Philip R. Glade

Anti-MIF activity of serum from a rabbit immunized with human B-lymphoid cell line migration inhibitory factor.

Abstract Antiserum with reactivity for human B-lymphoid cell line MIF has been developed after multiple injections of a rabbit with material obtained from large quantities of supernatant from the long-term cultured human lymphoid cell line PGLC-33H. The migration inhibitory factor (MIF) for immunization was initially separated by Sephadex G-75 gel filtration and the active fraction between molecular weight markers of 25,700 and 14,300 was further purified by preparative polyacrylamide-gel electrophoresis. The antiserum has no direct cytotoxicity for MIF target cells and will not directly block the effects of active MIF on these cells. Reactivity for MIF was detected by the capacity of immunoglobulins to alter the elution profile of MIF on Sephadex G-75 and to remove MIF activity when active supernatants were exposed to immunoabsorbents prepared with antisera. This reactivity resided in the 50% (NH4)2SO4 precipitates and DEAE-separated IgG fractions of the antiserum but not in the DEAE-separated IgM fraction. Immunoelectrophoresis of the partially purified MIF with diffusion against the antiserum resulted in a unique precipitin arc in the β2 region of the gel. The β2 precipitin showed no cross-reactivity to human transferrin, hemopexin, C ′3 C ′3 c , κ light chain, λ light chain, μ heavy chain, β2-glycoprotein II, β2-glycoprotein II, β2-glycoprotein III, and β2-microglobulin.

The role of cellular immunity in neoplasia.

32. This paper was presented at a Symposium on Childhood Cancer at the Annual 34. Requests for reprints should be addressed to: A. G. Knudson, Jr., M.D., Meeting of the American Pediatric Society, Denver. Colorado. April 17. 1975. Graduate School of Biomedical Sciences, University of Texas, Health Sciences 33. This research was supported in part by Medical Genetics Center Grant no. Center, Houston, Texas 77030 (USA). Center. Houston. Tex. 77030 (USA). 35. Accepted for publication July 25. 1975.

Antigen responsiveness of an established human B-lymphoid cell line I. Evidence for a surface receptor for Candida antigen detected by direct migration inhibition

Abstract Responsiveness to a soluble crude Candida albicans antigen (CaAg) by a long-term human lymphoid cell line with B-cell characteristics (PGLC-33H) is demonstrated by the direct migration inhibition (DMI) assay. An inability to detect comparable CaAg responses in other tested B- and T-lymphoid cell lines suggests that this may be a specific response for PGLC-33H cells. CaAg responsiveness is completely abrogated by trypsinization of cells. Regeneration of CaAg responsiveness is temperature dependent, detectable in 8–14 hr in cells maintained at 37°C. Trypsinized cells kept at 4°C do not regain CaAg response. The rate of cell-CaAg interaction is rapid and not temperature dependent. Cells pulsed with CaAg at 37, 25, and 4°C demonstrate comparable DMI responses following a 2-min pulse with CaAg. These findings suggest that PGLC-33H cells have surface receptors for CaAg which result in the inhibition of cell migration upon exposure to antigen.

Intracellular Localization of the Migration Inhibitory Factor (MIF) in a Long-Term Human Lymphoid Cell Line

Subcellular fractions of the human lymphoid cell line PGLC-33H were obtained by N2 cavitation and differential centrifugation. The purity of the fractions was assessed by the use of the following marker enzymes: beta-glucuronidase for the lysosomal-intermediate fraction; a nonspecific esterase for the microsomal fraction; and LDH for the supernatant fraction. These subcellular fractions were studied for MIF activity utilizing human lymphoid cells from established lines as target cells. MIF activity was most consistently found in the microsomal fraction. Also, MIF activity was closely associated with the relative specific activity of exterase. No such correlation with MIF activity could be demonstrated for beta-glucuronidase or LDH.

Antigen responsiveness of an established human B-lymphoid cell line. II. Loss of Candida antigen responsiveness following exposure of cells to specific anti-human immunoglobulin antisera.

Abstract PGLC-33H, a human B-lymphoid cell line, has demonstrated responsiveness to a soluble crude yeast antigen (CaAg), by the direct migration inhibition assay. Complete loss of CaAg responsiveness in PGLC-33H cells is detected following cocultivation of cells with anti-human μ chain antiserum and to a lesser extent with antihuman κ chain antiserum. Exposure of cells to other anti-human heavy and light chain antisera had no effect on CaAg responsiveness of the PGLC-33H cells. Following a 20-min exposure to anti-human μ chain antiserum at 37°C, but not at 0°C, PGLC-33H cells lost CaAg responsiveness. Regeneration of CaAg responsiveness is detectable within 24 hr in cells maintained at 37°C. These results suggest that the μ heavy chain and to a lesser extent the κ light chain or structures closely associated with these molecules are the CaAg membrane receptors on PGLC-33H cells.

Evidence for a surface receptor for human migration inhibitory factor on a B-lymphoid cell line.

Abstract Reception by PGLC-33H target cells for the migration inhibitory factor (MIF) produced by this established line has been investigated by pulse time and temperature dependence, MIF absorption, and abrogation by trypsinization. PGLC-33H supernatants containing MIF were concentrated 5× with Carbowax and dialyzed against serum free RPMI-1640 before use. Prior to standard capillary migration assay a minimum 30 min pulse of MIF at 37 °C is required for significant migration inhibition (MI > 20%). No significant MI is observed when cells are pulsed at 4 °C for up to 2 hr. Preincubation with PGLC-33H for 1 hr at 37 °C reduces activity of supernatants from 38 to 13% MI; at 4 °C to 27% MI. Trypsinization of target cells for 30 min at 25 °C abrogates response to MIF (43 to −14% MI). Trypsinized cells did not reduce activity of supernatants. MIF activity is abolished (32 to 3% MI) in samples preincubated with supernatants of the trypsinized cells inactivated with serum. These data suggest that cells from the human B-lymphoid cell line PGLC-33H have a surface receptor for human MIF.

High pyridoxine diet in the rate: Implications for clinical megavitamin therapy

Megavitamin therapy has found increased clinical application. Although the toxicity of fat soluble vitamins is well established, little has been learned of the actions and fate of massive doses of the water soluble vitamins. Current interest in the use of vitamin B6 in the treatment of homocystinuria due to cystathionine synthase deficiency prompted us to explore its mode of action and metabolic side effects in the rat. Large amounts of pyridoxine in the diet had no effect on appetite but resulted in a 20% increase in body weight and 40% in liver weight of pair-fed controls. The fat/protein ratio and the percentage of each in liver was unchanged despite the increased liver weight. The weights of brain, pancreas and kidney were unaffected. There appeared to be an increase in peritoneal fat, however, the epiyimal fat pads were significantly smaller in the high-pyridoxine group becuase of smaller cell size. Concentrations of free amino acids were unchanged as were the activities of cystathionine synthase and cystathionase. Incorporation of 35S from both methionine and cystine into the proteins of various organs were also unchanged. In the rats on high-pyridoxine diets there was greater incorporation of 35S from cystine into reduced glutathione and less into oxidized glutathione. Since the amounts of pyridoxine consumed by the experimental group was of the order of magnitude as that currently used in the treatment of homocystinuria, further studies are needed before it can be assumed that massive doses of water soluble vitamins can be used with impunity.