Phenotypic and molecular analysis of six human cell lines derived from patients with plasma cell dyscrasia.

Cell lines RPMI 8226, JJN3, U266 B1, NCI-H929 (all EBV-) and ARH77 and HS-Sultan (both EBV+) have been extensively characterized in this study. EBV- lines expressed the phenotype (CD138-, CD19+, CD20+) whereas EBV+ were (CD138+, CD19-, CD20-). CD56 expression was restricted to EBV- cell lines, with the exception of U266 B1, whereas PCA-1 was strongly expressed on five of the six cell lines. Only EBV+ cell lines bound peanut-agglutinin (PNA). However, all cell lines bound the lectin Jacalin that binds the same receptor as PNA, irrespective of the receptors sialylation status. By RT-PCR and direct sequencing of their IgH V/D/J domains, ARH77 was demonstrated to use the germline sequence VH4-34/dm1/JH6b, whereas no arrangement was demonstrated for RPMI 8226, suggesting IgH gene deletion or mutation. HLA class I and II antigens were detected using HLA typing on all cell lines warranting their use as suitable targets for HLA-restricted cytotoxic T cells. By sensitive RT-PCR, mRNA for IL-6, IL-6R and TNFbeta was found expressed in all cell lines. IL-1 mRNA expression was predominantly associated with the EBV+ phenotype. Although mRNA for IL-3 and GM-CSF was never detected, transcripts for c-kit ligand and, more commonly, its receptor were. Likewise GM-CSF, M-CSF and erythropoietin mRNA transcripts were detected in the majority of cell lines.

Dendritic cells generated from the blood of patients with multiple myeloma are phenotypically and functionally identical to those similarly produced from healthy donors.

Using a combination of GM-CSF, SCF, flk-2/flt-3 ligand, and IL-4, dendritic cells (DC) have been generated in vitro from the adherent fraction of mononuclear cells isolated from the blood of patients with MM. Analysis of cell yield showed no significant difference in DC yield (numbers or percentage of leucocytes) or total number of leucocytes generated in myeloma cultures compared to similar cultures prepared using mononuclear cells from the blood of healthy donors. The mean number of DC produced after 10d of culture were 8.19 x 10(5) and 9.87 x 10(5) cells (41% and 51% of all leucocytes) for the myeloma and normal cultures respectively. Flow cytometry investigation of phenotypic markers including CD1a, HLA-DR, CD80 (BB1/B7.1) and CD86 (B70/B7.2), and functional status (stimulatory potential in allogeneic mixed leucocyte reactions (MLR)) confirmed the generation of cells phenotypically identified as cultured DC. In addition, these cells were more effective than PBMC at stimulating allogeneic PBMC proliferation. These data demonstrate no difference between DC generated from patients with MM and healthy donors. This study was considered a prerequisite for future investigations directed towards developing effective immunotherapies for myeloma.

A comparison between the platelet activating properties of different contrast media used in radiology and MRI.

Previous studies have demonstrated that some intravascular radiographic contrast media (CM) used in angiography, especially non-ionic monomers, may cause platelet activation. This study was designed to elucidate which properties of the CM were responsible for this activity. Platelet activation engendered by CM was studied using flow cytometry to detect platelet degranulation (as CD62 expression) and fibrinogen binding. In order to elucidate the relevant characteristics of the CM responsible, contrast agents of differing structures, properties, formulations and osmolalities were studied; ionic and non-ionic, monomeric and dimeric. Gadolinium chelate MR enhancing agents and saline solutions of differing osmolalities were also investigated. Ionic dimeric sodium meglumine ioxaglate, non-ionic dimeric iodixanol and non-ionic dimeric iotrolan did not produce increased degranulation compared with saline controls. However, all agents produced a mild increase in bound fibrinogen. Experiments using saline solutions demonstrated that these effects are not attributable to the high osmolality of some CM. The broad comparison facilitated by this study shows that previous generalizations regarding platelet activation by CM, based on an ionic-non-ionic division, are not valid. We presume that some chemical structural property of the compounds is responsible and it is of note that the chemically distinct gadolinium chelates, gadolinium DTPA and gadolinium DTPA-BMA, also caused platelet activation to a similar degree. CD62 expression correlated with fibrinogen binding suggesting that at least one common pathway of platelet activation is involved.

Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids.

Inhibition of apoptosis (genetically programmed active cell death) by p210 BCR-ABL expression is a mechanism that might contribute to clonal expansion in chronic myeloid leukaemia (CML). Since cell death following exposure to ionizing radiation and many chemotherapeutic agents can occur by the apoptotic pathway, inhibition of apoptosis would be expected to confer a relative resistance to these treatments. Similarly, cells deprived of growth factors in vitro die by apoptosis, and inhibition of apoptosis would therefore be expected to allow cells to survive better in growth factor-deprived conditions. We found that the survival of normal and CML myeloid progenitors was the same after in vitro incubation in deprived conditions and after treatment with X-irradiation or glucocorticoids. We also found that mature cells in colonies produced by CML progenitors (CFU-GM) did not survive better than those produced by normal progenitor cells. Flow cytometric analysis of propidium iodide-stained cells provided a direct indication that the degree of apoptosis may correspond to the degree of deprivation. These results suggest that inhibition of apoptosis may not be the primary mechanism whereby BCR-ABL influences the expansion of the malignant clone in CML.

The effects of IgG and immune complexes on the endotoxin-induced cytokine response.

Cytokine induction following stimulation by endotoxin (LPS) was investigated in human peripheral whole blood. Blood cells were suspended in autologous plasma diluted in basal medium (BM-plasma) or Compound Sodium Lactate Solution (CSL-plasma), or in autologous serum diluted in CSL. Induction of interleukin 1 beta, interleukin 6 and tumour necrosis factor alpha (IL-1 beta, IL-6 and TNF-alpha, respectively) was investigated following incubation of blood cells with LPS, IgG (Sandoglobulin) alone, or preformed LPS/IgG immune complexes. All three cytokines were induced by LPS alone. With 30 mg/ml Sandoglobulin alone, IL-1 beta production changed little from control, whilst IL-6 production increased markedly in CSL-serum only. TNF-alpha production increased slightly in BM-plasma and CSL-plasma, but not in CSL-serum. Lower concentrations of Sandoglobulin did not affect cytokine production. Upon stimulation with LPS/Sandoglobulin immune complexes, a trend in cytokine production was seen compared with the response to LPS alone: IL-1 beta and IL-6 production increased, whilst TNF-alpha production decreased. This only occurred in CSL-plasma and CSL-serum. Complement activation was present only in CSL-plasma and CSL-serum. Thus in the presence of complement activation and/or serum, Sandoglobulin can induce IL-6 production whilst suppressing the small TNF-alpha response it otherwise stimulates. In addition, when LPS is presented in the form of IgG immune complexes, dissociation of IL-1 beta and IL-6 production from TNF-alpha production is seen but only in the presence of complement activation.(ABSTRACT TRUNCATED AT 250 WORDS)

An assessment of myeloid colony-forming-cell generation in liquid human bone marrow cultures: influence of accessory cells and cytokines IL-1 alpha, beta and IL-6.

Liquid culture of Ficoll isolated human marrow mononuclear cells followed by secondary clonogenic assays has been used to study the involvement of specific accessory cells and cytokines on the kinetics in vitro of human granulocyte-macrophage-colony forming cells (CFU-GM). Primary cultures produced an oscillation in CFU-GM numbers, typified by increases in CFU-GM after 4 and 9 days and reductions on days 2 and 4. Cultures of marrow cells depleted of mononuclear phagocytes produced a steady decline in CFU-GM number over a similar period. Removal of T cells was without effect. Comparison of colony assays using conditioned medium (CM) from the human bladder carcinoma cell line 5637 and 5637CM plus interleukin (IL)-3 revealed the decline of CFU-GM in mononuclear phagocyte depleted cultures due to lack of maturation of early myeloid progenitors. The cytokines IL-6 and IL-1 beta were detectable in the supernatant from whole marrow cultures and from cultures depleted of T cells but not in cultures depleted of mononuclear phagocytes. Using neutralizing polyclonal antibodies directed against IL-1 and IL-6, singly or in combination, in whole marrow cultures we showed that anti-IL-6 antibody alone had no effect on the number of colonies detected in the secondary clonogenic assay whilst anti-IL-1 and anti-IL-1 plus anti-IL-6 reduced colony counts by 44 and 83% respectively. Thus, endogenously produced IL-1 and IL-6 are involved in the dynamic changes in CFU-GM numbers when marrow mononuclear cells are cultured. Possible synergistic interactions are discussed.

TDM35--a new monoclonal antibody to the XH1 cervical carcinoma cell line. Characterization and immunoperoxidase localization in benign and malignant tissues.

The murine monoclonal IgG1 kappa antibody TDM35 was raised against the cervical carcinoma cell line XH1. The antibody recognizes 18.5-66 kDa NCA-like glycoproteins and immunostains a variety of formalin-fixed, paraffin-embedded normal, benign, and malignant tissues. It is of value in the diagnosis of carcinoma of the exocrine pancreas and it identifies foci of squamous and glandular differentiation in other tumours. TDM35 should form a useful addition to a panel of antibodies for the evaluation of epithelial lesions.

The International Reference Reagent for insulin-like growth factor-I.

Three preparations of recombinant DNA-derived insulin-like growth factor-I (IGF-I) were obtained, prepared in ampoules coded 86/522, 86/720 or 87/518, and evaluated as candidate International Reference Reagents in an international collaborative study (nine laboratories in four countries) in response to a request by the World Health Organization (WHO). Immunoassay dose-response curves for each of the three preparations did not in general differ significantly from those of local standards or from those of ampouled preparations of serum-derived IGF-I which were included in the study. The estimates of ampoule contents in terms of local standards showed considerable heterogeneity; the between-laboratory variability of estimates in terms of local standards was ten times greater than the inherent variability of estimates from these systems as estimated from comparisons of coded duplicates. Bioassay data were limited, and those available were inconsistent with immunoassay data. Of the three preparations, ampoules coded 86/720 were derived from an IGF-I preparation that was heterogeneous by high-performance liquid chromatography, and stability data for the preparation 86/522 were anomalous. As a result, the ampouled preparation coded 87/518 has been established by WHO as the International Reference Reagent for IGF-I for immunoassay, with an assigned ampoule content of 3.1 micrograms/ampoule, and is available from the National Institute for Biological Standards and Control.

Detection of host-derived contaminants in products of recombinant DNA technology in E. coli: a comparison of silver-staining and immunoblotting.

Contamination of medicines produced in E. coli by recombinant DNA methodology with host-cell proteins is considered a potential problem with this type of method. In this report techniques for the detection of trace quantities of host-cell proteins in SDS-gel electrophoretograms were examined. Detection of E. coli proteins by immunoblotting, using antisera raised in rabbits to lysates of E. coli, was compared with detection using the ultrasensitive silver stain. Silver staining detected a larger number of E. coli proteins in a one-dimensional electrophoresis system than did immunoblotting. Proteins that were markedly antigenic in the rabbit were detected at a greater sensitivity by the immunoblotting approach. Both techniques detected contaminant proteins in a preparation of methionyl human growth hormone produced in E. coli known to be contaminated with host-cell proteins. No contaminating proteins were seen by either technique in more rigorously purified preparations of growth hormone. A combination of these two approaches would provide useful evidence of purity of medicines produced by recombinant DNA technology, and is potentially applicable to a wide range of host-vector systems.