A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75‐kbp plasmid from a toxigenic non‐sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.

Autoimmunity develops when self-tolerance mechanisms are failing to protect healthy tissue. A sustained reaction to self is generated, which includes the generation of effector cells and molecules that destroy tissues. A way to restore this intrinsic tolerance is through immune modulation that aims at refurbishing this immunologically naïve or unresponsive state, thereby decreasing the aberrant immune reaction taking place. One major cytokine has been shown to play a pivotal role in several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS): tumor necrosis factor alpha (TNFα) modulates the induction and maintenance of an inflammatory process and it comes in two variants, soluble TNF (solTNF) and transmembrane bound TNF (tmTNF). tmTNF signals via TNFR1 and TNFR2, whereas solTNF signals mainly via TNFR1. TNFR1 is widely expressed and promotes mainly inflammation and apoptosis. Conversely, TNFR2 is restricted mainly to immune and endothelial cells and it is known to activate the pro-survival PI3K-Akt/PKB signaling pathway and to sustain regulatory T cells function. Anti-TNFα therapies are successfully used to treat diseases such as RA, colitis, and psoriasis. However, clinical studies with a non-selective inhibitor of TNFα in MS patients had to be halted due to exacerbation of clinical symptoms. One possible explanation for this failure is the non-selectivity of the treatment, which avoids TNFR2 stimulation and its immune and tissue protective properties. Thus, a receptor-selective modulation of TNFα signal pathways provides a novel therapeutic concept that might lead to new insights in MS pathology with major implications for its effective treatment.

The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) inClostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes inClostridium, i.e., uviA from C. perfringens and txeR from C. difficile. We report for the first time the transformation of C. tetani by electroporation, which permitted us to investigate the function of tetR. Overexpression of tetR inC. tetani induced an increase in TeTx production and in the level of the corresponding mRNA. This indicates that TetR is a transcriptional activator of the TeTx gene. Overexpression ofbotR/A (60% identity with TetR at the amino acid level) inC. tetani induced an increase in TeTx production comparable to that for overexpression of tetR. However,botR/C (50% identity with TetR at the amino acid level) was less efficient. This supports that TetR positively regulates the TeTx gene in C. tetani and that a conserved mechanism of regulation of the neurotoxin genes is involved in C. tetaniand C. botulinum.

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-γ were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.

Numerous studies have revealed the pleiotropic functions of tumor necrosis factor alpha (TNF-α), and have linked it with several neurodegenerative disorders. This review describes the signaling pathways induced by TNF-α via its two receptors (TNFR1 and TNFR2), and their functions in neurodegenerative processes as in Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and ischemic stroke. It has become clear that TNF-α may exert divergent actions in neurodegenerative disorders, including neurodegenerative and neuroprotective effects, which appear to depend on its signaling via either TNFR1 or TNFR2. Specific targeting of these receptors is a promising therapeutic strategy for many disorders.