Mohsen Abolhassani

Purification of a suppressor lymphokine (SL) from a human T-cell line

Human T leukemia cell line 81-66-45 spontaneously releases into the medium a suppressor lymphokine (SL), able to inhibit PHA-stimulated normal peripheral blood T cell proliferation. Ion exchange and gel filtration chromatography were used successfully to isolate and purify this immunosuppressive lymphokine from culture supernatants. When the purified suppressor lymphokine was characterized with SDS-polyacrylamide gel electrophoresis under reducing conditions, it was found to be a single protein chain of 66,000 daltons. Titration curves of the purified suppressor lymphokine indicated that the inhibitory activity is dose dependent. The suppressor lymphokine is cytostatic and its addition to the peripheral blood lymphocytes (PBL) did not change the cell number or cell viability. This factor was stable at pH 2.0-8.5 and at 56 degrees C for 30 minutes. The structural relationship of this lymphokine with other T cell factors is discussed.

Purification and characterization of a human leukemia cell-derived immunosuppressive factor

A human leukemia cell-derived suppressor factor (LDSF) capable of suppressing in vitro proliferation and activation of normal human lymphocytes was purified from human leukemic HL-60 cells. LDSF is constitutively produced by the cells and was purified from serum free culture supernatant by a combination of ion-exchange chromatography, gel filtration and electrophoresis. Purified LDSF was determined to be a single chain protein with an apparent molecular mass of 66,000 daltons. LDSF was not cytolytic to lymphocytes, was heat stable at 70 degrees C, and did not have any effect on IL-2 or transferrin receptor expression.

Effect of Sendai inducing virus in cytopathic effect inhibition assay of bovine leukocyte interferon.

Various methods for removal or inactivation of Sendai inducing virus from natural bovine leukocyte interferon (IFN) preparations were compared and the effects of Sendai virus in microtiter cytopathic effect (CPE) inhibition assay were investigated. Ultraviolet light and ultracentrifugation at either 108,000 X g or 145,000 X g for 2 h proved to be inadequate methods for complete removal of inducing virus from IFN preparations, as judged by the effects of residual virus in CPE inhibition assay. Optimal methods for inducer virus removal/inactivation were ultracentrifugation at 175,000 X g or dialysis against pH 2.0 buffer. Sendai virus itself was capable of inducing IFN production in assay cells at concentrations commonly found in virus-induced IFN preparations.

Identification of a co-stimulatory factor for human T cell proliferation

A factor enhancing proliferation of human peripheral blood lymphocytes (PBL) was identified in the culture supernatant of a human myeloid cell line HL-60 after ion exchange separation. Partially purified lymphocyte growth enhancing factor (LGEF) was found to enhance mitogen-stimulated PBL or purified T cell proliferation in a dose dependent fashion. LGEF alone did not stimulate PBL or T cell proliferation and its activity was dependent on the presence of mitogen. LGEF mediated T cell proliferation was neither accompanied by an increase in the level of IL-2 receptor expression, nor was dependent on the presence of monocytes. In ELISA assay antibodies against IL-1, IL-2, IL-3 and IL-6 did not recognize the LGEF preparation. The comparison of LGEF with other lymphokines is discussed.

Mechanism of regulating human leukemia cell growth and differentiation by a lymphokine

Human myelogenous leukemia cells can be induced to differentiate in vitro along the monocyte-macrophage pathway by a T cell lymphokine maturation inducer. Maturation inducer has now been purified from a human T cell line and determined to be a single chain protein with an approximate molecular weight of 53,500. It induces the differentiation and proliferation of human leukemic HL-60 promyelocytes in a dosage-dependent fashion. Initial interaction with cells at G1 phase induced the cells to enter proliferating S phase. Subsequent differentiation from S phase was dependent on an optimal inducer quantity (18.7 pM - 18.7 nM) which mediated growth cessation and termination differentiation to monocytes-macrophages. When inducer quantity was more or less than this optimal range, the cells did not undergo differentiation but were continuously stimulated to proliferate. This may represent an important proliferation mechanism of leukemic cells.

Two separate differentiation inducing proteins for human myeloid leukemia cells and their isolation from normal lymphocytes.

Conditioned medium from mitogen stimulated normal peripheral blood lymphocytes (PBL) has been demonstrated to contain a maturation inducer activity mediating the differentiation of human myeloid leukemia cells to monocytes and macrophages. The maturation inducer activity was isolated by salt precipitation, Sepharose CL-6B ion exchange and affinity chromatographies and electrophoresis. Two separate activities with M.W. ranges of 52-56 and 32-35 kDa capable of mediating the terminal differentiation of leukemic HL-60 promyelocytes to monocytes and macrophages were detected. The higher molecular weight species was determined to be a 54 kDa single polypeptide and was found to be distinct from IL-3 and IL-6 by ELISA and differentiation blocking assay. The inducing activity of the 32-35 kDa material was largely neutralized after treatment with anti-IL-3, but not with other anibodies. Employing the immuno-fluorescent antibody technique, the 54 kDa protein was detected on the surface membranes of PBL. The proportions and number of maturation inducer bearing lymphocytes in patients with acute myelogenous leukemia (0.4% and 35/mm , respectively) were significantly lower than that of healthy donors (7.9% and 178/mm ) The role of these physiological factors in leukemia cell differentiation is discussed.

Comparative Tryptic Peptide Analysis Of Rabbit Heavy Chain Allotypes By Hplc

Abstract Reversed-phase, high pressure liquid chromatography (HPLC) in a modified TFA/H2O-TFA/acetonitrile gradient has been successfully applied to the structural analysis of serologlcally defined rabbit immunoglobulln heavy chains. This mobile phase modification yields a relatively flat baseline at high UV sensitivity. Approximately 40 distinct tryptic pep-tides were resolved from each heavy chain, representing the VHa+ (a), a2, and a3) and VHa− (y33,30 and y33,−) immunoglobulin allotypes. About 30 peptides were shown to be derived from the Fc region (CH2 and CH3), and 8–10 peptides from the Fd region (VH1 and CH1). Seven Fd peptides were shared by all VHa+ and VHa− heavy chains. The al and a2 digests each displayed one allotype-specific peptide, whereas no allotype-specific peptides were observed for the a3 heavy chain. No differences were detected between the y33,30 and y33,− peptides; however, both expressed a common y-specific pep-tide. Amino acid analysis of purified al-specific and y-specific pept...

Purification of an acid-stable bovine leukocyte interferon

Bovine leukocyte interferon (BoL-IFN), produced in bovine peripheral blood leukocytes after priming and induction with Sendai virus, was concentrated by precipitation with KSCN (pH 3.5) and purified by gel column chromatography. Recovery of BoL-IFN from precipitation was higher when crude BoL-IFN containing more fetal bovine serum (FBS) was used. However, purity of BoL-IFN recovered from the gel filtration column was highest when crude BoL-IFN with no FBS was used. The use of 25% ethylene glycol in the column elution buffer resulted in over 93% recovery of the applied IFN activity, versus only 25% when buffer contained no ethylene glycol. Column-purified BoL-IFN was further concentrated by ultrafiltration and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in denaturing buffer. When crude BoL-IFN containing no FBS was used for purification, BoL-IFN from a selected column fraction applied to SDS-PAGE resulted in a single narrow band with an apparent molecular weight (MW) of 19,000 Da. Extraction of the SDS-PAGE gel resulted in a single peak of IFN activity indicating identity of the activity and the polypeptide. This proved to be a practical method for obtaining sufficient quantities of purified natural BoL-IFN for use in the production of monoclonal antibodies to BoL-IFN and other biological experiments.

Serologic and biochemical analysis of latent a1 IgG

Immunization of rabbits with anti-allotype antibody (Ab) induces at least two populations of highly cross-reactive molecules. One of these populations bears the nominal VHa allotype of the producing rabbit and is designated the VHa-positive anti-IdX Ab. The other population lacks the expected nominal allotype and thus could represent an induced latent allotype-bearing Ig. To define better the putative latent allotypes, they were subjected to serologic, electron microscopic and biochemical analyses. The induced latent-like population was compared to nominal a1 and found to be indistinguishable in inhibition assays incorporating both rabbit anti-a1 Ab and mouse monoclonal anti-a1 Ab. In contrast, the latent-like Ig was less inhibitory than normal a1 Ig in assays with goat and guinea-pig anti-a1 Ab. The isolated anti-IdX population was less inhibitory than either nominal or latent a1 Ig in all assays. Immunoelectron microscopic analysis indicates that complexes composed of latent-like a1 molecules and Fab anti-a1 ab resemble allotype/anti-allotype (i.e. a1/anti-a1) complexes. Tryptic digests of the putative latent a1 H-chains reveals that these molecules share an a1-specific peptide with digests of nominal a1 H-chain. The peptides from both nominal and latent a1 IgG appear to have blocked N-terminal residues and have a similar though not identical amino acid composition. The composition of these peptides is correlated with the first nine amino acids of the nominal a1 H-chain. The data suggest that the induced latent a1-like molecules share the same major a1 epitope with nominal a1 but may differ in some subtle respects. The possible genetic bases for these observations are discussed.