Immunoglobulin motif DNA recognition and heterodimerization of the PEBP2/CBF Runt domain.

The polyomavirus enhancer binding protein 2 (PEBP2) or core binding factor (CBF) is a heterodimeric enhancer binding protein that is associated with genetic regulation of hematopoiesis and osteogenesis. Aberrant forms of PEBP2/CBF are implicated in the cause of the acute human leukemias and in a disorder of bone development known as cleidocranial dysplasia. The common denominator in the natural and mutant forms of this protein is a highly conserved domain of PEBP2/CBF alpha, termed the Runt domain (RD), which is responsible for both DNA binding and heterodimerization with the beta subunit of PEBP2/CBF. The three-dimensional structure of the RD bound to DNA has been determined to be an S-type immunoglobulin fold, establishing a structural relationship between the RD and the core DNA binding domains of NF-kappaB, NFAT1, p53 and the STAT proteins. NMR spectroscopy of a 43.6 kD RD-beta-DNA ternary complex identified the surface of the RD in contact with the beta subunit, suggesting a mechanism for the enhancement of RD DNA binding by beta. Analysis of leukemogenic mutants within the RD provides molecular insights into the role of this factor in leukemogenesis and cleidocranial dysplasia.

A two-part phase I trial of high-dose interleukin 2 in combination with soluble (Chinese hamster ovary) interleukin 1 receptor.

Our purpose was to determine the maximum tolerated dose and toxicity associated with soluble Chinese hamster ovary [s(CHO)] recombinant human interleukin (IL) 1 receptor (IL-1R; Immunex, Seattle, WA) administration in humans and to determine the effective biological dose and/or maximum tolerated dose of the s(CHO) IL-1R in combination with high-dose IL-2 as determined by reduction in IL-2 toxicity and modulation of its biological effects. Twenty-seven patients with metastatic cancer were treated with escalating doses of s(CHO) IL-1R at 1, 1, 5, 10, 20, 40, and 55 mg/m2 i.v. on days -6 (except cohort 2), 1, and 15 and IL-2 at doses of 300,000 IU/kg (cohort 1) and 600,000 IU/kg (cohorts 2-7) i.v. every 8 h on days 1-5 and 15-19. No toxicity directly attributable to s(CHO) IL-1R was observed. The median number of IL-2 doses was 23. Hypotension and neurotoxicity were the major dose-limiting toxicities for the IL-2/s(CHO) IL-1R combination. Of the 24 patients treated with full-dose IL-2, there were six responses, three complete and three partial (response rate, 25%). Three patients developed thyroid dysfunction, and all 3 responding melanoma patients exhibited vitiligo. The t1/2 of s(CHO) IL-1R alone was 24-30 h and was not significantly altered by coadministration with IL-2. Whole-blood functional assays indicated that sufficient s(CHO) IL-1R was present in the circulation at top dose levels to inhibit the in vitro effects of IL-1beta on IL-8 induction; however, no effect on IL-2-induced IL-8 induction, or on the IL-1beta- or IL-2-induced tumor necrosis factor production, was observed. Suppression of IL-2-mediated tumor necrosis factor alpha and IL-6 induction in vivo during the first 24 h after IL-2 administration was observed, and the neutrophil chemotactic defect normally seen with IL-2 was not observed. IL-1R antagonist induction far exceeded that seen previously with IL-2 alone. No inhibition of either serum C-reactive protein induction or enhanced urinary nitrate excretion and no consistent effect on IL-2-related changes in peripheral blood mononuclear cell phenotype or endothelial adhesion molecule expression were seen. The coadministration of s(CHO) IL-1R produced no apparent reduction in IL-2 clinical toxicity manifested by either the ability to administer more IL-2 than anticipated or a reduction in the toxicity associated with a given amount of IL-2. Therefore, no effective biological dose could be identified for the s(CHO) IL-1R.

Phase I trial of interleukin 2 in combination with the soluble tumor necrosis factor receptor p75 IgG chimera.

Our purpose was to determine the effective biological dose and/or maximum tolerated dose of recombinant human tumor necrosis factor receptor:IgG chimera (rhuTNFR:Fc; Immunex, Seattle, WA) in combination with interleukin 2 (IL-2) with regard to reduction in IL-2 toxicity and modulation of biological effects of high-dose IL-2 administration. Twenty-four patients with metastatic cancer were treated with escalating doses of rhuTNFR:Fc at 1, 1, 5, 10, and 20 mg/m2 i.v. on days 1 and 15 (dose levels 1-5) or 10, 20, and 30 mg/m2 days 1 and 15 plus 50% dose on days 3, 5, 17, and 19 (dose levels 6-8) prior to IL-2 at doses of 300,000 IU/kg (dose level 1) and 600,000 IU/kg (dose levels 2-8) i.v. every 8 h on days 1-5 and 15-19. The t1/2 of rhuTNFR in patients receiving IL-2 was 72 h. The median number of IL-2 doses was 24, and central nervous system, skin, and cardiac arrhythmias were the major dose-limiting toxicities. TNF bioactivity was inhibited, and the polymorphonuclear leukocyte chemotactic defect normally seen with IL-2 was not observed. Increases in C-reactive protein, IL-6, IL-8, and IL-1 receptor antagonist levels were partially suppressed relative to historical controls, whereas peripheral blood mononuclear cell phenotypes, urinary nitrate, endothelial adhesion molecule expression in skin biopsies, and cellular infiltrates in tumor biopsies were consistent with findings in patients treated with IL-2 alone. Four patients developed thyroid dysfunction. There were five responses: two complete responses (both melanoma) and three partial responses (response rate, 21%). rhuTNFR:Fc may modulate the toxicity and some of the biological effects of IL-2 while preserving antitumor activity. Dose level 6 (10 mg/m2 on days 1 and 15, and 5 mg/m2 on days 3, 5, 17, and 19) has been chosen for a randomized, double-blind, placebo-controlled trial of IL-2 with and without rhuTNFR:Fc.

MRI of silicone vitreous ocular implants with phantom correlation.

We imaged two patients who had silicone oil injected their vitreous cavities. The images demonstrated an increased chemical shift artifact. We developed and tested an in vitro silicone oil model in an attempt to explain this. To our knowledge, this artifact has not been previously described. We constructed a phantom using medication cups containing water, lard and two types of silicone. Background control baths of water and lard were used to simulate different local environments. The phantoms were imaged with T1-and T2-weighted and T1-weighted fat suppression sequences. The artifacts were due to the pronounced chemical shift of silicone in relation to intraconal fat. Our model accurately reproduced our clinical images. Spectral analysis of the silicone model revealed different resonating frequencies for fat/silicone, thereby explaining the pronounced chemical shift artifact.

Very rapid lactate measurement in ischemic perfused hearts using 1H MRS continuous negative echo acquisition during steady-state frequency selective excitation.

Using 1H MRS continuous negative echo acquisition during steady-state frequency selective excitation (CASTLE) myocardial lactate accumulation was followed in a globally ischemic perfused rat heart model. 1H MRS CASTLE derived lactate determinations were verified biochemically and were measured during ischemia and reperfusion (both in the absence and in the presence of a known inhibitor of glycolysis). In addition, using the Bloch equations modified for the effect of diffusion in the presence of a magnetic field gradient the theoretical dependency of measurements made with CASTLE upon T1, T2 and the flip angle alpha were demonstrated. It was found that 1H MRS CASTLE allowed for rapid identification of the lactate -CH3 resonance in an isolated perfused heart with little shimming required, and excellent water and lipid suppression. Measurements of lactate using this technique reflected a true difference in myocardial lactate as evidenced by biochemical analysis and the expected changes in tissue lactate that accompanied reperfusion and ischemia in the presence of a glycolytic inhibitor. Theoretical calculation demonstrated that the dependency of the relative signal intensity obtained with 1H MRS CASTLE was a complex function of T1, T2, and alpha. These calculations also demonstrated the theoretical feasibility of applying 1H MRS CASTLE to localized spectroscopy using a surface coil.

Protein and lysozyme content of adult human nucleus pulposus.

A radiologically normal human nucleus pulposus was extracted with 4 M guanidinium chloride and the non-collagenous proteins separated from the proteoglycans by dissociative density gradient centrifugation. Lysozyme was identified as a matrix constituent of the normal, mature human nucleus pulposus.