Kwong Y. Tsang

In vitro restoration of immune responses in aging humans by isoprinosine.

The in vitro effects of isoprinosine (ISO) on the immune responses of aging humans were investigated. 64 healthy elderly humans (65 yr of age or over) were included in this study. Four immune parameters were measured, namely, Concanavalin A (ConA)-induced lymphocyte proliferation, natural killer cell (NK) activity, neutrophil chemotaxis, and interleukin-2 (IL-2) production. The ConA-induced lymphocyte proliferation was depressed in 55 of the 64 individuals (85.9%%), while the NK activity was depressed in 41 of the 64 individuals (64%). Neutrophil chemotaxis was depressed in 52 of the 64 individuals (81.1%) and IL-2 production was depressed in 35 of the 64 individuals (54.6%). In the presence of ISO, ConA-induced lymphocyte proliferation, NK activity, neutrophil chemotaxis, and IL-2 production were restored to normal or near normal levels in 50 of the 55 (90.0%), 35 of the 41 (85.3%), 44 of the 52 (84.6%), and 25 of the 35 (71.4%) aging humans, respectively. Our results indicate that ISO acts as an immune potentiator in these in vitro immune assays.

Purification of IgM monoclonal antibody from murine ascitic fluid by a two-step column chromatography procedure

Two IgM monoclonal antibodies (Mabs) were purified from murine ascitic fluid by a two-step procedure involving pseudoligand affinity chromatography on immobilized Cibacron Blue F3GA and gel filtration chromatography on ACA-22. The purified IgM from one ascites sample, H-1, was determined to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while ascites containing E3.1 IgM Mab, was purified to greater than 95% IgM. The retention of biological activity was confirmed by immunofluorescence staining of peripheral blood lymphocytes.

Induction of immunity to a human osteosarcoma-associated antigen in mice using anti-idiotypic antibodies

Polyclonal rabbit anti-idiotypic antibodies (Ab2) were produced and analyzed for their ability to stimulate humoral immunity against a human tumor-associated antigen (TAA) in BALB/c mice. Murine monoclonal antibody (Mab) OSA-1 recognizes an 85,000-Da TAA present on both human osteosarcoma tissue and osteosarcoma cell lines. Rabbits were immunized with OSA-1 (Ab1) to produce Ab2. The polyclonal Ab2 were shown to react against an idiotope located at or near the antigen combining site of Ab1. Ab2 were demonstrated to be potent inhibitors of TAA binding to Ab1. BALB/c mice were immunized with this Ab2 preparation and then tested for the presence of osteosarcoma TAA reactive antibodies. Sera from Ab2-immunized mice were shown by Western blot to contain antibodies whose specificity resembled Ab1. Thus, immunization with polyclonal rabbit Ab2 was shown to stimulate production of Ab3 in mice which reacted against a human osteosarcoma TAA.

Isoprinosine effects on interleukin-1 production in acquired immune deficiency syndrome (AIDS).

The in vitro effects of isoprinosine (ISO) on the production of IL-1 in AIDS patients and normal controls were investigated in this study. IL-1 production from adherent cells was measured by an indirect method using EL-4 cells. Five of eleven AIDS patients had depressed IL-1 production. Various concentrations of ISO were used to treat the adherent cells in vitro and the optimal concentration for stimulating IL-1 production was determined to be 100 micrograms/ml/10(6) cells. IL-1 production was augmented to normal or to near normal levels in four of five AIDS patients. Our results indicate that depressed immunity seen in some AIDS patients may be partly due to the depressed in IL-1 production and also that ISO can act as an immune potentiation in enhancing the production of this lymphokine in vitro.

Altered configuration of Gc on the plasma membrane of transformed and malignant human B lymphocytes

Normal human peripheral blood B cells exhibit strong membrane fluorescence for Gc (vitamin D-binding protein), and this protein can form a close spatial relationship with integral membrane immunoglobulin (mIg) with evidence of codistribution in the lipid bilayer. In contrast, fluorescence for both Gc and mIg has been found in this study to be weak or absent in several B lymphoblastoid cell lines and in chronic lymphocytic leukemia B cells. Moreover, the comobility of these components, where detectable, was also impaired. In abnormal B cells, the intensity of membrane fluorescence for Gc was substantially increased after crosslinking of mIg with antibody, and the latter was also associated with increased specific radioiodination of Gc by lactoperioxidase. These results indicate that Gc can apparently become displaced under certain circumstances within or through the lipid bilayer. The altered content or membrane topography of Gc in such abnormal B cells might be associated with impaired expression and mobility of mIg.

Inhibition of interleukin-2-induced T-cell proliferation by sera from patients with the acquired immune deficiency syndrome.

Sera from 22 patients with either lymphadenopathy syndrome (LAS), acquired immune deficiency syndrome (AIDS)-related complex (ARC), or acquired immune deficiency syndrome were examined for their effect on the interleukin-2 (IL-2)-induced proliferative response of an IL-2-dependent cytotoxic T-cell line, CTL-20. All of the patient sera included in this study were positive for the presence of antibodies against human T-cell lymphotropic virus type III (HTLV-III) as determined by an HTLV-III-specific enzyme-linked immunosorbent assay (ELISA). Eighteen of the 22 patient sera examined (81.8%) exhibited at least a modest suppressive effect on the proliferative response of CTL-20 cells. The inhibitory effect was dose-dependent and varied in intensity for each individual serum. In many cases, the magnitude of suppression was absolute in that it totally abrogated IL-2-induced DNA synthesis. Normal human serum (NHS) exerted no suppressive influence on the IL-2-induced proliferative response of identical control cultures. This same panel of 22 patient sera exhibited no significant inhibitory effects on the levels of protein synthesis in cultures of a non-IL-2-dependent human T-cell line, CCRF-HSB-2, indicating that the suppressive effect was not mediated by nonspecific serum cytotoxicity. The inhibitory effect of patient sera in the IL-2-dependent target cell assay correlated with the ability of these same sera to suppress the mitogen-induced proliferative response of normal human peripheral blood lymphocytes (PBL). These observations are particularly striking in view of the recognized defects of IL-2-dependent effector T-cell functions in AIDS.

Effect of isoprinosine on sialylation of interleukin-2☆

The effects of Isoprinosine (ISO) on interleukin-2 (IL-2) production by human peripheral blood mononuclear cells (PBMC) were investigated. Treatment (of human PBMC) with ISO enhanced IL-2 production by PBMC from 7 of 10 normal individuals. However, no augmentation of IL-2 production was observed when cultures of HUT-78 cells, a human leukemic T cell line, were treated with ISO. IL-2 purified from supernatants of human PBMC treated with ISO exhibited pI values of 5.5 and 6.4. IL-2 prepared from untreated PBMC exhibited a single pI value of 8.2. The pI value of IL-2 prepared from ISO-treated PBMC shifted to 8.2 after treatment with neuraminidase, demonstrating that the IL-2 molecules isolated from ISO-treated PBMC possessed sialic acid. The pI values of the IL-2 isolated from ISO-treated and untreated HUT-78 culture supernatants were identical (pI = 7.8) and were not modified by neuraminidase treatment. These results suggest that the increase in IL-2 production following treatment of PBMC with ISO may be mediated through the activation of a distinct subset of IL-2 producing cells. Furthermore, the sialylation of IL-2 may be of physiologic and immunopharmacologic importance.

Partial purification and characterization of B cell growth factor constitutively secreted by human T-cell hybridoma

Human T cell hybridomas were established by fusion of PHA-activated PBL with the 8-azaguanine resistant human T-leukemic cell line CEM-CM3. High levels of B cell growth factor (BCGF) activity were detected in the supernatants of hybridoma C8-2B2 and its subclones. Hybridoma C8-2B2, in addition to the Leu 3a, also expressed the OKT11 surface marker which was not detectable on the parent CEM-CM3 cells. BCGF from the culture supernatant was purified by combined use of salt fractionation and gel filtration to 36.6 fold with 23.9% recovery of activity. The BCGF produced by hybridoma C8-2B2 has a molecular weight range of 16,000-20,000 in two major electrophoretically different forms with pI values of 6.4 and 7.4.

Production of antibody to human osteosarcoma associated antigens by continuous human lymphoblastoid cell lines

Human lymphoblastoid cell lines that produce specific antibody against human osteosarcoma associated plasma membrane antigens have been established by preselecting OSAA binding human B lymphocytes from an osteosarcoma patient followed by Epstein-Barr virus (EBV) transformation. Four cell lines ( Kla -1, Kla -2, Kla -3, Kla -4) were obtained and cloned. The antibodies were lambda light containing IgM. The specificities of the antibodies were confirmed by immunofluorescence assays using tumor cell lines of various histologic types. Positive immunofluorescence was observed with all human osteosarcoma cell lines tested except one but not with tumor cell lines of other histologic types.

Cultivation of human osteosarcoma cell lines in serum-free hormone supplemented medium

SummaryTwo human osteosarcoma cell lines (TE-85, LM-1) were adapted to grow in serum-free hydrocortisone supplemented medium. Human osteosarcoma associated antigens were detected both on the cell membrane as well as in the concentrated culture medium. TE-85 cells produced bone specific alkaline phosphatase and LM-1 cells produced both bone specific and placental-like alkaline phosphatases when cultured in this medium. Successful proliferation of human osteosarcoma cell lines in serum-free medium is valuable in human osteosarcoma research.