Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line.

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l(-1) human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11(*) cells which had been engineered to express a humanised antibody, CAMPATH(*)-1H, were routinely grown using serum-containing medium. From a seeding density of 10(5) cells ml(-1), cells grown in static culture with serum reached a maximal cell density of 6.5×10(5) cells ml(-1) after 6 days in culture and produced a maximal antibody concentration of 69 mg l(-1) after 11 days in culture. CHO 3D11(*) cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10(5) cells ml(-1) reached a maximal viable cell density of 2.16×10(6) cells ml(-1) after 108 h in culture and a maximal antibody concentration of 131.1 mg l(-1) after 122 h in culture.

Immunohistochemical analysis of CDw52 antigen expression in non-Hodgkin's lymphomas.

AIM: To determine the antigen expression of CDw52 using Campath-1 antibodies in a series of non-Hodgkin's lymphomas (NHLs). METHODS: Tissue sections of lymphoma were stained immunohistochemically using rat Campath-1G and humanised Campath-1H with avidin-biotin-peroxidase complex techniques. Fifty-two fresh frozen lymphomas and a further 26 paraffin wax embedded sections were studied. RESULTS: Thirty-seven out of 41 B cell lymphomas were positive with Campath-1H in frozen sections (low grade, 24 of 24; high grade, 13 of 17) as were three out of five T cell lymphomas. Reed-Sternberg cells in six cases of Hodgkin's disease did not react. Eleven out of 16 high grade B cell lymphomas also stained positively with Campath-1G in paraffin wax sections as did five out of 10 T cell lymphomas. CONCLUSIONS: The Campath-1 antibodies showed that CDw52 antigen expression was present in all cases of low grade B cell NHL examined. Immunohistochemical staining in high grade B cell NHL and in T cell NHL was variable. These findings may be relevant to patient selection when considering treatment with Campath-1 antibodies.

Regulation of tumor necrosis factor production by adrenaline and beta-adrenergic agonists.

The effects of adrenaline and isoproterenol, a specific beta-adrenergic agonist, on TNF production were investigated. Both agents inhibited the production of TNF by human blood and THP-1 cells stimulated by LPS. The effect of adrenaline was prevented by a beta-receptor antagonist, but not by an alpha-receptor antagonist. Levels of TNF mRNA were not reduced by adrenaline. Inhibition of TNF production was observed only if cells were first exposed to adrenaline or isoproterenol at about the same time as to LPS; incubation of THP-1 cells with isoproterenol for 24 h before LPS stimulation dramatically increased response, and prevented suppression of TNF production by a second dose of isoproterenol. Intracellular cAMP levels were increased by adrenaline and isoproterenol, at concentrations that inhibited TNF production. However, prolonged incubation of THP-1 cells with isoproterenol resulted in depression of cAMP concentrations to below basal levels. These data suggest that TNF production can be regulated by beta-receptor stimulation, that such regulation is mediated by changes in intracellular cAMP concentrations and is exerted at a posttranscriptional level. Adrenaline may be an important endogenous regulator of TNF production in sepsis.

A convenient human whole blood culture system for studying the regulation of tumour necrosis factor release by bacterial lipopolysaccharide.

Bacterial lipopolysaccharide (LPS, endotoxin) induces a dose-dependent release of TNF in whole human blood which has been diluted five-fold. It is modulated by interferon-gamma, prostaglandin E2 and indomethacin in the same manner as observed with tumour necrosis factor (TNF) release from human monocyte/macrophage cells cultured in vitro. The whole blood culture system (WBCS) can provide up to 250 samples from 10 ml of venous blood and enables an individual blood to be assessed in terms of TNF inducibility and its modulation by other biological agents. The whole blood culture system was used to demonstrate the individual variation between blood donors. The results demonstrated that the information provided by induced cytokine release and its regulation in the ex vivo system would be a valuable addition to that obtained from in vitro methods.

A comparison of specificity and biological activity of polyclonal and monoclonal antibodies raised against Salmonella minnesota R595 lipopolysaccharide.

Murine monoclonal antibodies (MAbs) and immune rabbit serum were raised against the rough mutant of Salmonella minnesota strain R595. These antibodies were tested for their ability to inhibit LPS-induced B-cell mitogenicity and neutralise LPS toxicity in chick embryos. Immune rabbit serum inhibited both mitogenicity and LPS lethality. None of the MAbs or a cocktail of antibodies were able to neutralise LPS lethality in chick embryos. However, they were able to inhibit mitogenicity by varying degrees.

Comparison of the opsonic activity of polyclonal and monoclonal antibodies raised against Salmonella minnesota strain R595.

Murine monoclonal antibodies and immune rabbit serum were raised against the rough mutant Salmonella minnesota strain R595. These antibodies were tested for their opsonic activity against the homologous strain and the smooth wild type S. minnesota by luminol-dependent chemiluminescence and a microscopic assessment of phagocytosis. Immune rabbit serum opsonised both strains. Treatment with normal rabbit serum inhibited the phagocytic uptake of S. minnesota R595. None of the monoclonal antibodies RE01 (anti-KDO), RE12 (anti-KDO) and RE23 (anti-lipid A) were opsonic. Unopsonised S. minnesota R595 stimulated marked chemiluminescence possibly because of its hydrophobic surface, but this was not reflected in increased uptake by phagocytic cells. Results obtained with luminol-dependent chemiluminescence should be interpreted with caution when the opsonisation of rough bacterial strains or those with high surface hydrophobicity is being investigated.

Increase and inhibition of pre-B cell proliferation in culture, by T cells.

T cells may either increase or decrease in vitro proliferation of marrow pre-B cells from patients with acute lymphatic leukaemia after withdrawal of successful treatment. There is less proliferation when T cells are removed by E rosetting, and repletion of T cells restores proliferation. When additional T cells from the patients were added to the patients' marrows, proliferation was increased more effectively than with T cells from healthy subjects; there was no evidence of an allogeneic effect. In contrast, normal T cells stimulated with concanavalin A suppress proliferation. There was no evidence of differentiation into B cells.

Growth of pre-B cells in cultures of bone marrow from children with acute lymphoblastic leukaemia and other diseases.

Pre-B cells from the bone marrow of children with acute lymphoblastic leukaemia (ALL) survived up to 144 hr after the completion of treatment and divided in culture with maximum cell numbers at 24 hr. There was no rise in B cell number and no evidence of differentiation from pre-B to B cells. Binucleated pre-B cells in cultures containing cytochalasin B confirmed that pre-B cell division was occurring. Cycloheximide reduced cell numbers in culture but bromodeoxyuridine did not. Pre-B cell numbers also increased in culture of morphologically normal marrows from treated and untreated patients with solid tumours, and probably in normal marrows from patients with non-malignant diseases.

Immunosuppression and serious infections in children with acute lymphoblastic leukaemia: a comparison of three chemotherapy regimes.

Eighty-four children presenting with acute lymphoblastic leukaemia were entered into a trial designed to test the effect on host toxicity of regular drug-free periods during chemotherapy. Patients received the same total dose of drugs either continuously (daily), intermittently (a 5 d course every 3 weeks) or in an intermediate way between these two (a 14 d course followed by a 7 d gap). Mean neutrophil counts were lower in the intermittent group and fell significantly at 6 week intervals, after courses which included prednisolone and vincristine in addition to methotrexate and 6-mercaptopurine. Mean lymphocyte counts, mitotic response to phytohaemagglutinin and plasma immunoglobulin levels were significantly lower in the continuous group. The results in the intermediate group fell between those of the other two groups. All six remission deaths occurred in the 42 patients in the continuous group, who had a much higher incidence of infections (mostly viral and protozoal) than the other two groups. It is concluded that the intermittent chemotherapy schedule permits the maintenance of a lymphocyte population size and function which provides a satisfactory level of defence against infection without prejudice to its anti-leukaemic effect.

Pre-B and B cells in children on leukaemia remission maintenance treatment.

The percentage of pre-B cells, mature B cells and IgM plasma cells were reduced in the marrow of children receiving continuous cytotoxic drug treatment to maintain leukaemia remission, compared with children receiving intermittent drug treatment in the UKALL V trial or untreated controls. When treatment was ended, the proportion of marrow pre-B cells rose above that of the controls and remained elevated for more than 6 months. These observations define more precisely the cellular basis of suppression of antibody immunity by cytotoxic drugs. They also suggest the existence of a complex feedback control of pre-B cell numbers and B cell differentiation during recovery.

Expression of hexosaminidase isoenzymes in childhood leukemia.

Because differences in hexosaminidase isoenzyme profiles of granulocytes and lymphoyctes suggested that such profiles might help to distinguish between various types of leukemia, was examined leukocyte extracts from 55 untreated children and 12 controls by automated anion-exchange chromatography. In 23 of 27 cases of the common form of childhood acute lymphoblastic leukemia, the activity of hexosaminidase component I was greatly increased; the activity ratio of hexosaminidase I to hexosaminidase A was greater than 0.5 in 18 of these cases, whereas for other types of leukemia and for all normal cells tested, the ratio was less than 0.2. A raised hexosaminidase I was shown to be associated with leukemic cells by the finding of normal isoenzyme profiles of bone-marrow cells from 17 patients in remission who had such an increase either at diagnosis or in relapse. Hexosaminidase isoenzyme analysis had diagnostic value and may provide a new marker for study of leukocyte differentiation.

Acute lymphoblastic leukaemia in children: Classification and prognosis.

Immunological (surface-marker) tests have been used to define four subgroups of acute lymphoblastic leukaemia (A.L.L.) in childhood: T-A.L.L., B-A.L.L., common-A.L.L., and null-A.L.L. A study of 94 children shows that the common-A.L.L. subgroup achieves a much longer duration of remission than T-A.L.L.; our findings also confirm the association of some clinical features with T-cell A.L.L. Within the common-A.L.L. subgroup, initial white-cell count correlates with prognosis.