Browsing by Author "Gedamu, Lashitew"
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- ItemOpen AccessAssessment of the immunogenicity and protective efficacy of leishmania donovani superoxide dimutase and peroxidoxins and the role of peroxidoxin gene cluster in parasite survival(2009) Daifalla, Nada Sulaiman; Gedamu, Lashitew
- ItemOpen AccessCathelicidin Mitigates Staphylococcus Aureus Mouse Mastitis and Prevents Bacterial Invasion in Mammary Epithelial Cells(2019-08-23) Cavalcante, Paloma; Cobo, Eduardo; Barkema, Herman; De Buck, Jeroen; Jenne, Craig; Gedamu, LashitewStaphylococcus aureus is an important cause of mastitis, increasingly problematic due to the presence of bacterial strains resistant to conventional antibiotics. The ability of S. aureus to invade host cells is key to its ability to escape immune defense and antibiotics. This study focused on functions of cathelicidin, a small cationic peptide secreted by epithelial cells and leukocytes, in the pathogenesis of S. aureus mastitis. We determined that endogenous murine cathelicidin (CRAMP; Camp) was key in controlling S. aureus infection, as cathelicidin knockout mice (Camp-/-) inframammary challenged with S. aureus had higher bacteria burden and more severe mastitis compared to wild-type mice. Both human and murine cathelicidins, LL37 and CRAMP, respectively, reduced invasion of S. aureus in mammary epithelia. This function was independent of TLR2 and CD36 cell surface expression. LL-37 internalized into mammary epithelial cells and impaired S. aureus growth in vitro. We conclude that cathelicidins are promising anti-infectious therapeutic targets in mastitis; endogenous or exogenous cathelicidins protected against S. aureus infection by preventing internalization and potentially by directly killing this pathogen.
- ItemOpen AccessCharacterization of peroxidoxins in leishmania chagasi: role in parasite survival(2003) Barr, Stephen; Gedamu, Lashitew
- ItemOpen AccessCloning and characterization of iron superoxide dismutase cDNAs of Leishmania donovani chagasi and Trypanosoma cruzi(1996) Ismail, Said Osman; Gedamu, Lashitew
- ItemOpen AccessDevelopmental regulation of cysteine protease, iron superoxide dismutase, and peroxidoxin genes of Leishmania donovani complex(2005) Hamilton, Janice; Gedamu, Lashitew
- ItemOpen AccessDifferential Immune Response against Recombinant Leishmania donovani Peroxidoxin 1 and Peroxidoxin 2 Proteins in BALB/c Mice(2015-08-25) Daifalla, Nada S.; Bayih, Abebe Genetu; Gedamu, LashitewWe assessed the immune response against recombinant proteins of two related, albeit functionally different, peroxidoxins from Leishmania donovani: peroxidoxin 1 (LdPxn1) and peroxidoxin 2 (LdPxn2) in BALB/c mice. We also evaluated the effect of coadministration of TLR agonists (CpG ODN and GLA-SE) on the antigen-specific immune response. Immunization with recombinant LdPxn1 alone induced a predominantly Th2 type immune response that is associated with the production of high level of IgG1 and no IgG2a isotype while rLdPxn2 resulted in a mixed Th1/Th2 response characterized by the production of antigen-specific IgG2a in addition to IgG1 isotype. Antigen-stimulated spleen cells from mice that were immunized with rLdPxn1 produced low level of IL-10 and IL-4 and no IFN-γ, whereas cells from mice immunized with rLdPxn2 secreted high level of IFN-γ, low IL-4, and no IL-10. Coadministration of CpG ODN or GLA-SE with rLdPxn1 skewed the immune response towards a Th 1 type as indicated by robust production of IgG2a isotype. Furthermore, the presence of TLR agonists together with rLdPxn1 antigen enhanced the production of IFN-γ and to a lesser extent of IL-10. TLR agonists also enhanced a more polarized Th 1 type immune response against rLdPxn2.
- ItemOpen AccessEngineering Streptavidin with Switchable Ligand Binding Affinity Using Disulfide Bonds at the Biotin Entry Gateway(2020-07-22) Marangoni, Jesse M; Wong, Sui-Lam; Ng, Kenneth; Evans, Stephen; Schriemer, David; Ro, Dae-Kyun; Gedamu, LashitewStreptavidin is widely used in biotechnological applications for its specific, high-affinity interaction with its natural ligand biotin, a small vitamin that can be easily conjugated to biomolecules of interest. However, its utility is often limited since many applications require reversibility in binding whereas others require higher affinity than that offered by wild-type streptavidin. For some applications, it would even be beneficial to allow extremely tight binding and subsequent ligand release within a single protocol. To combine both extremely tight and reversible binding into a single protein, two streptavidin muteins, designated M88 and M112, were engineered to each contain a distinct disulfide on opposite sides of a flexible loop critical for ligand binding. Each disulfide bond has markedly different effects on protein structure and binding kinetics. While the disulfide in M112 caused a detrimental conformational change which decreased biotin binding affinity, oxidized M88 showed a ~250-fold decrease in off-rate constant at 21°C and increased thermostability when compared to wild-type streptavidin. Furthermore, reduction of the disulfide bond increased the off-rate constant ~19,000-fold compared to the oxidized form, reducing the half-life for dissociation from 50 years to 1 day. Increasing the temperature to 50°C allows ligand release from the reduced form with a half-life of 9 minutes. M88 thus displays redox and temperature dependent ligand binding, both of which can be used to switch between high- and low-affinity states. M88 coupled to a matrix can be used to capture and release biotinylated biomolecules. For applications where increased temperature is not viable, further engineering of M88 has been used to reduce ligand binding affinity. The relative ease of controlling protein disulfide bond redox state with mild chemical agents allows switchable affinity of M88 towards biotin.
- ItemOpen AccessEvaluation of serum antibody titers, proliferative responses and cytokines after prime-boost immnization of balb/c mice with different formulations of pxn-1 and sod-b1 vaccine candidates from leishmania donovani complex parasites(2008) Tegegne, Mulugeta Gelaw; Gedamu, LashitewLeishmaniasis is a vector borne disease that 1s caused by a protozoan parasite, Leishmania. With an estimated 350 million people at risk, a worldwide prevalence of 12 million and 2 million new reports each year, the world health organization (WHO) marked leishmaniasis as one of the most serious, epidemic prone infectious diseases of our time. Because of lack of effective treatment and the issue of feasibility with other control measures, vaccination remains the best hope for control of all forms of leishmaniasis. Peroxidoxins and Superoxide Dismutases are antioxidants, and are potential vaccine candidates tested in many disease systems including leishmaniasis. However, there are no reports on Pxnl and SODBl from L. donovani complex parasites that cause fatal visceral leishmaniasis, as vaccine candidates. Here in our study we demonstrated that a prime/boost strategy based on Pxnl and SODBl from L. donovani complex parasites, both in DNA/DNA and DNA/Protein strategies (for Pxnl) and DNA/Protein strategy (for SODBl ) is immunogenic and showed humoral and specific T cell, and cytokine responses suggestive of a predominant Thl, but not Th2, like response in Balb/c mice. In both strategies, albeit a varying scale of response, a Thl phenotypic response, characterized by in vitro lymphocyte proliferation, IFN-y production and IgG2a antibodies, was observed with negligible IL-10 production. Future protection studies in mice and antigenic studies on human samples are required to expand the results.
- ItemOpen AccessEvaluation of the Efficacy of Fusion or Combination Leishmania Donovani Peroxidoxin 1 and Superoxide Dismutase B1 Vaccine Candidates against Leishmaniasis in BALB/c Mice: Role of Granulocyte Macrophage Colony Stimulating Factor.(2013-09-09) Bayih, Abebe Genetu; Gedamu, LashitewLeishmaniasis is a vector-borne infectious disease that affects millions of people worldwide. Human leishmaniasis appears in three major clinical forms, cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL). Visceral leishmaniasis is the deadliest form with a mortality rate of about 100% in untreated clinically overt cases. The fact that people who are cured from CL develop durable protective immunity to re-infection has led to the assumption that developing effective vaccine to the disease should be feasible. However, there is no universally effective vaccine yet. Previous studies in our lab have demonstrated that amastigote-specific Leishmania donovani peroxidoxin 1 (LdPxn1) and iron superoxide dismutase B1 (LdFeSODB1) induce specific immune response and partially protect BALB/c mice when administered together with adjuvants. In this study, it was hypothesized that fusing these antigens or using them in a form of cocktail vaccine would further increase the immunogenicity and protective efficacy of the antigens. In addition, two forms of immunization strategies were compared; heterologous DNA/protein prime-boost and homologous protein/protein immunizations. Murine granulocyte macrophage colony-stimulating factor (mGMCSF) adjuvant was used in tandem fusion with the DNA vaccines. Generally, the fusion/cocktail vaccine significantly increased immunogenicity of the vaccines in both immunization protocols. However, the high immunogenicity result was not directly reflected in the protection. In DNA/protein approach, the fusion vaccine was found to be more protective than LdFeSODB1 but not LdPxn1. In protein/protein immunization, the cocktail vaccine showed lower protection than each of the individual antigens. As demonstrated by multiparameter flow cytometry, the increased immunogenicity and protection in DNA/protein immunization was correlated to induction of significantly higher number of antigen-specific CD4+ helper T cells that individually express IFN-γ, TNF-α, and IL-2 cytokines. In addition, the presence of mGMCSF adjuvant in DNA antigens generally increased immunogenicity and protective efficacy of individual or fusion vaccines as compared with the corresponding antigens without mGMCSF. Taken together, these results suggest that heterologous DNA/protein immunization with the fusion vaccine in the presence of mGMCSF adjuvant is more efficacious than protein/protein immunization with the cocktail vaccine.
- ItemOpen AccessFunctional analysis of quantitative differential human metallothionein I gene expression(1990) Shworak, Nicholas W.; Gedamu, Lashitew
- ItemEmbargoHeavy metal induced gene expression in fish cell lines(1986) Price-Haughey, Janet; Gedamu, Lashitew
- ItemOpen AccessHost responses in laying hens following infectious bronchitis vaccination: Comparison of two vaccination strategies(2021-06-03) Buharideen, Sabrina Marsha; Abdul Careem, Mohamed Faizal; Czub, Markus; Niu, Dongyan; Gedamu, LashitewThe infectious bronchitis virus (IBV) causes infectious bronchitis (IB) and causes nephritis and reproductive tract abnormalities depending on the infecting IBV strain. Vaccination for the control of IB has been practiced for decades. Although it has been shown that administration of inactivated vaccine following priming with live attenuated vaccines in pullets induces protection of laying hens against IB, the immunological basis of this protective response has not been investigated adequately. The first objective was to inactivate and formulate the IBV Massachusetts (Mass) variant that was isolated from a flock with shell-less egg syndrome (SES) as an in-house adjuvanted vaccine and test whether it induces an antibody-mediated immune response. Although we observed that the in-house adjuvanted inactivated IBV Mass variant vaccine induces a positive antibody-mediated immune response, it does not induce an antibody-mediated immune response similar to that of a commercial inactivated IBV Mass vaccine. The second objective of the study was to compare two vaccination strategies adopted by the Canadian poultry industry in terms of their ability to induce an adequate immune response in the IBV-impacted tissues in laying hens. Vaccination strategy 1 (multiple live attenuated vaccines) and vaccination strategy 2 (combination of live attenuated vaccines given multiple times and one inactivated vaccine) were given to pullets between 3 and 16 weeks of age. Serum anti-IBV antibodies, recruitment of T cell subsets, and interferon (IFN)-γ mRNA expression were measured at 10 weeks post-last vaccination, in selected tissues. We observed that vaccination strategy 2 induced higher serum anti-IBV antibody response and IFN-γ mRNA expression in the lungs, kidneys and reproductive tract. Both vaccination strategies 1 and 2 recruited CD4+ T cells in the lungs and isthmus and CD8+ T cells in all the examined tissues except the uterus. Serum collected from chickens vaccinated with vaccination strategy 2 was able to neutralize the IBV Mass variant that caused SES in Western Canadian layers, indicating the potential ability of vaccination strategy 2 to protect laying hens against this IBV variant. Overall, our findings indicate that administration of live attenuated vaccines followed by an inactivated vaccine, induces better host responses in laying hens.
- ItemOpen AccessImmune Response and Protective Efficacy of a Heterologous DNA-Protein Immunization with Leishmania Superoxide Dismutase B1(2017-11-22) Bayih, Abebe Genetu; Daifalla, Nada S.; Gedamu, LashitewGrowing evidence shows that antioxidant proteins of Leishmania could be used as vaccine candidates. In this study, we report the efficacy of Leishmania donovani iron superoxide dismutase B1 (LdFeSODB1) as a vaccine antigen in BALB/c mice in a DNA-protein prime-boost immunization regimen in the presence or absence of murine granulocyte macrophage colony stimulating factor (mGMCSF) DNA adjuvant. The expression study confirmed that LdFeSODB1 is expressed in mammalian cells and mGMCSF fusion mediates the secretion of the recombinant protein. Heterologous immunization with LdFeSODB1 induced a strong antibody- and cell-mediated immune response in mice. Immunization triggered a mixed Th1/Th2 response as evidenced by the ratio of IgG2a to IgG1. Antigen-stimulated spleen cells from the immunized mice produced high level IFN-γ. Multiparametric flow cytometry data showed that immunization with LdFeSODB1 induced significantly higher expression of TNF-α or IL-2 by antigen-stimulated T cells. Eight weeks after L. major infection, immunization with the antigen shifted the immune response to a more Th1 type than the controls as demonstrated by IgG2a/IgG1 ratio. Moreover, IFN-γ production by antigen-stimulated spleen cells from immunized mice remained high. The footpad swelling experiment showed that immunization with LdFeSODB1 resulted in partial protection of mice from a high dose L. major infection.
- ItemOpen AccessInvestigating the role of the cell envelope in E. coli relating to silver sensitivity and resistance(2018-05-11) Westersund, Connor; Turner, Raymond Joseph; Gedamu, Lashitew; Harrison, Joe J.Silver (I) is an antimicrobial agent that has established antimicrobial activity, yet the mechanism of action is unclear. This thesis follows up on an observation that Ag+ ions cause the cell membrane to separate from the cell wall in Escherichia coli. Data from a chemical genomic screen was utilized, identifying 6 Ag-responsive cells with single gene deletions (damX, rodZ, minC, sanA, ybhO, tolB), which are genes involved in maintenance of the cell envelope. When these mutants were grown in the presence of Ag+, cells demonstrated extensive cell envelope damage as seen by transmission electron microscopy. Evaluation of kill curves, some mutants conferred resistance and others sensitivity to silver in comparison to wildtype. From the data collected, it was recognized that functions around cell wall and transport across the cell membranes have roles in both silver resistance and sensitivity.
- ItemOpen AccessIron superoxide dismutase cDNAs of Leishmania chagasi: characterization and functional studies(1997) Paramchuk, Wendy Jo-Ann; Gedamu, Lashitew
- ItemOpen AccessIsolation and characterization of casein kinase II cDNA from Leishmania chagasi(1996) Bhatia, Ajay; Gedamu, Lashitew
- ItemOpen AccessIsolation and functional analysis of iron superoxide dismutase (FeSOD) genes in Leishmania(2002) Plewes, Katherine Ann; Gedamu, Lashitew; Storey, Douglas G.
- ItemOpen AccessIsolation, characterization and functional studies of Cathepsin L and B-like Cysteine Protease genes from Leishmania donovani complex(2002) Somanna, Ashwini; Gedamu, Lashitew
- ItemOpen AccessLeishmania donovani Cathepsin B: Impact on Proteins and ncRNAs of Small Extracellular Vesicles, and on Infection Metabolism(2023-02-17) dos Santos Meira, Camila; Gedamu, Lashitew; Buret, Andre; Muench, Douglas; Claire Arrieta, Marie; Jardim, ArmandoLeishmaniasis is a spectrum of chronic diseases caused by protozoan parasites of the Leishmania genus. These parasites can circumvent immune defenses and survive in phagolysosomes of host cells by disrupting signaling processes and cellular functions. Leishmania virulence factors including papain-like cysteine proteases, have been extensively implicated in immune evasion and parasite differentiation. The disruption of the cathepsin B-like cysteine protease of L. donovani was found to attenuate infectivity and induce proteome remodeling in these parasites, affecting the expression of metabolic enzymes and proteins released in extracellular vesicles (EVs). Here, the effects of L. donovani cathepsin B on EV-derived proteins and small non-coding RNAs (ncRNAs), and on metabolism of infected macrophages were investigated. L. donovani cathepsin B wild type (WT), cathepsin B knockout (KO), and episomally complemented knockout (CM) parasites were used to generate comparative proteomic and transcriptomic profiles of small EVs (sEVs), and of metabolites in the spent media of infected U937 cells. LC-MS/MS semi-quantitative analyses revealed the cathepsin B-induced modulation of diverse elements in sEVs, including metabolic enzymes, calpain cysteine proteases, kinases, and translation-related components. Protein-protein networks were remarkable in sEVs and cathepsin B-induced alterations in their proteomes seemed to differ from those previously observed in the parasites, indicating selective packaging of protein cargo in these vesicles. The exploration of the Illumina sequenced small RNAs (18-30nt) from sEVs showed striking abundances of tRNA, rRNA and snoRNA fragments originating from a small subset of genes and with suspected regulatory functions. Moreover, ncRNA genes were differentially expressed in WT and CM sEVs, while KO sEVs exhibited significant expression of coding genes. The bioinformatics predictions of miRNA-like and other regulatory elements for non-annotated transcripts identified in sEVs suggested potential non-canonical regulatory mechanisms in Leishmania. The mass-spectrometry-based metabolomics study indicated extensive differences in the metabolism of macrophages infected with L. donovani WT, KO and CM, especially in nucleotide, energy and carbon metabolisms. Several disturbances appeared to correlate with cathepsin B-induced modulation of metabolic enzymes. Overall, the data suggest the participation of cathepsin B in protein and RNA sorting/packaging into Leishmania EVs and demonstrate the potential mechanisms by which this protease affects host-parasite interaction.
- ItemOpen AccessLeishmania donovani iron superoxide dismutase a: determinants for mitochondrial targeting(2007) Getachew, Fitsum; Gedamu, Lashitew