Browsing by Author "Jain, Neeta"

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    Approaches towards therapeutic development against chronic brucellosis in a mouse model
    Jain, Neeta (Virginia Tech, 2012-01-19)
    Brucellosis is the most common zoonotic disease worldwide. The intracellular localization of Brucella hinders the action of drugs that poorly cross cell membrane barriers. Additionally, when the immune response fails to clear the infection, chronic brucellosis ensues that becomes more challenging to treat with antibiotics. Therefore, two approaches, intracellular drug delivery and immunostimulation, have been explored in this dissertation, with an aim to develop a better therapeutic against Brucella infection in mice. First, to overcome the cell membrane barriers, drug loaded nanoparticles were tested to treat B. melitensis infection in mice. Gentamicin loaded block-ionomer complexes (BICs) and magnetite block-ionomer complexes (MBICs) were tested in vitro and along with clusters of MBICs (MBIClusters) were tested in vivo as tools to deliver gentamicin intracellularly. While these complexes showed very high efficacy compared to free gentamicin against Brucella in macrophage cell culture, they failed to show similar efficacies in mice. Histopathological examination of kidneys from mice treated with MBICs or MBIClusters showed deposition of brown pigment-laden macrophages in peri-renal adipose tissue and the pigment was confirmed as MBICs or MBIClusters based on special staining for iron. Additionally, it was found that doxycycline-gentamicin (DG) treatment results in better clearance of Brucella from infected mice compared to doxycycline alone. Secondly, two vaccine candidates, irradiated B. neotomae (IBN) and outer membrane vesicles (OMVs), were tested as immunostimulants to treat chronic B. melitensis infection in mice in combination with antibiotics. The non-ionic block co-polymer Pluronic P85, when mixed with OMVs as an adjuvant showed significantly higher protection against B. melitensis challenge in vaccinated mice compared to those vaccinated with OMVs alone. When tested as immunostimulants, there was no additive effect of vaccines and antibiotics on Brucella clearance from mice. However, IBN enhanced the production of IFN-γ while OMVs were associated with enhanced antibody production. This enhancement in the immune system resulted in the control of Brucella growth after the end of treatment. When given without antibiotics, vaccine alone failed to clear any Brucella from infected mice. The use of these vaccine candidates in combination with antibiotics shows a potential to prevent relapses in cases of brucellosis.
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    Characterization of Outer Membrane Vesicles from Brucella melitensis and Protection Induced in Mice
    Avila-Calderon, Eric Daniel; Lopez-Merino, Ahidé; Jain, Neeta; Peralta, Humberto; Lopez-Villegas, Edgar Oliver; Sriranganathan, Nammalwar; Boyle, Stephen M.; Witonsky, Sharon G.; Contreras-Rodriguez, Araceli (Hindawi Publishing Corp, 2011-12-29)
    The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFa, and IFN? genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.
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    Identification of a Single-Nucleotide Insertion in the Promoter Region Affecting the sodC Promoter Activity in Brucella neotomae
    Moustafa, Dina A.; Jain, Neeta; Sriranganathan, Nammalwar; Vemulapalli, Ramesh (PLOS, 2010-11-24)
    Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.
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    Role of entF Gene in Iron Acquisition by Brucella abortus 2308
    Jain, Neeta (Virginia Tech, 2009-05-11)
    Brucella causes undulant fever in humans and uterine and systemic infection leading to abortions in domestic animals and wild life. For the acquisition of iron in mammalian hosts, species of Brucella are known to produce two siderophores, 2, 3-dihydroxy benzoic acid (2, 3-DHBA) and brucebactin. Inability to synthesize of 2, 3-DHBA affects the ability of pathogen to metabolize erythritol, replicate in trophoblast cells and cause abortion in pregnant ruminant host. The entF gene has been implicated in the unresolved pathway allowing brucebactin biosynthesis in Brucella. The research effort presented in this thesis tries to relate the role of entF in iron acquisition and potential relation with erythritol metabolism by wild type B. abortus 2308. An entF deletion mutant (BAN1) of B. abortus 2308, generated using cre-lox methodology was found to be growth inhibited in iron minimal media compared to wild type strain. Growth inhibition was further enhanced with the addition of an iron chelator or 0.1% erythritol. Compared to wild type strain, no growth inhibition of BAN1 mutant was found in murine J774A.1 macrophages, which suggests that Brucella could acquire iron inside mammalian cells. The entF gene complemented mutant strains of BAN1 (BAN2A and BAN2B) were found to be intermediate in their ability to grow in iron minimal media supplemented with 0.0.05% erythritol, when compared to wild type and BAN1 strain. The results from the present thesis demonstrate that entF gene plays an important role in iron acquisition and erythritol metabolism by B. abortus 2308 under iron limiting conditions.