Evgenia M. Dragunsky

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.

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.

Experimental evaluation of antitoxic protective effect of new cholera vaccines in mice

Intraperitoneal immunization of mice and subsequent challenge with purified cholera toxin (CT) were employed to evaluate the anti-cholera toxin protective effect of two new oral cholera vaccines, live CVD 103-HgR and killed B subunit-whole cell (BS-WC). CVD 103-HgR vaccine demonstrated 100% protection of mice against 2.25 LD50 and 70% against 3 LD50 of CT. Mice immunized with BS-WC vaccine were protected against 2.25 and 3 LD50 of CT in 88 and 62% of cases, respectively. All three killed parenteral vaccines failed to protect against CT. We suggest this mouse system for preliminary evaluation of the antitoxic protective activity of cholera vaccines.

Increased sensitivity of antithymocyte globulin-treated newborn rat assay for metastasis.

Tumorigenic and metastatic abilities of FL, transformed human amnion, Vero and LLC-MK2, continuous monkey kidney cell lines (CCLs), none of which produced lung metastases in nude mice, were studied in ATG-treated newborn Wistar rats. Effects of subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of 10(6) and 10(7) cells were compared. l.p. inoculation of the same number of FL cells produced larger primary tumors, increased incidence of lung metastasis and more metastases per rat than s.c. inoculation. The same was true of the higher inocula administered by either route as compared to the smaller inocula. The CCLs (Vero at passages 320 and 323, LLC-MK2 at passage 306) inoculated either s.c. or i.p. induced small tumors regressing in all animals. After s.c. inoculation of 10(6) Vero or LLC-MK2 cells lung metastases did not develop in any rats, but 10(7) inoculum and i.p. administration of both size inocula caused single lung metastases. Thus, increased sensitivity of the rat system was achieved by larger size of the s.c. inoculum or the i.p. route of inoculation. Highly sensitive assay is especially important for evaluation of possible metastatic abilities of CCLs.