Browsing by Author "Murano, Elsa Alina"
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- The effect of heat shock, growth atmosphere, and recovery atmosphere on the survival of Escherichia coli 0157:H7 to heatMurano, Elsa Alina (Virginia Tech, 1990)E. coli 0157:H7 is an important foodborne pathogen, responsible for several outbreaks of hemorrhagic colitis where improperly cooked hamburger meat was thought to be the vehicle. Various time/temperature combinations were used to determine the optimum conditions of heat shock which would result in the greatest number of survivors to a 55°C heat treatment. The optimum conditions were 42°C for 5 minutes and were used throughout the study. Heat shock of aerobically grown cells resulted in an increase in the mean D value after a 55°C heat treatment by a factor of 2.1 over nonheat-shocked controls. Heat shock of anaerobically grown cells also resulted ina significant increase in mean D value over nonheat-shocked controls. Anaerobic growth itself resulted in an increase in the ability of the cells to survive the 55°C heat treatment when compared with aerobically grown cells. Both heat-shocked and anaerobically grown cells contained a protein corresponding to a sigma³² subunit of RNA polymerase which has been identified as the 71,000 Galton heat shock protein characteristic of E. coli cells. Anaerobic plating resulted in a significant increase in the mean D values of both aerobically grown and anaerobically grown cells. The largest increase in mean D values was observed in aerobically grown non-heat-shocked cells, which increased by a factor of 2.3 when plated anaerobically instead of aerobically. The activities of catalase and superoxide dismutase in aerobically grown and anaerobically grown cells were studied to determine the reason why anaerobic plating enhanced recovery of cells. The activities of both enzymes were eliminated after heat treatment at 55°C for 20 minutes, regardless of whether the cells were heat-shocked or not. The ability of heat shock and anaerobic growth to protect the cells from a subsequent heat treatment was tested by measuring the rate of release of cell materials during heating at 55°C. Heat-shocking and anaerobic growth resulted in even faster release of cell materials during heating than controls, suggesting that neither of these stresses protected the cells against the effects of heat. The effect of heat shock on cell injury was studied. Heat shock of aerobically grown cells resulted in the greatest difference in log number of cells between cells plated in nonselective medium vs. selective medium. Thus, more cells were injured if heat-shocked than if not heat-shocked. Heat-shocking of anaerobically grown cells also resulted in more injured cells than non-heat-shocked controls.
- Role of polymorphonuclear leukocytes in the tumoricidal activity of Propionibacterium acnesMurano, Elsa Alina (Virginia Polytechnic Institute and State University, 1987)The mechanism responsible for the killing of tumor cells after injection of mice with a mixture of tumor cells and Propionibacterium acnes were investigated. Tumor cells were injected intramuscularly into Balb/c mice either alone or together with P. acnes vaccine. The tumor cells were then removed from the injection site 12 hours after injection, and transferred into fresh mice. Tumor cells from control animals given tumor cells only caused tumors when transferred into fresh mice 12 hours after injection whereas tumor cells from animals given both tumor cells and vaccine did not develop tumors in the fresh nice. ELISA tests were done to estimate the number of tumor cells in the lesions. In control animals given 10⁵ tumor cells the estimated numbers dropped to 10³ cells at 24 hours, but thereafter rose steadily. Palpable tumors were present 7-10 days later. In animals given 10⁵ tumor cells + 500 ug of P. acnes vaccine, estimated tumor cell numbers fell steadily, and could not be detected after 2 days. Palpable tumors never developed in these animals. These results indicate that tumor cells are killed, or rendered nontumorigenic, during the first 12 hours after co-injection into mice with P. acnes Histological studies showed that injection of P. acnes vaccine, with or without tumor cells, induced large numbers of polymorphonuclear leukocytes (PMNs) at 12 hours. To determine the role of PMNs in the killing of tumor cells, tumor cells were incubated with supernatant obtained from the phagocytosis mixture of PHNs and P. genes. After a 2- hr. incubation, the tumor cells were washed and injected into fresh mice. No tumors developed, indicating that a product of the phagocytosis of P. acnes by PMNs played a role in the killing of tumor cells. Bacterial vaccines such as P. freudenreichii, which are poorly protective against tuner cells, produced phagocytosis supernatants which were unable to kill tumor cells. Various oxygen radical scavengers/inhibitors were used to test their effect on the toxicity of the supernatant on tumor cells and chinese hamster ovary cells. Both azide and catalase rendered the supernatant nontoxigenic, suggesting that H₂O₂, produced by PHNs during phagocytosis of P. acnes, is responsible for the killing of tumor cells. However, the addition of catalase 30 minutes after the start of phagocytosis had no effect on the toxicity of the supernatant, suggesting that H₂O₂ is converted to other toxic radicals during the course of phagocytosis of P. acnes by PMNs. The oxygen consumption levels of PHNs during phagocytosis of P. acnes or other bacterial vaccines was measured and found to be similar regardless of the antitumor ability of the vaccine used. This suggests that the difference in the ability of various vaccines to protect mice against tumors may be in the production of a particular oxygen radical by PMNs during phagocytosis, and not in the production of different quantities of the same radicals.