Differing Alterations of Odor Volatiles Among Pathogenic Stimuli.

Alterations of the volatile metabolome (the collection of volatiles present in secretions and other emanations) that occur in response to inflammation can be detected by conspecifics and chemometric analyses. Using a model system where mouse urinary metabolites are altered by treatment with lipopolysaccharide (found in the outer cell membrane of gram-negative bacteria), we hypothesized that alteration of body odor volatiles will vary according to the pathogen responsible for inducing the inflammation. We tested this hypothesis by treating mice with different immunogens that engage different immune signaling pathways. Results suggest that alterations of body odor volatiles resulting from inflammation do contain detailed information about the type of pathogen that instigated the inflammation and these differences are not merely dependent on the severity of the inflammatory event. These results are encouraging for the future of differential medical diagnosis of febrile diseases by analysis of the volatile metabolome. In particular, our data support the possibility that bacterial infections can be differentiated from viral infections such that antibiotic drug stewardship could be drastically improved by reducing unneeded treatments with antibiotics.

Sharing an environment with sick conspecifics alters odors of healthy animals.

Body odors change with health status and the odors of sick animals can induce avoidance behaviors in healthy conspecifics. Exposure to sickness odors might also alter the physiology of healthy conspecifics and modify the odors they produce. We hypothesized that exposure to odors of sick (but non-infectious) animals would alter the odors of healthy cagemates. To induce sickness, we injected mice with a bacterial endotoxin, lipopolysaccharide. We used behavioral odor discrimination assays and analytical chemistry techniques followed by predictive classification modeling to ask about differences in volatile odorants produced by two types of healthy mice: those cohoused with healthy conspecifics and those cohoused with sick conspecifics. Mice trained in Y-maze behavioral assays to discriminate between the odors of healthy versus sick mice also discriminated between the odors of healthy mice cohoused with sick conspecifics and odors of healthy mice cohoused with healthy conspecifics. Chemical analyses paired with statistical modeling revealed a parallel phenomenon. Urine volatiles of healthy mice cohoused with sick partners were more likely to be classified as those of sick rather than healthy mice based on discriminant model predictions. Sickness-related odors could have cascading effects on neuroendocrine or immune responses of healthy conspecifics, and could affect individual behaviors, social dynamics, and pathogen spread.

Cytokine contributions to alterations of the volatile metabolome induced by inflammation.

Several studies demonstrate that inflammation affects body odor. Volatile signals associated with inflammation induced by pyrogens like LPS are detectable both by conspecifics and chemical analyses. However, little is known about the mechanisms which translate detection of a foreign molecule or pathogen into a unique body odor, or even how unique that odor may be. Here, we utilized C57BL/6J trained mice to identify the odor of LPS-treated conspecifics to investigate potential pathways between LPS-induced inflammation and changes in body odor, as represented by changes in urine odor. We hypothesized that the change in volatile metabolites could be caused directly by the pro-inflammatory cytokine response mediated by TNF or IL-1ß, or by the compensatory anti-inflammatory response mediated by IL-10. We found that trained biosensors generalized learned LPS-associated odors to TNF-induced odors, but not to IL-1ß or IL-10-induced odors. Analyses of urine volatiles using headspace gas chromatography revealed distinct profiles of volatile compounds for each treatment. Instrumental discrimination relied on a mixture of compounds, including 2-sec-butyl-4,5-dihydrothiazole, cedrol, nonanal, benzaldehyde, acetic acid, 2-ethyl-1-hexanol, and dehydro-exo-brevicomin. Although interpretation of LDA modeling differed from behavioral testing, it does suggest that treatment with TNF, IL-1ß, and LPS can be distinguished by their resultant volatile profiles. These findings indicate there is information found in body odors on the presence of specific cytokines. This result is encouraging for the future of disease diagnosis via analysis of volatiles.

Brain Injury Alters Volatile Metabolome.

Chemical signals arising from body secretions and excretions communicate information about health status as have been reported in a range of animal models of disease. A potential common pathway for diseases to alter chemical signals is via activation of immune function-which is known to be intimately involved in modulation of chemical signals in several species. Based on our prior findings that both immunization and inflammation alter volatile body odors, we hypothesized that injury accompanied by inflammation might correspondingly modify the volatile metabolome to create a signature endophenotype. In particular, we investigated alteration of the volatile metabolome as a result of traumatic brain injury. Here, we demonstrate that mice could be trained in a behavioral assay to discriminate mouse models subjected to lateral fluid percussion injury from appropriate surgical sham controls on the basis of volatile urinary metabolites. Chemical analyses of the urine samples similarly demonstrated that brain injury altered urine volatile profiles. Behavioral and chemical analyses further indicated that alteration of the volatile metabolome induced by brain injury and alteration resulting from lipopolysaccharide-associated inflammation were not synonymous. Monitoring of alterations in the volatile metabolome may be a useful tool for rapid brain trauma diagnosis and for monitoring recovery.

Mapping site-specific changes that affect stability of the N-terminal domain of calmodulin.

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the ¹H-¹5N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability.

Effects of stabilizers on the destabilization of proteins upon adsorption to aluminum salt adjuvants.

Excipients for protein-based vaccines are currently identified by evaluating the stability of the protein in solution. In most cases, however, the protein is adsorbed to the surface of an aluminum salt adjuvant in the final vaccine formulation. Previous studies showed that model protein antigens may be structurally altered and less thermally stable upon adsorption to aluminum salt adjuvants [Jones LS, Peek LJ, Power J, Markham A, Yazzie B, Middaugh CR, 2005, J Biol Chem 280:13406-13414]. The work presented herein provides evidence that compounds that stabilize the protein in solution also stabilize the adsorbed protein; however, the stability of the adsorbed protein in the presence of the stabilizer remains lower than that of the protein in solution. Potential implications of the reduced stability on the approach used to select excipients during formulation development are discussed.

Thermal stability of adenovirus type 2 as a function of pH.

Although several recent studies have focused on the characterization and formulation of adenovirus type 5, similar efforts focusing on adenovirus type 2 (Ad2) have been lacking. To this end, multiple biophysical techniques were employed to investigate the thermal stability of Ad2 as a function of pH. Highly cooperative thermally induced changes in capsid conformation were detected near 45 and 65 degrees C under neutral conditions. The first transition is attributed to the loss of the penton bases and their associated fibers followed by more complete physical degradation at higher temperatures. Data in this work as well as previous studies suggest that a common mechanism of icosahedral virus thermal degradation exists. Conformational changes detected in these studies occurred at increasingly higher temperatures with decreasing pH from 8 to 5 suggesting that the physical stability of Ad2 is enhanced under mildly acidic conditions. To consolidate the data generated in these studies, a multi-dimensional Eigenvector approach was employed to generate an empirical phase diagram (EPD) of Ad2. The EPD identifies conditions, or "phase boundaries," where structural integrity is altered, in addition to providing a tool that can be used to identify conditions under which forced degradation and excipient-screening studies can be conducted.