Relationship between tumor necrosis factor alpha and feline immunodeficiency virus expressions.

The presence of feline immunodeficiency virus (FIV) proviral DNA, expression of FIV p26 core protein, and production of tumor necrosis factor alpha (TNF-alpha) were assessed in sequential biopsies of spleen and lymph node sections, of mononuclear cells of the peripheral blood, and of the serum of specific-pathogen-free cats during the acute phase of FIV infection. A temporal relationship between TNF-alpha production and FIV p26 expression was noted. Two months following FIV infection, and preceding the detection of FIV viremia, levels of TNF-alpha in serum increased significantly (P = 0.04), and they remained elevated during FIV viremia in the third month postinfection. Immunoprecipitates representing expression of TNF-alpha and of FIV p26 were localized in common foci of lymph nodes of FIV-infected cats during this period of active viremia. With the advent of anti-FIV antibodies, circulating levels of TNF-alpha and p26 antigen and expression of TNF-alpha and p26 in the lymph nodes decreased during the fifth month postinfection, and p26 production became undetectable. With clearance of viremia, burden of proviral DNA in peripheral blood mononuclear cells became reduced (P = 0.041), with provirus remaining integrated principally within lymph nodes (P = 0.046). During aviremia, p26 expression was undetectable in any tissue but remained inducible in vitro. During acute FIV infection, TNF-alpha production and p26 expression are intimately linked.     

Novel muteins of human tumor necrosis factor alpha

For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Cloning and characterization of gene TNF alpha encoding equine tumor necrosis factor alpha.

We report the molecular cloning and nucleotide sequence of the equine gene encoding tumor necrosis factor alpha. The 2610-bp genomic sequence was derived from three overlapping polymerase chain reaction products.     

Binding of human tumor necrosis factor alpha to multimeric complementary peptides.

A peptide with binding properties for tumor necrosis factor (TNF alpha) sequence 144-157 has been designed, using a computer-assisted method able to create peptide sequences hydropathically complementary to a given sequence. The complementary peptide was synthesized in a multimeric form starting from an octadentate polylysine core, to facilitate its immobilization and to provide interaction multivalency. Once immobilized on a solid support to prepare an affinity column, it recognized the target TNF144-157 peptide selectively from crude peptide mixtures containing TNF fragments encompassing the entire TNF alpha sequence. Similar selectivity and specificity were shown for full-length recombinant TNF alpha, allowing its purification from crude Escherichia coli extracts. The octameric complementary peptide preserved its recognition properties for TNF alpha and biotinylated TNF alpha even after coating on microtiter plates. Competitive binding occurred with unlabeled TNF alpha in the range between 0.01 and 10 micrograms/ml, in the presence of detergent such as 0.05% Tween 20 and in the presence of 1% normal goat serum. The effect of complementary peptide multimerization was evidenced by its enhanced binding affinity for TNF alpha, which exists in solution as a trimer, while the target TNF[144-157] peptide was recognized with much lower strength. The dissociation constant for interaction with TNF alpha was close to 10 nM, allowing its easy detection by solid phase assays in concentrations as low as 10 pmol/ml.     

Dual role of tumor necrosis factor alpha in brain injury

Brain injury (ischemia, trauma) is among the leading cause of mortality and disability in the western world. It induces increased production of tumor necrosis factor (TNF alpha) by brain resident cells. There is conflicting evidence on the role of this response in the injured brain, showing its potential effect in both processes of repair and of damage. This review presents data from clinical and experimental studies on the stimulation of TNF alpha production in brain injury and on the deleterious consequence of this acute response. Its inhibition by pharmacologic agents, neutralizing antibodies or soluble receptors has protective effects. In contrast, there are reports (from in-vitro studies or knock-out mice) on the beneficial effects of TNF alpha. To reconcile these apparently conflicting reports, the exact timing and extent of TNF alpha activation must be taken into account, as well as the presence of other mediators such as reactive oxygen species. It is suggested that the appropriate context of mediators, at any given time after brain injury may well determine whether the effect of TNF alpha is protective or toxic.     

Apoptosis induced by tumor necrosis factor in cells chronically infected with feline immunodeficiency virus.

Tumor necrosis factor alpha (TNF-alpha) induced morphologic changes such as chromatin condensation and cell shrinkage in a feline fibroblastic cell line (CRFK) chronically infected with feline immunodeficiency virus (FIV) but not in uninfected CRFK cells. DNA extracted from TNF-alpha-treated CRFK cells infected with FIV showed a ladder of nucleosomal DNA, indicating that this cytocidal effect by TNF-alpha was due to programmed cell death, or apoptosis. These findings may have implications for understanding the pathogenesis of FIV infection and for the design of specific therapeutic strategies for AIDS in humans as well as cats.     

Neutralization of microcystin shock in mice by tumor necrosis factor alpha antiserum.

Microcystis aeruginosa is a common cyanobacterium in water blooms that appear widely in nutrient-rich, fresh, and brackish waters, and its toxic blooms cause the death of domestic animals. The administration of a crude toxic cell extract of M. aeruginosa K-139 to mice can produce tumor necrosis factor (TNF) and prompt severe physiological disturbances, especially liver damage, which can lead to death. The in vitro production of TNF-alpha by peritoneal macrophages was observed after stimulation with the cell extract or the purified toxin from K-139 cells. The expression of a TNF-alpha mRNA was also detected in spleen cells and peritoneal macrophages after stimulation with the cell extract. However, a previous injection of rabbit anti-murine TNF-alpha serum could prevent the liver damage to some extent and protect the mice from death. These findings indicate the involvement of TNF in microcystin shock.