Reactive nitrogen and oxygen species (O2*-, H2O2, NO* and ONOO-) have been strongly implicated in the pathophysiology of neurodegenerative and mitochondrial diseases. In the present study, we examined the effects of nitrosative and/or nitrative stress generated by DETA-NO {(Z)-1-[2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate}, SIN-1 (3-morpholinosydnonimine hydrochloride) and SNP (sodium nitroprusside) on U87MG glioblastoma cybrids carrying wt (wild-type) and mutant [A3243G (Ala3243-->Gly)] mtDNA (mitochondrial genome) from a patient suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). The mutant cybrids had reduced activity of cytochrome c oxidase, significantly lower ATP level and decreased mitochondrial membrane potential. However, endogenous levels of reactive oxygen species were very similar in all cybrids regardless of whether they carried the mtDNA defects or not. Furthermore, the cybrids were insensitive to the nitrosative and/or nitrative stress produced by either DETA-NO or SIN-1 alone. Cytotoxicity, however, was observed in response to SNP treatment and a combination of SIN-1 and glucose-deprivation. The mutant cybrids were significantly more sensitive to these insults compared with the wt controls. Ultrastructural examination of dying cells revealed several characteristic features of autophagic cell death. We concluded that nitrosative and/or nitrative stress alone were insufficient to trigger cytotoxicity in these cells, but cell death was observed with a combination of metabolic and nitrative stress. The vulnerability of the cybrids to these types of injury correlated with the cellular energy status, which were compromised by the MELAS mutation.

Inhalation of small numbers of Francisella tularensis subspecies tularensis (Ftt) in the form of small particle aerosols causes severe morbidity and mortality in people and many animal species. For this reason, Ftt was developed into a bona fide biological weapon by the USA, by the former USSR, and their respective allies during the previous century. Although such weapons were never deployed, the 9/11 attack quickly followed by the Amerithrax attack led the U.S. government to seek novel countermeasures against a select group of pathogens, including Ftt. Between 2005–2009, we pursued a novel live vaccine against Ftt by deleting putative virulence genes from a fully virulent strain of the pathogen, SCHU S4. These mutants were screened in a mouse model, in which the vaccine candidates were first administered intradermally (ID) to determine their degree of attenuation. Subsequently, mice that survived a high dose ID inoculation were challenged by aerosol or intranasally (IN) with virulent strains of Ftt. We used the current unlicensed live vaccine strain (LVS), first discovered over 70 years ago, as a comparator in the same model. After screening 60 mutants, we found only one, SCHU S4 ΔclpB, that outperformed LVS in the mouse ID vaccination-respiratory-challenge model. Currently, SCHU S4 ΔclpB has been manufactured under current good manufacturing practice conditions, and tested for safety and efficacy in mice, rats, and macaques. The steps necessary for advancing SCHU S4 ΔclpB to this late stage of development are detailed herein. These include developing a body of data supporting the attenuation of SCHU S4 ΔclpB to a degree sufficient for removal from the U.S. Select Agent list and for human use; optimizing SCHU S4 ΔclpB vaccine production, scale up, and long-term storage; and developing appropriate quality control testing approaches.