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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2014)

Biofilm is the virulence factor that is responsible for chronic infection in diseases such as Cystic Fibrosis (CF) and chronic wounds. In this thesis, we examine the role of AlgX, a required protein for alginate biosynthesis in P. aeruginosa. We show that the absence of AlgX resulted in the loss of mucoidy and in silico studies demonstrated that AlgX binds alginate. Alanine mutations demonstrated that K396, T398, W400, and R406 are important for alginate binding. Alginate rescue assays confirm the importance of these amino acid residues for alginate biosynthesis and acetylation. This is the first functional demonstration of AlgX role in alginate biofilm biosynthesis and acetylation.

Biofilm-producing bacteria and redox imbalance are the leading factors that turn acute wounds into chronic wounds. We demonstrated that LIGHT-/- mouse wounds contain elevated levels of reactive oxygen species (ROS). To see whether chronic wounds can be generated, we increased the redox imbalance in the LIGHT-/- wounds and infecting the wounds with biofilm-forming bacteria. We demonstrated that by using these conditions, we could induce chronic wounds in the LIGHT-/- mouse model 100% of the time. These wounds do not re-epithelialize, contain high bacterial burden, and sustained multi-species bacterial infections that are biofilm-forming and antibiotic-resistant.

We also demonstrated that increasing the redox imbalance was sufficient to turn db/db wounds into chronic wounds. These wounds sustained spontaneous biofilm- producing bacterial infections. To verify that redox imbalance is critical for chronicity, we treated chronic wounds with antioxidants and found that oxidative stress was highly reduced, biofilm production was decreased, and bacteria became more sensitive to antibiotics. This is the first demonstration that chronic wounds can be generated and reversed by manipulating the wound's redox microenvironment in an animal model.

Norspermidine has been shown to negatively affect the structure of extracellular polymeric substances (EPS). Using the bacteria that colonized db/db chronic wounds, we demonstrated that 2 mM norspermidine reduces the amount of adherent biofilms produced by exudate bacterial communities. Furthermore, application of norspermidine increases the bactericidal effect of gentamicin. We believe that norspermidine could be used in combination with bactericidal antibiotics to control biofilm infection in dermal chronic wounds.

    Cover page: The Role of Bacterial Biofilms in Chronic Infections
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2014)
    By 2025, more than 500 M people worldwide will suffer from diabetes; 125 M will develop foot ulcer(s) and 20 M will undergo an amputation, creating a major health problem. Understanding how these wounds become chronic will provide insights to reverse chronicity. We hypothesized that oxidative stress (OS) in wounds is a critical component for generation of chronicity. We used the db/db mouse model of impaired healing and inhibited, at time of injury, two major antioxidant enzymes, catalase and glutathione peroxidase, creating high OS in the wounds. This was necessary and sufficient to trigger wounds to become chronic. The wounds initially contained a polymicrobial community that with time selected for specific biofilm-forming bacteria. To reverse chronicity we treated the wounds with the antioxidants α-tocopherol and N-acetylcysteine and found that OS was highly reduced, biofilms had increased sensitivity to antibiotics, and granulation tissue was formed with proper collagen deposition and remodeling. We show for the first time generation of chronic wounds in which biofilm develops spontaneously, illustrating importance of early and continued redox imbalance coupled with the presence of biofilm in development of wound chronicity. This model will help decipher additional mechanisms and potentially better diagnosis of chronicity and treatment of human chronic wounds.
      Cover page: Generating and reversing chronic wounds in diabetic mice by manipulating wound redox parameters.
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2009)
      Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8(+) T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses ( rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8(+) T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8(+) T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.
        Cover page: A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human DiseaseCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2014)
        Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.
          Cover page: A Novel Model of Chronic Wounds: Importance of Redox Imbalance and Biofilm-Forming Bacteria for Establishment of Chronicity
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