Human Tumor Necrosis Factor Binding Research Articles

Effect of recombinant human TNF–binding protein-1 and GnRH antagonist on mRNA expression of inflammatory cytokines and adhesion and growth factors in endometrium and endometriosis tissues in baboons

To evaluate the mechanism of action of recombinant human tumor necrosis factor (TNF)-binding protein-1 by assessing differential expression of messenger RNA (mRNA) for cytokines, matrix metalloproteinases, and growth and adhesion factors in baboons. Analysis of gene expression in a prospective randomized study. University Fertility Center. In the in vivo study, 14 baboons were randomly and subcutaneously (SC) treated with either phosphate-buffered saline (PBS), GnRH antagonist, or recombinant human TNF-binding protein-1 at the time of induction. In the ex vivo study, 4 baboons were treated by menstrual endometrium that had been incubated randomly with either PBS or recombinant human TNF-binding protein-1 before intrapelvic injection. In the in vivo study, analysis of 11 endometrial and 10 endometriosis biopsies included either PBS (n = 5), GnRH antagonist (n = 8), or recombinant human TNF-binding protein-1 (n = 8). In the ex vivo study, 2 endometrial and 4 endometriosis biopsies were analyzed from 4 baboons. The mRNA expression of TNF-alpha, IL-8, IL-6, transforming growth factor-beta (TGF-beta), vascular endothelial growth factor, intercellular adhesion molecule-1, matrix metalloproteinase-1, and regulated on activation, normal T-cell expressed and secreted were investigated using real-time reverse transcriptase-polymer chain reaction (PCR). TGF-beta mRNA expression was decreased in endometriotic lesions from baboons treated with recombinant human TNF-binding protein-1 when compared with the placebo group. Except TGF-beta, mRNA expression of inflammatory cytokines and adhesion/growth factors is not affected in endometrial and endometriosis biopsies from baboons after induction of endometriosis combined with either systemic injection of recombinant human TNF-binding or GnRH antagonist or ex vivo treatment with recombinant human TNF-binding protein-1. Further studies are needed to elucidate the mode of action on how inhibition of TNF-alpha activity prevents the development of endometriosis.

Ability of recombinant human TNF binding protein-1 (r-hTBP-1) to inhibit the development of experimentally-induced endometriosis in rats

The aim of this study was to assess whether r-hTBP-1 (recombinant human tumor necrosis factor binding protein-1), the soluble form of tumor necrosis factor-α (TNF) receptor type 1 might be effective in counteracting the proliferation of ectopic endometrium using an in vivo experimental model of endometriosis. The in vivo model involved transplanting a square fragment of autologus uterine tissue onto the inner surface of the abdominal wall in rats. r-hTBP-1 was administered for 1 week at 10 mg/kg, s.c. divided into two daily injections. The gonadotropin-releasing hormone antagonist antide was used for reference and given at the dose of 2 mg/kg, s.c. every 3 days for 1 week. The animals were killed 2 and 9 days after the last treatment and the size of endometriotic implants measured. Blood samples and spleens were also taken for assessment of estradiol-17β levels and natural killer (NK) activity in vitro against murine YAC cells, respectively. The results of this study indicate that r-hTBP-1 is effective in reducing the size of the endometriotic-like foci mainly at the later sacrifice time-point when they were significantly decreased by 64% as compared to control animals. As expected, antide induces an almost complete and statistically significant remission both at the 2-day (94%) and the 9-day (88%); sacrifice time-point. Histological examination indicates that, compared to controls, r-hTBP-1 induces a slightly increased degeneration of the stromal tissues of the implants at both examination times and, limitedly to the earlier observation time, of the mucosal epithelium. No differences in the spleen cell NK activity were observed at either sacrifice time-points in any treatment group. Estradiol-17β concentrations are significantly decreased in the antide-treated groups only at 9 days while no statistically significant changes are found in the animals receiving r-hTBP-1. The results of this study carried out in a rat experimental model of endometriosis provide evidence of the potential effectiveness of r-hTBP-1 in this pathological condition and support the role of TNF in its development.

Interleukin 1 Induces HIV-1 Expression in Chronically Infected U1 Cells: Blockade by Interleukin 1 Receptor Antagonist and Tumor Necrosis Factor Binding Protein Type 1

Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.

Glycosylated recombinant human tumor necrosis factor binding protein-1 reduces mortality, shock, and production of tumor necrosis factor in rabbit Escherichia coli sepsis.

To examine the effect of glycosylated recombinant human tumor necrosis factor binding protein-1 (r-hTNF binding protein-1), the extracellular domain of the tumor necrosis factor receptor p55 produced in mammalian cells, in a rabbit model of circulatory shock due to Escherichia coli. Prospective, randomized, controlled trial. University hospital research laboratory. Eighteen female, New Zealand white rabbits. Anesthetized rabbits, infused with E. coli (10(9) organisms/kg), were pretreated with either r-hTNF binding protein-1 or saline. Mean arterial pressure, central venous pressure, cardiac output, and heart rate were recorded every 20 mins for 1 hr before, and for 4 hrs after, the infusion of E. coli. Blood samples were obtained at 1-hr intervals for platelet count and white blood cell count, r-hTNF binding protein-1, and tumor necrosis factor (TNF) measurements. Administration of r-hTNF binding protein-1 resulted in improvement of mean arterial pressure, cardiac output, and systemic vascular resistance, as compared with the vehicle-treated group (p < .05). Treatment with r-hTNF binding protein-1 was associated with 100% survival, as compared with 55.6% of the saline-treated rabbits (p < .05). Approximately 85% of r-hTNF binding protein-1 was cleared from the circulation 1 hr after the bolus injection (from 171 +/- 27 micrograms/mL at time = 0, to 27 +/- 4 micrograms/mL at 60 mins, decreasing to 6 +/- 2 micrograms/mL for the next 3 hrs). The r-hTNF binding protein-1-treated rabbits had lower serum TNF bioactivity during the first 2 hrs (p < .01). The decreased bioactivity of TNF was confirmed by a specific radioimmunoassay for rabbit TNF. However, at 4 hrs, the vehicle-treated rabbits had lower serum bioactive TNF concentrations (p < .05). The decrease in TNF concentrations in the r-hTNF binding protein-1-treated rabbits resulted from decreased production and, in part, from carry-over of r-hTNF binding protein-1 into the bioassay. Treatment with r-hTNF binding protein-1 improved hemodynamic variables and survival of E. coli-challenged rabbits. Administration of r-hTNF binding protein-1 suppressed bioactivity of TNF in the circulation of these rabbits, and the production of TNF as well.

Design and performance of an ultraviolet resonance Raman spectrometer for proteins and nucleic acids

We describe an ultraviolet resonance Raman (UVRR) spectrometer appropriate for structural studies of biological macromolecules and their assemblies. Instrument design includes the following features: a continuous wave, intracavity doubled, ultraviolet laser source for excitation of the Raman spectrum; a rotating cell (or jet source) for presentation of the sample to the laser beam; a Cassegrain optic with f/1.0 aperture for collection of the Raman scattering; a quartz prism dispersing element for rejection of stray light and Rayleigh scattering; a 0.75-m single grating monochromator for dispersion of the Raman scattering; and a liquid-nitrogen-cooled, charge-coupled device for detection of the Raman photons. The performance of this instrument, assessed on the basis of the observed signal-to-noise ratios, the apparent resolution of closely spaced spectral bands, and the wide spectrometer bandpass of 2200 cm-1, is believed superior to previously described UVRR spectrometers of similar design. Performance characteristics of the instrument are demonstrated in UVRR spectra obtained from standard solvents, p-ethylphenol, which serves as a model for the tyrosine side chain, the DNA nucleotide deoxyguanosine-5'-monophosphate, and the human tumor necrosis factor binding protein, which is considered representative of soluble globular proteins.

Administration route dependency of distribution of PEGylated recombinant human tumor necrosis factor binding protein (rhTNFbp-PEG20K dimer) following i.v. and s.c. injection in rats

Administration route dependency on the distribution of PEGylated recombinant human tumor necrosis factor binding protein (rhTNFbp-PEG20K dimer) was observed following a subcutaneous (sc) and an intravenous (iv) administrationin rats. rhTNFbp-PEG20K dimer is composed of two rhTNFbp molecules (molecular weight 18,278 daltons each) joined by polyethylene glycol 2000 (PEG20K). The steady state distribution volume of rhTNFbp-PEG20K was 55 ml/kg and 359 ml/kg following the i.v. and s.c. administrations, respectively. These results suggest that the distribution of rhTNFbp-PEG20K is limited within the capillary space after i.v. administration, while rhTNFbp-PEG20K can distribute into a space (35.9% of body weight) which is between extracellular space and total body water. A lymphatic absorption may play a role in the distribution of rhTNFbp-PEG20K dimer following the sc administration. The present study suggests that the administration route of large protein molecule should be determined depending upon target sites.

A monoclonal antibody-based enzyme immunoassay for quantitation of human tumor necrosis factor binding protein I, a soluble fragment of the 60 kDa TNF receptor, in biological fluids

Three hybridoma cell lines secreting monoclonal IgG antibodies specific for human tumor necrosis factor-binding protein I (TNF-BP I), the extracellular domain of the 60 kDa TNF receptor, were developed by fusion of spleen cells from mice immunized with TNF-BP I purified from urine. The antibodies recognize three different epitopes on TNF-BP I. Two of the antibodies were used to develop a two-site (‘sandwich’) enzyme immunoassay with horseradish peroxidase as the marker enzyme. The assay was able to measure TNF-BP I in serum, urine and cell culture supernatants with a sensitivity of about 200 ng/1 and a precision better than 10%. TNF-BP I was detected in the serum of healthy individuals at a mean concentration of 2.1±1.0 μg/l(mean± standard deviation; range, 0.52–5.4 μg/l, n=42); no significant difference was seen in patients with chronic polyarthritis (2.3±0.79 μg/l; n=15). Serum TNF-BP I was significantly elevated in patients with burns (6.5±1.7 μg/l; n=10) and markedly increased in patients with renal failure (49±17 μg/l; n=6). TNF-BP I was also detectable in urine from normal individuals (2.2±1.2 μg/l; range 0.78−4.3 μl; n=16). Culture supernatants of several human tumor cell lines also contained TNF-BP I. The assay will be a useful tool to detect activation of the TNF receptor by the physiological ligands, TNF-α and TNF-β, as well as transmodulation by other mediators in various pathological conditions.