Inhibition of Fc epsilon RI-mediated activation of mast cells by 2,3,4-trihydropyrimidino[2,1-a]isoquinolines.

Assays based on reporter gene technology represent today an important tool in the pharmaceutical industry for discovering novel compound classes interfering with the activation and signaling of target cells after stimulation. Here we describe a reporter gene assay targeting mast cell activation of IgE plus antigen, established in an attempt to identify substances preventing type I allergy (allergic rhinitis, allergic conjunctivitis, allergic asthma, and acute and chronic urticaria). The assay is based on a murine mast cell line designated CPII, stimulation by IgE plus antigen, and a reporter gene construct with the TNF alpha promoter linked to luciferase as a read-out system. Via screening about 50,000 substances, compound 2 was found to inhibit the reporter gene induction in the submicromolar range in this assay. Analogues of compound 2 of the 2,3,4-trihydropyrimidino[2,1-a]isoquinoline type were synthesized starting from 2-alkyl-substituted benzonitriles via aminolysis with 1,3-diaminopropane, dimetalation of 2-substituted 2-phenyl-1,4,5,6-tetrahydropyrimidines with n- and sec-butylithium, reaction with carboxylic acid methyl esters, and finally acidic dehydration. From about 50 derivatives, compound 41 was selected as a lead structure with an IC50 of 0.2 microM and a TC50 of 2.7 microM. In a first profiling in secondary assays, it effectively interfered with the production of mediators such as TNF alpha, IL-4, IL-6, IL-13, and leukotriene synthesis as measured by the corresponding ELISAs. In addition, a passive cutaneous anaphylaxis in mice (a typical type I reaction) is inhibited to more than 90% by compound 41, when administered intradermally 90 min before challenge.

Accelerating the pace of luciferase reporter gene assays.

Luciferase reporter gene assays have gained more importance because of their easy readout, high sensitivity and lack of environmental waste disposal problems. However, several obstacles remain that have prohibited a wider use and the implementation of this type of assay in high-throughput screening programs: (i) Measurements need to be carried out within an active enzyme reaction, and the assessment of such reactions are time-dependent; (ii) the signal produced has a "flash" type characteristic and therefore requires specialized equipment for measurement; and (iii) side-reactions can occur that interact with the signal readout of the assay in a non-reproducible way. These hurdles make an otherwise convenient assay principle troublesome for larger-scale screening use. We have attempted to overcome these problems by different means, leading to the development of LucLite, a stable signal homogeneous reagent system. This system allows use in a higher throughput screening capacity and enables the use of standard scintillation/luminescence instruments.

Establishment and phenotypic characterization of three new human myeloma cell lines (U-1957, U-1958, and U-1996).

Three new human myeloma cell lines (U-1957, U-1958, and U-1996) have been established in vitro. The cell lines are Epstein-Barr virus (EBV) negative, monoclonal, and aneuploid and should thus represent malignant cell populations and not EBV-carrying non-neoplastic B lymphoblastoid cell lines. The myeloma origin of the cell lines is also suggested by their capacity for production of monoclonal complete immunoglobulin (Ig) molecules (U-1957 and U-1958) or IgG light chains (U-1996) of the same type as the myeloma protein in vivo. All the cell lines have morphological features of plasmablasts-plasma cells but appear to represent slightly different stages of B cell differentiation. Thus, the U-1958 has plasma cell morphology, expresses only PCA-1 and OKT-10 but no other B cell antigens, and secretes 1.5 micrograms/mL of IgG/10(6) cells/24 hours. The U-1957 has plasma cell morphology and expresses Fc receptors and the LB-1 antigen in addition to the PCA-1 and OKT-10 antigens. This line produces only minimal amounts of IgG, which appears not to be secreted. The U-1996, finally, is a kappa light chain producer, has a plasmablast morphology, and expresses LB-1 in addition to the PCA-1 and OKT-10 antigens. All three cell lines are chromosomally heterogeneous and contain several markers with a 14q+ abnormality as a common characteristic abnormality. These new myeloma lines have been in continuous culture for approximately 3 years and are instrumental in studies of various aspects of the biology of human myeloma.

Construction of a large scale membrane reactor system with different compartments for cells, medium and product.

A flat membrane reactor system has been designed where different membranes separate cells from medium and cells from product, respectively. By the use of this system the product can be enriched and partially purified within the reactor. Like other membrane systems perfect protection from mechanical stress of surface adherent as well as suspension type-cells is achieved. The scale up of the design is possible in a wide range. The prototype construction corresponds to a conventional reactor of appr. 300 l and contains a membrane area of 25 m2. Beside the three chamber operation mode it is possible to operate the system as a two chamber reactor. With slightly modified gaskets the reactor resembles a new type of tube reactor, where the cells can be refed on their way through the 50 m long tube.

Use of a rotating wire cage for retention of animal cells in a perfusion fermentor.

Separation of cells within a perfused suspension system is still a problem. We modified the rotating steel wire cage described by other researchers (2) for separation of single cells from suspension. The system was mounted to the stirrer shaft of a circular-loop fermenter. After optimization of fermenter design experimental data showed good separation of cells at useful perfusion rates. Data of a 5 l-prototype show with a mammalian tumor cell line a retention rate of better than 95% at a perfusion rate of D = 0.05. Scaling-up studies show a good scaling-up potential and a wide range of possible applications.

Use of a dynamic filtration method for separation of animal cells.

For solving the problem of concentrating and washing of viable cell suspensions a dynamic filter device has been evaluated. Results show that concentrating of cells under sterile conditions can be performed. With a laboratory scale filter (Sulzer-Biodruckfilter BDF-01) a 9-fold concentration was achieved, the filtration speed was about 15 l/h. The viability did not change significantly. Details of experiments are given and advantages are discussed.

A convenient system for the serial quantitation of viable cells measuring luminescence of ATP.

Measurement of intracellular ATP using luminescence offers an alternative method for the routine determination of viable animal cells in a large number of cultures. Optimal conditions of the new technology are described as is its application in various test systems.

Production kinetics of monoclonal antibodies.

Two typical patterns of kinetics of monoclonal antibody (mAb) production are observed. Hybridomas which produce high mAb-titers do not show any feed back inhibition upon secretion of mAb into the culture supernatant over long periods of cultivation. "Low producing hybridomas" generally display high initial rates of mAb production, however, after a few hours the mAb secretion is stopped at a certain low concentration level in the order of some 100 mg mAb per ml. To illustrate the case that the high initial production rates of mAb of low producing hybridomas could be maintained for a period of at least 24 hours, mAb titers comparable to those produced by high-mAb-titer producing hybridomas were achieved.

A laboratory fermentor for agarose immobilized hybridomas to produce monoclonal antibodies.

Mass culture of immobilized cells in airlift-fermenters usually ends up with the beads accumulating in the foamy layer on the surface of the reactor fluid or, in stirred tankreactors, with partial destruction of the beads. We tried to use an airlift fermenter vessel for growing cells, immobilized in agarose beads. Instead of using the gas for driving, we mounted a slowly turning marine type impeller within the drought tube. Oxygen was supplied on occasional demands by the original sparger. This set up leads to sufficient operational characteristics of the reactor without accumulation of the beads in the foamy layer and without mechanical destruction. Different productivities of either immobilized cells or cells in free suspension culture are reported.

Entrapment of animal cells for production of monoclonal antibodies and other biomolecules.

Animal cell technology is attracting considerable interest because of the capacity of animal cell cultures to synthesize or transform complex compounds such as virus vaccines, immunochemicals, hormones or enzymes. For the growth of surface-dependent cells, microcarrier technology is gaining importance. Here, we have attempted to immobilize surface-independent cells, normally grown in suspension, by entrapping them in polymer microbeads. Such entrapment should give increased stability to the normally fragile animal cells, allow for high cell densities to be achieved within the beads and make such preparations suitable for continuous operation. At the same time, the need for separation of the desired product from the cells is obviated. With the model systems studied, we showed that hybridoma, as well as other cell lines entrapped in agarose microbeads, remained viable. Both immunoglobulins and lymphokines were exported through the microbeads into the medium for 1-3 weeks, at levels corresponding well to those produced with free cells.

Interleukin-2 production in continuous culture.

The increasing interest in the generation of lectin or antigen-activated (human) T cells by means of Interleukin-2 (TCGF) has led to numerous attempts to produce this substance. The simplest procedure is to use conditioned medium (CM) from IL-2 producing primary cells or cell lines with considerable activity. We describe a method of continuous CM-production in a 2-litre fermentation apparatus using a primate lymphoid cell line (MLA 144) which excretes IL-2 spontaneously. It was possible to harvest large amounts of homogeneous, lectin free material with similar production rates under different culture conditions. The activity of our CM was determined in various specific systems and proven to be comparable to that of supernatants of lectin-stimulated human primary lymphocytes.

Entrapment of animal cells for the production of biomolecules such as monoclonal antibodies.

An important problem in the production of monoclonal antibodies is the large-scale cultivation of hybridoma cells in vitro. Fragility of cells and suboptimal in vitro cultivation methods have led to poor results in larger scale production up to now. To lower the mechanical stress on the cells we tried to entrap the cells into microspheres made of polymer material. In addition to other materials, agarose as embedding medium was investigated and results with hybridoma and other, non anchorage-dependent cell lines are given. The conclusion of the results is that encapsulation of living cells is possible and entrapped cells remain viable and continue to produce the desired substance for at least several weeks. The substances are secreted through the polymer matrix. Handling of microspheres is shown to be easy and simple fermentation apparatus may be used for the production on a reliable technical scale. Some problems remain unsolved, such as the determination of viable cell count within the microspheres and cultivation in columns which seems to be the simplest form of continuous production process.

Simple test system for interleukin-2.

A simple system was developed to evaluate tests using the principle of the multiplication of specific cells. Of the possible application such as hormones, growth factors, etc., we show here the determination of IL-2 activity. The test is performed in flat bottom microplates wherein the sample is diluted with standard microtiter systems and cells are added. After the incubation period cells are fixed to the surface and stained. The absorbed stain is eluted and the optical density measured with a Titertec Multiscan photometer. Results correspond well to commonly used radiotracer systems and are as sensitive as those but much easier, cheaper and quicker to perform.

Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

Alteration of a human lymphoblastoid cell line, REH6 during subsequent cultivation.

Chromosomal and isoenzyme alteration of a human leukemia cell line, REH6, is shown during 80 subsequent passages. No correlation between these properties could be found.

Experience with testing for mycoplasma with five different methods.

Five methods for mycoplasma-content detection in cell-cultures are established in this laboratory. Results with about 20 different cell lines continuously grown in this laboratory in some determinations indicate that four methods give good correspondence. A microbiological method gave contrary results in many cases. It is possible that new infections will grow on mycoplasma broth but the older infections are adapted to cell-culture and give negative results on artificial medium. New low-level infections cannot be detected by the other methods which do not include any efficient enrichment step. The most convenient method in our opinion is a DNA-staining method according to Chen because this is a very quick, inexpensive and easily performable process. In practice it seems necessary to check cell cultures in two ways, firstly with a quick method and additionally with the microbiological test to ensure the detection of new low-level infections.

Experience with an image-analyzing computer in virus plaque measurements.

Due to the extremely fast and reliable plaque-number determinations, the image-analyzing computer is most useful for large screening programs involving plaque-reduction tests.