neuronal cells always exhibited a much greater susceptibility to infection and a greater propensity to sustain viral growth. By immunofluorescence, 90-100% of neurons commonly had viral inclusion bodies, while doses of the virus three to four orders of magnitude higher still left greater than 99% of astrocytes, in brain cell cultures and 90 +/- 5% of the non-neuronal cells in DRG cultures without any obvious signs of rabies virus. Neuroblastoma cells (95 +/- 5% with viral antigens) produced viral yields about four orders of magnitude higher than glioma cells (10 +/- 5% with viral antigens). Though the overall infectivity of street virus was lower than that of fixed virus strains, a significantly higher viral tropism for neurons than for glia was maintained. Thus, primary neuronal cultures offer a means of exploring molecular events in rabies virus infection and their role in pathogenesis.

ATP release from pure cholinergic synaptosomes is not blocked by tetanus toxin.

Tetanus toxin (TeTx) is a neurotransmission impairing toxin that acts on several neurotransmitter systems. TeTx also inhibits the K+-induced release of acetylcholine (ACh) from synaptosomes isolated from the electric organ of Torpedo. Neither the membrane potential and depolarization, nor the depolarization-induced calcium uptake into cholinergic nerve terminals is modified after TeTx poisoning. On the other hand, it is known that, when cholinergic nerve terminals are stimulated, there is a release of ATP associated with the release of ACh. We have explored the action of TeTx on this co-release, and have found that there is no action of TeTx on the nucleotide release. Thus, TeTx blocks ACh release without modifying ATP release.

A 2-year follow-up of an anti-HIV immune reaction in HIV-1 gp160-immunized healthy seronegative humans: evidence for persistent cell-mediated immunity.

The first trial of an anti-HIV immunization, using a recombinant vaccinia virus expressing gp160 (rV) for priming and paraformaldehyde-fixed rV-infected PBLs and soluble gp 160 for boosting, clearly showed an in vitro HIV-protective immune reaction. This result led us to carry out an additional 2 year Phase I clinical trial in 25 HIV-seronegative volunteers, using HIV gp 160 antigens for immunization in four different protocols. The 2 year trial showed (a) the safety of the preparations, (b) a transient humoral immunity following each boost, and (c) a long-lasting memory T-cell response. Memory cytotoxic T-lymphocytes (CTLs) induced by gp 160 antigen with or without vaccinia vector lysed HLA class I restricted target cells expressing HIV-1 env antigens. These results are consistent with CTLs being an effective component of an AIDS vaccine to control cell-to-cell viral replication, dissemination in the organism, and subsequent evolution toward AIDS.

One-year follow-up of vaccine therapy in HIV-infected immune-deficient individuals: a new strategy.

Immunization of AIDS/ARC patients with autologous cells expressing HIV antigens, although providing clinical and biological benefits, fails to restore cellular immunity. The latter result is due partly to the antiproliferative effect of HIV-1 on activated T-cells (immune suppression), which leads to blockade of specific immune reactions. To overcome immune suppression, a new vaccine strategy was designed consisting of an immunization against HIV-1 combined with components of the T-cell-suppressive (antiproliferative) network. This new vaccine treatment proved to be innocuous in mice, monkeys, and two non-HIV-infected humans. A Phase I clinical trial was performed in six patients previously under cellular immunotherapy and still presenting a cellular immune defect. Preliminary results confirmed, after a 1-year follow-up of the patients, the safety of the new vaccine, which also partially restored the cellular immune response, including anti-HIV HLA-restricted cell-mediated cytotoxicity, delayed hypersensitivity to recall antigens, and proliferation of T-cells specifically activated by recall antigens.

The use of antibody Fab fragments specifically directed to two different complementary parts of the tetanus toxin molecule for studying the mode of action of the toxin.

The injection of 500 minimal lethal doses (MLDs) of tetanus toxin into mice routinely causes a flaccid-type paralysis and death within 8 h. Non-precipitating antibody fragments (Fab) directed against each of two papain cleavage products of tetanus toxin (Ibc and IIc) were used to study this botulinum toxin-type effect of tetanus toxin. Ibc (100,000 daltons) is a toxic fragment which does not bind to gangliosides but will produce a flaccid type paralysis when injected into mice. Treatment of intact tetanus toxin (500 MLDs) with Fab-Ibc prevents the flaccid type paralysis and such mice will die from a spastic paralysis after about 24 h. IIc (50,000 daltons) is an atoxic fragment of tetanus toxin which binds tightly to gangliosides. Treatment of tetanus toxin with Fab-IIc prior to intracerebral injection converts the characteristic spastic paralysis to a flaccid paralysis. It is proposed that the botulinum toxin-type effect of tetanus toxin complexed to Fab-IIc results from the inability of such complexes to be transported to the central nervous system. Moreover, the ability of Fab-Ibc to prevent flaccid paralysis, but not spastic paralysis, suggests that both types of paralysis may be mediated by the same portion of the tetanus toxin molecule.

Papain-derived fragment IIc of tetanus toxin: its binding to isolated synaptic membranes and retrograde axonal transport.

The papain-derived fragment IIc of tetanus toxin, which is immunologically identical to the B-IIb fragment, has previously been shown to bind gangliosides. As could be expected from its analogy with the B-IIb fragment, the IIc fragment was also found to bind to isolated synaptic membranes and to be transported retrogradely from the axonal endings within muscle to the motoneuronal perikarya. It is concluded that the IIc fragment--like the B-IIb fragment--might also serve as a specific carrier for chemical and chemotherapeutical agents into the central nervous system.

A correlation between the appearance and the evolution of tetanus toxin binding cells and neurogenesis.

The ontogenesis of cells expressing surface membrane binding sites for tetanus toxin (Tt) was studied in the mouse nervous system. Cells were labeled shortly after the tissue dissociation and the toxin bound was revealed by immunofluorescence. In the brain, spinal cord and dorsal root ganglia the toxin binding cells (TBC) are found as of very early stages of nervous system organogenesis, i.e. at 10 days of gestation. There is a close temporal correlation between the pattern of emergence and accumulation of TBC and the known pattern of appearance of post-mitotic neurons in mouse cerebral cortex, cerebellum and spinal cord. The curves of TBC abundance as a function of fetal age in various nervous system areas are different. They show regional fluctuations in the proportion of TBC that reflect the cumulative changes in the dynamics of neuronal subpopulations. The results indicate that Tt can be used as an ontogenetically early marker of neuronal differentiation and that the acquisition of Tt receptors may represent one of the earliest detectable characteristics of the developing neurons.

Concanavalin A inhibits nicotinic acetylcholine receptor function in cultured chick ciliary ganglion neurons.

The effects of various lectins and toxins on neuronal nicotinic acetylcholine receptor function have been studied in primary cultures of chick ciliary ganglion neurons. Neuronal response to acetylcholine receptor activation was measured by a cation flux method at 4 degrees C in a high potassium-low sodium medium designed to stabilize membrane potential near zero, with acetylcholine as the agonist and cesium-137 as the tracer ion. Exposure to 1 mM acetylcholine for 30 s produced a 5-10-fold stimulation of cesium-137 influx. Acetylcholine-stimulated influx was inhibited more than 95% by 10 microM D-tubocurarine, but was insensitive to both 1 microM tetrodotoxin and 1 microM alpha-bungarotoxin. Concanavalin A (50 micrograms/ml) inhibited agonist-induced ion flux by 80% at 4 degrees C. Succinyl-concanavalin A was ineffective at concentrations up to 250 micrograms/ml, and could not protect against the concanavalin A inhibition. However, inhibition by concanavalin A was eliminated by prior incubation of the lectin with 0.2 M alpha-methyl-D-mannoside and subsequent co-incubation with the sugar. Wheat germ agglutinin, lentil lectin, cholera toxin and tetanus toxin were without effect at either 4 degrees C or 37 degrees C. These results suggest a specific interaction between concanavalin A and neuronal nicotinic acetylcholine receptors.

Use of the B-IIb tetanus toxin derived fragment as a specific neuropharmacological transport agent.

It has been previously demonstrated that the non-toxic B-IIb tetanus toxin-derived fragment is transported retrogradely within neurons of the rat peripheral nervous system. In the present work we have shown that when a foreign polypeptide, in this case the Ibc tetanus toxin fragment, is coupled to B-IIb by a disulfide bond, it is transported retrogradely from the axonal endings within muscle to the motoneuronal perikarya. In contrast, Ibc fragment alone was found not to be transported. From these results we draw the conclusion that fragments like B-IIb may serve as specific carriers for chemical and chemotherapeutic agents into the central nervous system.

Prenatal and postnatal ontogenesis of neurotransmitter-synthetizing enzymes and [125I]tetanus toxin binding capacity in the mouse hypothalamus.

In order to estimate the early neuronal maturation in the hypothalamus, we followed the development of 3 neurotransmitter-synthetizing enzymes (TH, GAD, ChAT) and a neuronal cell surface marker (tetanus toxin) in the hypothalamus as compared to cerebral hemispheres. This showed that TH, ChAT and GAD activities were present in both structures on the fourteenth fetal day. Yet, before birth, whereas GAD and ChAT activities remained low and followed a similar development in the 2 structures, TH activity was important and higher in hypothalamus than in brain hemispheres. After birth the activities of the 3 enzymes increased rapidly between day 5 and day 20, but their evolution in the hypothalamus always preceded that in the cerebral hemispheres. Tetanus toxin binding capacity was present on the thirteenth fetal day in the 2 structures, but during fetal life in the hypothalamus, the level of toxin binding was always higher than in the cerebral hemispheres. After birth, the toxin binding increased between day 2 and day 10 and reached adult level at the same time in both structures, around day 15. We conclude that neuronal maturation proceeds earlier in the hypothalamus than in brain hemispheres, and that the differentiation of a neuronal surface marker appears concomitantly with that of specific intraneuronal enzymes.

Experimental modification of postnatal cerebellar granule cell migration in vitro.

Histotypic migration of [3H]thymidine pulse-labeled granule cell neurons in cerebellar folium explants was monitored in the presence of antibodies to cell adhesion molecules and quantified by automatic image analysis. When explants were cultured in the presence of monovalent antibody fragments to cell adhesion molecules L1 and N-CAM, an inhibition of cell migration of 33.3 +/- 4.4% and 13.9 +/- 2.1%, respectively, was observed. In the presence of an equimolar mixture of monovalent antibody fragments to L1 antigen and N-CAM no additive effects in inhibition of cell migration were seen. Antibodies to the L2 carbohydrate epitope which is common to L1, N-CAM and other cell surface glycoproteins showed a similarly small effect on cell migration as antibodies to N-CAM. Monoclonal antibodies to cell surface antigen M2 and polyclonal antibodies to mouse liver membranes reacting with the surface of all cerebellar cell types did not alter the migratory behavior of granule cells. Cultivation of explants in the presence of neuraminidase, ganglioside binding toxins, as well as glycosaminoglycans and glycosaminoglycan degrading enzymes, also did not modify the extent of cell migration under the culture conditions used.

Tetanus toxin inhibits spontaneous quantal release and cleaves VAMP/synaptobrevin.

Tetanus toxin decreased the frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocytosis and peptidase activity on synaptobrevin, is discussed.

Transsynaptic retrograde labeling in the oculomotor system of the monkey with [125I]tetanus toxin BIIb fragment.

The injection of radioactive [125I]tetanus toxin BIIb fragment into the extraocular eye muscles of monkeys led to strong retrograde labeling of motoneurons. In addition, two patterns of weaker labeling were found: (1) discrete deposits of silver grains associated with cell soma, and (2) diffuse deposits unrelated to cell bodies. The discrete cell soma labeling was found in all areas known to make synaptic contact with the retrogradely filled motoneurons, and is indicative of transsynaptic retrograde transport. The use of tetanus toxin as a transsynaptic retrograde tracer substance is shown here for the first time at the light microscopic level.

The effect of tetanus toxin on in vitro synaptogenesis.

Cultures of spinal cord neurons and cocultures of rat embryo neurons and muscle cells have been studied in the presence of tetanus toxin (TT) at a concentration of 40 micrograms/ml of medium. TT strongly stimulated neurite outgrowth, notably branching from the cell bodies. In addition it induced a marked, overall increase in acetylcholine receptor (AChR), but inhibited focalisation of AChR and acetylcholinesterase (AChE) at the synaptic sites. TT seems to act on neurite emergence, on the neuronal factor(s) controlling AChE and AChR concentrations, and on the factor(s) modulating degradation and/or synthesis of AChR.

Defining a region on tetanus toxin responsible for neuromuscular blockade.

Administering high doses of tetanus toxin to animals produces neuromuscular blockade. Previous studies, in which specific F(ab) antibody fragments were used to mask the 50,000 MW COOH-terminal portion of the heavy chain (fragment c) on the toxin molecule, have shown that the paralyzing effect of the toxin was most probably located in an area comprising the light chain and the 50,000 MW NH2-terminal portion of the heavy chain (Fragment Ibc). In our study, the toxin was also complexed with F(ab) fragments directed to the light chain (alpha), heavy chain (beta), beta minus IIc, and with monoclonal antibodies to epitopes on IIc and beta minus IIc. Investigating the effect of the resulting complexes both in mice and on the sphincter pupillae muscle in rabbits permitted us to circumscribe further the tetanus toxin neuromuscular blocking activity in a region of the NH2-terminal fragment (Mr = 50,000) of the heavy chain (fragment beta minus IIc). Our results are consistent with the assumption that the beta minus IIc fragment is critical for the neuromuscular blockade activity of tetanus toxin. However, it cannot be ruled out that both the peripheral and central effects of the toxin result from the same portion of the toxin molecule, the nature of the action depending on where the toxin is carried after its introduction into the organism.

Tetanus toxin blocks potassium-induced transmitter release and rearrangement of intramembrane particles at pure cholinergic synaptosomes.

We have studied the action of tetanus toxin on the release of acetylcholine from a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. We have also studied the morphological changes induced by chemical stimulation on the presynaptic plasma membrane of poisoned synaptosomes. These changes were studied by means of freeze-fracture techniques. We found that tetanus toxin blocks the release of acetylcholine from isolated nerve terminals in a dose-dependent manner. The maximal inhibition is achieved at a concentration of 12.5 nM in 10 min. This effect is prevented by tetanus toxin antiserum. Tetanus toxin also blocks the rearrangement of intramembrane particles at plasma membrane of poisoned synaptosomes, specifically the decrease of small (less than or equal to 9.5 nm diameter) intramembranous particles at the protoplasmic hemimembrane leaflet and the increase of large (greater than 9.5 nm diameter) intramembrane particles at the external hemimembrane leaflet induced by potassium stimulation. These results suggest that intramembrane particle rearrangement could be related to acetylcholine secretion.

Fragment A-B of tetanus toxin does not block neuromuscular transmission in the goldfish.

While 4 micrograms of Fragment A-B of tetanus toxin (which lacks the binding site for nervous tissue) causes flaccid paralysis and death in mice, 26 micrograms has no toxic effect in goldfish. Antibodies to either A-B or to fragment C (which contains the binding site) block the paralytic effect of whole toxin in goldfish. It is concluded that binding is necessary for the neuromuscular blocking action of the toxin in goldfish.