Inessa S. Levenbook

Tumorigenicity of Vero cells

One of the current criteria for evaluating the acceptability of cell lines for use in vaccine production is lack of tumorigenicity. Vero cells represent an example of a class of cells known as continuous cell lines. They were derived from African green monkey kidney, and their growth properties and culture characteristics have many advantages over other cell substrates for use in vaccine production. We have tested Vero cells for tumorigenicity in nude mice and in a human muscle organ culture system, and found a significant increase in their tumorigenic potential with increasing passage numbers. Cells at passage 232 and higher produced nodules in all nude mice inoculated. Histologically the nodules were well defined, anaplastic tumors, which exhibited some of the characteristics of renal adenocarcinomas. In about 6 to 8 days all of the nodules began to regress. Data were obtained that suggested an immune mechanism was the basis for the regression phenomenon.

Transgenic mice as an alternative to monkeys for neurovirulence testing of live oral poliovirus vaccine: validation by a WHO collaborative study

OBJECTIVE Extensive WHO collaborative studies were performed to evaluate the suitability of transgenic mice susceptible to poliovirus (TgPVR mice, strain 21, bred and provided by the Central Institute for Experimental Animals, Japan) as an alternative to monkeys in the neurovirulence test (NVT) of oral poliovirus vaccine (OPV). METHODS Nine laboratories participated in the collaborative study on testing neurovirulence of 94 preparations of OPV and vaccine derivatives of all three serotypes in TgPVR21 mice. FINDINGS Statistical analysis of the data demonstrated that the TgPVR21 mouse NVT was of comparable sensitivity and reproducibility to the conventional WHO NVT in simians. A statistical model for acceptance/rejection of OPV lots in the mouse test was developed, validated, and shown to be suitable for all three vaccine types. The assessment of the transgenic mouse NVT is based on clinical evaluation of paralysed mice. Unlike the monkey NVT, histological examination of central nervous system tissue of each mouse offered no advantage over careful and detailed clinical observation. CONCLUSIONS Based on data from the collaborative studies the WHO Expert Committee for Biological Standardization approved the mouse NVT as an alternative to the monkey test for all three OPV types and defined a standard implementation process for laboratories that wish to use the test. This represents the first successful introduction of transgenic animals into control of biologicals.

Propagation and properties of Kaposi's sarcoma-derived cell lines obtained from patients with Aids: similarity of cultured cells to smooth muscle cells

Cells derived from Kaposis sarcoma (KS) were propagated in vitro using conditions which resulted in elimination of contaminating fibroblasts and the emergence of homogeneous cell populations which morphologically resembled smooth muscle cells and had neoplastic characteristics. In long-term culture, they differentiated into large ribbon-like cells with longitudinal fibrillarity of their cytoplasm. These fibrils stained red by Masson trichrome staining, and were reactive with antibodies to desmin. Dense bodies typical of myoblasts were observed in some cells by electron microscopy. The cells did not form capillary structures like endothelial cells, they lacked Weible-Palade bodies, and did not express the blood-clotting Factor VIII-related antigen or receptors for the lectin Ulex europaeus agglutinin I. They did express four other antigens, however, in common with endothelial cells. The cells did not form tumors in athymic nude mice; however, they formed colonies in soft agar, manifested tumor-like growth on muscle organ cultures, and were invasive in an artificial basement membrane invasion assay. The results indicate that a component of KS is closely related to leiomyoblasts and and has neoplastic properties.

In vitro characterization of Salmonella typhi mutant strains for live oral vaccines

Several Salmonella typhi attenuated mutant strains, suggested as candidates for live oral vaccine, were examined for their characteristics in vitro in comparison with parental strains Ty2 and CDC10-80. Three methods were used: interaction of bacteria with the human monocyte-macrophage U937 cell line evaluated by microscopic examination, bacterial growth in the cell culture medium estimated by absorbance and bacterial resistance to human plasma assessed by the viable count technique. The most informative data were obtained in the test with U937 cells. Ty2 penetrated almost 100% of the cells, multiplied rapidly and caused death of the cells. CDC10-80 infected about 30% of the cells, multiplied slightly and did not kill the cells. The Ty2 mutant galE via EX462 behaved like CDC10-80. Bacteria of the galE Ty21a, Vi + Ty21a, 541 Ty and 543 Ty, found in only 3-4% of the cells, did not multiply within the cells and decreased in number with time. These findings correlate with the reported virulence of these strains for humans. With the second method, the rate of bacterial growth in cell culture medium did not differentiate Ty2, CDC10-80 and EX462. They grew at the same rate and faster than the remaining mutants. The plasma resistance test did not discriminate between EX462 and other mutants. These tests did not reveal any difference between Vi + Ty21a and Vi-Ty21a.

Effects of γ-IFN and NGF on subpopulations in a human neuroblastoma cell line: Flow cytometric and morphological analysis

SummaryNeuroblastomas are neural crest-derived tumors that contain neuronal, melanocyte, and Schwann cell precursors. We examined the effects of treatment with γ-interferon (γ-IFN) and nerve growth factor (NGF), alone, and in combination, on these progenitor subpopulations in the human neuroblastoma cell line, SH-SY5Y. Using fluorescence-activated flow cytometry (FACS), changes in expression of three differentiation-specific or-associated marker proteins, the 200 kD neurofilament protein, the myelin basic protein, and the S-100 protein, were analyzed. Growth rates and morphological changes associated with each treatment over the 2-wk incubation period were noted. The greatest effects were observed with combined IFN +NGF treatment. These were significant increases in expression of all three proteins distinctive morphological signs of differentiation, and extensive inhibition of proliferation compared to control cultures. Treatment with NGF alone resulted in increased neurofilament protein expression and in the length and number of neurite extensions, but there was no effect on the growth rate. IFN induced striking morphological changes, significant inhibition of growth, and changes in protein expression that correlated with neuronal to non-neuronal subpopulation shifts due to the death of differentiated cells. When treatment was discontinued after 15 d, the morphological changes induced by NGF were reversed within 2–3 d, while those induced by IFN±NGF were present up to 4 wk post-treatment. Small, neuroblastic colonies were observed throughout the treatment period and within 4–6 wk after the cessation of treatment this cell-type fully reconstituted the cultures suggesting the presence of a stem cell. Our results indicate that treatment with γ-IFN±NGF can regulate growth and induce, either stem cells or progenitor neuronal, Schwann and melanocyte subpopulations in the SH-SY5Y cell line to irreversibly differentiate.

Human muscle: a model for the study of human neoplasia

SummaryHuman muscle (HM) was used in an organ culture system to study the growth of human tumor cells and to test an antitumor drug. The HM system mimicked the in vivo situation regarding the behavior of neoplastic versus normal cells in that tumor cells proliferated extensively and invaded, while normal cells showed only a limited proliferative potential and a limited invasion was observed with fibroblasts but not with epithelial cells. In addition, when human plasma (HP) was used in place of fetal bovine serum (FBS) and cell culture medium as a source of nutrients, the tumor cells displayed a more aggressive histopathologic pattern. The HM system, as illustrated by the 5-FU results, allows the direct visualization of the effect to an antitumor agent not only on tumor cell growth but also on a range of histopathologically evaluable characteristics of the interaction of tumor cells with the host tissue. The HM system provides for the first time an in vitro experimental model using easily accessible adult human tissue to study cancer and its treatment.

Salmonella typhi vaccine strain in vitro; low infectivity in human cell line U937

Salmonella typhi strain Ty21a has been used for live oral vaccine. The infectivity of Ty21a, in comparison with S. typhi Ty2, was evaluated using the human monocyte-macrophage cell line U937. Assays were performed by quantitative microscopy and viable count technique. Ty2 infected approximately 100% of the cells, multiplied extensively within these cells and caused cell death. The same dose of Ty21a infected only about 15% of the cells, resulting in a low number of intracellular bacilli and cell survival. The use of gentamicin in the test confirmed intracellular multiplication of Ty2 but not Ty21a. The system described may be suitable as a test system for characterization of the degree of virulence of Ty21a and other live, oral typhoid vaccines.

Tumorigenicity testing of primate cell lines in nude mice, muscle organ culture and for colony formation in soft agarose

Primate neoplastic and finite cell lines were tested in one in vivo and two in vitro test systems: adult nude mice, muscle organ culture (MOC) and soft agarose (SA). Comparison of the sensitivity of the systems indicated that nude mice were inferior to either in vitro system: WI-38 VA13 (an SV40 transformed cell line) did not cause tumours in these animals yet it behaved as if it were neoplastic in MOC and formed colonies in SA. There was complete correlation between results obtained in MOC and SA. All cell lines which produced tumors in vivo were positive in both in vitro test systems. None of the lines which showed normal patterns in MOC and in SA was tumorigenic in nude mice. Since testing in vitro is simpler, faster, and is thought to be reliable, we recommend SA followed by MOC as the initial assays for determining tumorigenicity of cells.

Development of a transgenic mouse neurovirulence test for oral poliovirus vaccine: international collaborative study 1993-1999.

The neurovirulence safety of oral live poliovirus vaccine (OPV) has been tested in monkeys, because only primates are sensitive to all three types of poliovirus. The genetic engineering of transgenic mice susceptible to poliovirus led to studies on the suitability of these mice for a neurovirulence test of OPV. A WHO international collaborative study started with type-3 OPV in 1993 and was completed in 1999. The study produced a voluminous set of data proving that the TgPVR21 mice, inoculated with OPV samples into the lumbar cord, provided a test for neurovirulence of OPV as sensitive and reproducible as the monkey test. A statistical decision model for acceptance/rejection of type-3 vaccines using the transgenic mouse test has been developed. The mouse neurovirulence test showed a number of essential advantages over the monkey test. This is the first example of a successful introduction of transgenic animals into control of biologicals. In October 1999, the WHO Expert Committee on Biological Standardization approved TgPVR21 mice as alternative to the monkey model for neurovirulence testing of OPV type 3. A final step of the collaborative study with OPV types 1 and 2 is in progress, and data obtained so far are promising. Two breeding stations for production of TgPVR21 mice are being established in Asia and Europe.

Comparison of the colony forming ability and invasive potential of six primate cell lines treated with retinoic acid

SummaryFour human and two nonhuman primate cell lines were studied to determine their growth characteristics in soft agar, and for invasive characteristics in a muscle organ culture assay system. The cell lines ranged from normal human diploid to frankly tumorigenic in animal models. Additional studies were performed to assess the effects of retinoic acid (RA) on colony forming ability and invasion. The results showed a wide range of cloning efficiencies among the cell lines tested, as well as the degree of invasiveness. Of the soft agar growth parameters studied, colony growth index correlated best with invasion in the organ culture assay. RA significantly inhibited the growth of the tumor cells in soft agar, but failed to inhibit invasion in the organ culture assay although there was inhibition of surface proliferation. The data are consistent with the suggestion that cell proliferation and invasion are not necessarily linked characteristics.