Double-blind, randomized, placebo controlled pilot study evaluating efficacy and reactogenicity of an oral ETEC B-subunit-inactivated whole cell vaccine against travelers' diarrhea (preliminary report).

Diarrhea caused by enterotoxigenic E.coli (ETEC) is an important health problem in developing countries and in travelers to these areas. In previous trials formulations of ETEC vaccines containing the B-subunit of cholera toxin, which is antigenically similar to the heat labile enterotoxin of ETEC, and the most prevalent colonization factor antigens of ETEC, were shown to stimulate relevant mucosal immune responses in volunteers from Sweden and Egypt.

High yield production of an inactivated coxsackie B3 adjuvant vaccine with protective effect against experimental myocarditis.

Dilated cardiomyopathy, perhaps chronic postviral fatigue syndrome as well as juvenile diabetes could be triggered by enteroviral infections. The frequency of sudden death after myocarditis and its relationship to enteroviral infections is disputed. Neonatal enteroviral disease is rare, but can be severe. It is also possible that enteroviruses pose a threat to immunocompromised patients, like bone marrow transplant recipients. Consequently, the emergence of chronic enteroviral diseases as a concept, prompted our attempts to produce an enteroviral vaccine. 1. Live attenuated enterovirus strains were previously in some cases shown to be suitable as vaccine candidates. We obtained neutralizing antibody titres ranging from 40-2560 against Coxsackie B3 virus (RD strain). Animals were protected to 90% against challenge infection. 2. Inactivated whole vaccine. We used beta-propiolactone to inactive Coxsackie B3 virus. 74% of the animals survived if the vaccine was prepared with Quil A matrix as adjuvant. The neutralisation antibody titres varied from < 5 to 320. By comparison aluminium hydroxide (p = 0.06) and Freund's adjuvant were inferior (p < 0.01). 3. Subunit vaccines. We have previously used the ISCOM (immunostimulatory complex) technology to produce a Coxsackie B3 subunit vaccine. High levels of neutralizing antibodies were obtained (512)-comparable to natural infection. All animals survived challenge infection after two booster doses with 16 nanogram of the ISCOM preparation. Limiting for this technique was the availability to include sufficient amount of antigenic protein material. In addition to neutralizing antibodies a cellular response might be obtainable. In conclusion we have shown that vaccine can be made against Coxsackie B3 virus with good protective effect and significant neutralisation antibody titre.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a U-937 subline which can be induced to differentiate in serum-free medium.

We report the selection and characterization of a U-937 subline which is capable of long-term growth in serum-free medium and can be induced to differentiate. The subline (U-937-1SF) can be maintained in standard RPMI-1640 medium supplemented by antibiotics only. As compared to the serum-dependent U-937 parental cell line, U-937-1SF produced lower amounts of lysozyme and elastase and had a decreased surface expression of complement receptor 1 (CD35) and myeloid antigens CDw17 and CD38. Apart from these alterations, the U-937-1SF cells appear to be morphologically, cytogenetically and phenotypically similar to the parental U-937 clone-1 cells. The capacity of U-937 clone-1 cells to undergo phorbol myristic acid (PMA)-, vitamin D3 (VitD3)- and retinoic-acid (RA)-induced differentiation was retained in the U-937-1SF cells as evidenced by the induced growth arrest, development of a monocyte/macrophage morphology and increased expression of differentiation-associated antigens, e.g. CD11b, CD11c, CD14 and CD18. The growth-inhibitory response to cytokines involved in the activation and differentiation of monocytes, IFN-gamma, TNF-alpha, IL-1 beta, IL-6 and GM-CSF, was normal. Our results suggest that the U-937-1SF subline can be used as a serum-free model system for studies on various aspects of monocyte differentiation.

Serum-free cell culture.

Cell culture is one important tool when studying cellular functions and molecular biology. It is also a basic method in most virological investigations. Serum has been an obligatory component in most cell culture media. During the last decades serum-free, chemically defined media have been developed, that are supplemented with a number of substances with specific cellular activities. The main developments of defined media are presented. Examples are given of investigations with different cell types.

Serum-free media for lymphoid culture.

A serum-free medium is described that we have been using since almost ten years when growing a number of lymphoid cell lines. Serum-free media offer several advantages before media containing even well standardized animal sera. As an example of studies where serum-free media offer advantages we present data demonstrating the use of a defined medium when monitoring glycosidase enzyme levels in lymphoid cells.

Patterns of glycosidases in the histiocytic cell line U-937. Effects of agents inducing cell differentiation.

1. The cell bound glycosidases in sublines and clones of the histiocytic cell line U-937 have previously been shown to display characteristic patterns. 2. In this paper the effects of differentiation inducing agents upon glycosidase patterns of one subline, U-937 GTB, are presented. 3. Teleocidin, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), dimethyl-sulfoxide (DMSO), dihydroxyvitamin D3 and supernatants from mixed lymphocyte culture (MCL) all induce cellular differentiation of U-937 GTB. 4. Significant changes of the levels of cell bound glycosidases were seen after addition of inducing agents. 5. Alterations have been monitored as relative effects upon the absolute glycosidase activities and as effects upon selected ratios of different glycosidases. 6. The separate inducing agents show distinct enzyme patterns.

Glycosidase and phosphatase activities in U-937 and some clones and sublines.

1. The activities of nine glycosidases, lysozyme and acid and alkaline phosphatases were compared in the histiocytic lymphoma cell line U-937 and a set of clones and sublines derived from this line. 2. The patterns of the different enzyme activities and selected enzyme ratios have been used as a method to distinguish between different clones and sublines. 3. Sublines with high lysozyme levels were rich in most cell-bound glycosidases. 4. During long-term growth distinct enzyme patterns of individual lines were preserved. 5. The enzyme pattern during a cell culture growth cycle was basically stable.

Significance of glycosidases and phosphatases in Cercopithecus and Macaca cells.

When selected ratios of different glycosidases and phosphatases from primary monkey kidney cells or from monkey kidney cell lines are presented graphically, characteristic patterns do evolve. Three different subtypes of Vero cells show similar glycosidase patterns. The Vero subtypes tested show glycosidase patterns that are closely similar to those of primary cells of Cercopithecus aethiops. Glycosidase patterns of BS-C-1 and CV-1 cells are less similar to those of primary Cercopithecus cells than are those of Vero cells. Primary kidney cells from Macaca cynomolgus show significantly different glycosidase patterns compared with those of different Cercopithecus cells. The distinct glycosidase patterns can be used to classify the tested cell lines in relation to each other.

Serum free cultivation of lymphoid cells for virus and interferon production.

Minor modifications of standard media have given a defined serum free medium for the production of polio virus and measles virus in lymphoblastoid cells and of Namalva interferon in lymphoma cells. By usage of slightly modified standard roller equipment in the order of 2 X 10 12TCID-50 of polio virus and 4 X 10(9) IU of interferon can be produced on one roller rack. The produced poliovirus does comply with requirements for inactivated poliovaccine.

Significance of glycosidase patterns in lymphoid cells.

Characteristic patterns of cell bound lysosomal glycosidases were found in different lymphoblastoid cell lines derived from Epstein-Barr virus-transformed lymphocytes. The enzyme pattern resembled that found in normal lymphocytes from healthy individuals except for a marked increase in alpha-L-fucosidase. beta-D-Glucuronidase on the contrary markedly decreased in the lymphoblastoid cells. Burkitt's lymphoma cells on the other hand showed glycosidase patterns that were quite distinct from those in lymphoblastoid cells. Each lymphoma cell line showed a characteristic pattern. This is one indication of a heterogeneous origin of these tumors. Glycosidase patterns may be used to roughly distinguish different lymphoid cell lines.

Propagation of large quantities of poliovirus in human lymphoblastoid cells grown in a serum-free medium.

All three types of poliovirus replicated to good titers in Epstein-Barr virus-transformed human lymphoblastoid cells isolated from patients with infectious mononucleosis. Cells were grown in a serum-free medium, which consisted of enriched Eagle's medium with addition of 0.1% albumin, 0.1% Intralipid and 1 mg/l of transferrin. Large quantities of virus could be produced in 2- to 20-l roller flasks or in spinner flasks. The propagated poliovirus gave an antibody response that complies with requirements for vaccine production.

Glycosidases from the interferon producing lymphoid cell line Namalva and from three other lymphoid cell lines.

1. The occurrence of lysozyme, neuraminidase and fourteen other glycosidases was investigated in the three lymphoma cell lines Namalva, Raji and Daudi derived from a Burkitt's lymphoma and the lymphoblastoid cell line Robinson from Epstein-Barr virus transformed normal peripheral blood lymphocytes. High activity of beta-N-acetyl-D-glucosaminidase was found in three of the cell lines, which also showed fairly high activities of beta-N-acetyl-D-galactosaminidase, alpha-D-mannosidase and beta-D-mannosidase. In Daudi the highest glycosidase activity was found for beta-D-mannosidase. 2. Neuraminidase and lysozyme were not detected in any of the four cell lines. 3. These cell lines showed characteristic enzyme patterns and enzyme ratios which may be used for the identification of the cell lines. 4. When calculated on a protein basis no statistically significant change in glycosidase activities of the cells could be recorded during interferon production.