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Membrane-bound/soluble IL-2 receptor (IL-2R) and levels of IL-1 alpha, IL-2, and IL-6 in the serum and in the PBMC culture supernatants from 17 patients with hematological malignancies.

The present study investigated the peripheral blood mononuclear cells (PBMC) blastic responses to PHA, PHA plus recombinant IL-2 (rIL-2) and rIL-2 alone; the expression of membrane-bound IL-2R on PHA-stimulated PBMC; and the levels of IL-1 alpha, IL-2, IL-6, and sIL-2R in serum and in culture supernatants from PHA-stimulated PBMC in 17 patients with with non-Hodgkin's lymphoma (NHL), 4 with Hodgkin's lymphoma (HL), 5 with Hairy cell leukemia, 1 with chronic myelogenous leukemia, and 1 with chronic lymphocytic leukemia. The patients with HL and NHL with active disease (AD) were separated from those in clinical remission. The patients with AD were studied at diagnosis (obviously before therapy) and the patients in clinical remission were out of therapy since at least 6 mo. The lymphocyte blastogenic response to PHA was significantly lower in patients with HL and NHL with AD than in the control group. The response to rIL-2 alone was in the same range in the control group and in HL and NHL AD patients. By adding rIL-2 to PHA there was an increase of the blastogenic response of the same patients. The percentage of CD25 expressed on PHA-stimulated lymphocytes from patients with HL and NHL AD and from normal subjects is in the same range. Serum levels of IL-2, IL-6, and sIL-2R were significantly higher in HL and NHL AD patients than in controls as well as in all other hematological malignancies. Supernatants derived from PHA-stimulated PBMC were assessed for the presence of cytokines and sIL-2R by ELISA. The levels of IL-2, IL-6, and sIL-2R were significantly lower in HL and NHL AD patients than in controls as well as in all other hematological malignancies.

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Syngeneic anti-idiotypic antibodies eliminate excess radiolabeled idiotypes at experimental radioimmunolocalization.

Significant improvements in tumor/nontumor ratio can be achieved by injections of nonlabeled anti-idiotypic monoclonal antibodies (MAbs) during radioimmunolocalization and radioimmunotherapy using MAbs to target experimental tumors. The in vivo effects of an anti-idiotypic MAb (alpha H7) against a radioiodinated, high affinity, low dissociation rate, monoclonal antiplacental alkaline phosphatase antibody (H7) was investigated. Following in vivo injection of the anti-idiotypic MAb, the radioactivity in experimental tumors was found to decrease only 25% while the reduction of corresponding radioactivity in nontumor tissues amounted to 65-85%, compared to the group receiving no anti-idiotypic MAbs. These results indicate that it is possible to partially clear the circulation and nontumor tissues from excess of radiolabeled idiotypic antibody, without significant decrease in specific tumor localization, increasing the tumor/nontumor ratio three- to fourfold. Circulating nontumor targeting radiolabeled antibodies is one of the major limiting factors in radioimmunotherapy today. Injection of anti-idiotypic MAbs could selectively significantly reduce the radiation dose to radio-sensitive tissues, i.e., bone marrow and intestine, thus improving efficiency in radioimmunoscintigraphy and radioimmunotherapy.

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Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.

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Determination of the kinetics of permeation of dimethyl sulfoxide in isolated corneas

Corneal cryopreservation requires that endothelial cells remain viable and intercellular structure be preserved. High viability levels for cryopreserved endothelial cells have been achieved, but preserving intercellular structure, especially endothelial attachment to Descemet's membrane, has proved difficult. Cell detachment apparently is not caused by ice, suggesting osmotic or chemical mechanisms. Knowledge of the permeation kinetics of cryoprotectants (CPAs) into endothelial cells and stroma is essential for controlling osmotic and chemical activity and achieving adequate tissue permeation prior to cooling. Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess the permeation of dimethyl sulfoxide (DMSO) into isolated rabbit corneas. Corneas with intact epithelia were exposed to isotonic medium or 2.0 mol/L DMSO for 60 min and subsequently transferred to 2.0 or 4.0 mol/L DMSO, respectively, at 22, 0, or -10 degrees C. DMSO concentration in the cornea was measured vs time. The Kedem-Katchalsky model was fitted to the data. Hydraulic permeability (m3/N.s) is 7.1 x 10(-13) + 216%-11% at 22 degrees C, 8.2 x 10(-13) + 235%-21% at 0 degree C, and 1.7 x 10(-14) + 19%-16% at -10 degrees C. The reflection coefficient is 1.0 + 2%-1% at 22 degrees C and 0 degree C, and 0.9 +/- 5% at -10 degrees C. Solute mobility (cm/s) is 5.9 x 10(-6) + 6%-11% at 22 degrees C, 3.1 x 10(-6) + 12%-11% at 0 degree C, and 5.0 x 10(-8) cm/s + 59%-40% at -10 degrees C.

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