Browsing by Author "Gonzalez-Juarrero, Mercedes, advisor"
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Item Open Access A QuPath workflow utilizing machine learning to analyze homing protein specificity and penetration into lung granulomas of Mycobacterium tuberculosis infected mice(Colorado State University. Libraries, 2024) Patterson, John, author; Gonzalez-Juarrero, Mercedes, advisor; Lyons, Mike, committee member; Ghosh, Soham, committee memberTargeted delivery of drugs to the lungs can improve TB chemotherapy and thus our goal is to develop TB-drug loaded nanoparticles tagged to pulmonary homing peptides. In a previous study, homing peptides to the lungs of TB diseased animals were identified using preclinical TB models (Balb/c and C3HeB/FeJ mice). The selection of homing peptides was carried out using a phage library containing peptides with known homing affinity in other diseases (e.g. cancer). Having identified and selected the homing peptide PL1 (PPRRGLIKLKTS) to granulomas present in the lungs of murine TB models, the PL1 peptide and a negative control (scrambled LinnTT peptide) were tagged to Fluorescein Amidites (FAM). To facilitate tracking in vivo of the nanoparticles to be loaded with TB drugs, silver nanoparticles (SNP) were conjugated to Cy3 fluorochrome, a fluorescent marker used in in vivo tracking studies, followed by functionalization with the PL1 homing peptide (PL1-SNP) or biotin as negative control (Ctrl-SNP). Tracking and homing of the PL1 peptide to granulomas was possible after in vivo administration via intraperitoneal (IP) or intravenous (IV) route of either the FAM tagged synthetic peptides or Cy3-SNPs to Mycobacterium tuberculosis (Mtb) infected C3HeB/FeJ mice. Visualization of the fluorescence-tagged carriers within the lungs was performed using microscopic slides affixed with lung sections from each mouse followed by whole slide imaging. The semi-quantitative analysis of the fluorescence whole slide images performed using the QuPath workflow confirmed that PL1-FAM, or PL1-SNP homed to the granulomas. Thereafter, a QuPath workflow was developed that uses machine learning approaches (MLP) for unbiased identification of tissue types. Other tools were used for characterization and quantification of FAM (synthetic peptides) and Cy3 (SNP) positive cells within granulomatous lesions of the C3HeB/FeJ TB mouse model. Moreover, it was important to quantify the penetration capacity of the FAM tagged peptide as well as the peptide coated SNP into granulomas. QuPath also includes a built in MLP pixel classifier for unbiased segmentation of the whole slide. In addition, a modified QuPath script was developed to segment the granulomas into concentric regions (outer, inner and center) followed by detection and quantification of positive cells for either fluorochrome within each region. Specific colocalization of PL1 with its known receptor (FN-EDB), either as a synthetic peptide or coupled to the SNP, was also studied using lung sections from mice treated with PL1-FAM or PL1-SNP and counter stained with Alexa 647 conjugated anti-FN-EDB monoclonal antibodies. The modified QuPath script was trained to quantify fluorescence from Alexa 647 in cells within granulomas and the Pearson coefficient and QuPath script was used to assess PL1 and FN-EDB colocalization within each region of the granuloma. The results demonstrated that when compared to their respective control samples, the IP route of administration provides equal or better homing of PL1 peptide to the granulomas than the IV route. Both the PL1-FAM and PL1-SNP home to the granulomas and specifically colocalize with its receptor FN-EDB. The FAM tagged peptide and SNP penetrate to the inner and center regions of the granuloma whereas the control SNP were unable to penetrate the barrier in the outer region of the granulomas. The QuPath workflow developed here can be used for tracking and quantification of other homing peptides and nanoparticles for development of new TB therapeutics.Item Open Access An evaluation of biological responses to model biomaterials in vivo and in vitro(Colorado State University. Libraries, 2009) Chamberlain, Lisa M., author; Gonzalez-Juarrero, Mercedes, advisorThe use of in vitro and in vivo models to study inflammatory responses is extremely common in the pre-clinical evaluation of implantable materials and anti-inflammatory drugs. In this body of work we performed comparative studies of the inflammatory responses elicited by different biomaterials when interacting with cells in the implanted host. The results demonstrate first the non-equivalence between immortalized cell lines and primary-derived cell types in the inflammatory response and second temporal effects on differences in responses. Additionally, differences between in vivo and in vitro models are clearly demonstrated, and potential differences between our in vivo and other published models are seen. The results obtained from this comparative study will help to explain many discrepancies found between previous studies reported in the literature.Item Open Access Characterization of Brucella infection in ruminant hosts: disease pathogenesis, immunology, and epidemiology(Colorado State University. Libraries, 2015) Higgins, Jennifer, author; Bowen, Richard A., advisor; Gonzalez-Juarrero, Mercedes, advisor; Nol, Pauline, committee member; Callan, Robert J., committee memberBrucellosis is one of the most common zoonotic diseases worldwide, with endemic disease areas in the Middle East, Mediterranean Basin, Central Asia, Africa, and Central and South America. Disease is caused by various species of the gram negative bacteria Brucella. Infection in humans results primarily from contact with infected livestock or consumption of contaminated livestock products; cattle, small ruminants, and swine are the primary reservoir hosts. Although the Brucella bacterium was discovered over a century ago, control of disease remains a major challenge in many areas worldwide. Research on this pathogen has mostly been conducted in mouse models, which are naturally resistant to infection. Little is known of the immune response of natural ruminant hosts to Brucella infection. Here we report an epidemiological study of brucellosis in Mongolia, as well as an experimental infection study of pregnant goats with two strains of B. melitensis – 16M, a fully virulent strain, and Rev. 1, a reduced virulence vaccine strain. Design of the experimental infection study was influenced by findings from field research in an endemic disease region. The objectives of the experimental challenge study were to characterize clinical disease, shedding, and tissue burdens in infected animals. The cellular immune response was then compared in animals infected with the two B. melitensis strains with the aim of identifying components of the protective response induced by the Rev. 1 vaccine strain and deficits in the immune response elicited by infection with virulent B. melitensis 16M. A fluorescence polarization assay was utilized to identify antibodies in milk samples and estimate the proportion of Brucella positive cattle, yak, and hybrids in three regions of Mongolia. Additionally, prevalence of brucellosis in herd owners was assessed via questionnaire. Information was also collected from herd owners regarding animal husbandry practices and herd health in order to identify individual-and herd-level characteristics that are predictive for brucellosis. The study indicates that brucellosis remains endemic in cattle, yak, and hybrids within Bulgan and Khuvsgul provinces of Mongolia despite a national control program. Herd level prevalence was determined to be 10.4% in the 77 herds tested. High levels of human disease were also reported. Results of the study indicate that the Mongolian brucellosis control program must be critically evaluated if the national goal of obtaining brucellosis-free status by 2021 is to be realized. In an experimental challenge study, pregnant does infected with B. melitensis 16M at midgestation had an 86% abortion rate, while no Rev. 1-infected does aborted. Fetal infection rate was 92% and 43% in kids of 16M- and Rev. 1-infected does, respectively. Widespread tissue colonization was noted in 100% of 16M-infected does, and all of these animals shed brucellae in milk and vaginal secretions. Infection in does inoculated with Rev. 1 was more variable with only one animal showing generalized infection and colonization at levels similar to that of 16M-infected animals. Other Rev. 1-innoculated animals showed low levels of focal infection and shedding. Here we report the first isolation of B. melitensis from muscle tissue of experimentally infected goats. Milk was also found to pose a significant public health risk with three 16M-infected animals consistently shedding brucellae at levels of 104 – 107 CFU/ml over the four days on which samples were collected postpartum. Despite the clear differences in clinical disease resulting from infection with the two strains of B. melitensis, protective versus deficient components of the immune response elicited by these two strains remain undefined. A pro-inflammatory response characterized by increases in granulocytes, monocytes, and CD4+ lymphocytes was identified by flow cytometric analysis of blood from 16M-infected does. In comparison cell numbers remained consistent with pre-infection levels in Rev. 1-inoculated animals. Limited production of IFN-γ and low level expression of the CD25 activation marker indicate a potential anergic state of CD4+ T cells in B. melitensis-infected goats. Increased numbers of IFN-γ producing WC1+ gamma-delta T cells at 28 days post-infection in Rev. 1-inoculated goats in comparison to 16M-infected animals may suggest a role of this cell type in the protective response elicited by the Rev. 1 vaccine strain. The research presented in this dissertation builds upon current knowledge of Brucella epidemiology, pathogenesis, and immunology in natural ruminant hosts. The work provides a strong framework from which further comparative investigations of immune response to virulent B. melitensis and the reduced virulence B. melitensis vaccine strain, Rev. 1, can be conducted with the ultimate goal of defining components of a protective versus deficient response to Brucella in a natural host. This will ultimately aid in development of improved vaccines facilitating control of disease in endemic areas like Mongolia.Item Open Access Inhalational antibiotic therapy for treatment of chronic pulmonary Mycobacterium abscessus disease in mice(Colorado State University. Libraries, 2019) Pearce, Camron, author; Gonzalez-Juarrero, Mercedes, advisor; Jackson, Mary, committee member; Volckens, John, committee memberMycobacterium abscessus (M. abscessus) is a nontuberculous mycobacterium that causes chronic pulmonary infections. Due to M. abscessus's intrinsic antibiotic resistance, treatment is often complex with low cure rates. Tigecycline, a glycylcycline class antibiotic, demonstrates bactericidal effects against M. abscessus without eliciting bacterial resistance mechanisms, however, this antibiotic requires intravenous administration and causes significant side effects that limit its use. Here, we tested the hypothesis that tigecycline administered via inhalation has the potential to maximize the bactericidal effect while reducing side effects. GM-CSF knockout mice with pulmonary M. abscessus infection were treated by intrapulmonary tigecycline aerosols in 0.25 mg, 1.25 mg, and 2.50 mg doses for 28 days. Assessment of pulmonary bacterial burden after full treatment duration shows that inhaled tigecycline is highly effective, dose-dependent, and well tolerated. We concluded that inhaled tigecycline represents a viable treatment option for M. abscessus pulmonary disease. Future studies should address the pharmacokinetics, and ultimately, translation into clinical trials.Item Open Access Minipigs as a neonatal animal model for TB vaccine efficacy(Colorado State University. Libraries, 2016) Ramos Arriaza, Laylaa, author; Gonzalez-Juarrero, Mercedes, advisor; Bowen, Richard, committee member; Izzo, Angelo, committee member; Guth, Amanda, committee member; Dow, Steven, committee memberCurrently, the only vaccine available to prevent tuberculosis (TB) is Bacillus Calmette-Guerin (BCG). The vaccine lacks efficacy against pulmonary disease or reactivation of latent TB but prevents disseminated TB in children and is thus widely used in countries with endemic TB as part of the neonatal vaccine regimen. There are several new vaccines that have shown efficacy against TB in adult animal models yet fail to protect infants from TB disease in clinical trials. Failure in the development of new pediatric vaccines may be due to incomplete knowledge in the elicited immune response to BCG vaccination and testing of vaccine efficacy in adult rather than neonatal animal models. In this novel approach, we used the mini-pig as a neonatal animal model for evaluation of immune responses to BCG vaccine. We demonstrate young mini-pigs are susceptible hosts to the highly virulent Mycobacterium tuberculosis (Mtb) strain, HN878 and that the pathological course of infection resembles that seen in human TB. In this study we longitudinally monitored the immune response of neonatal mini-piglets vaccinated with BCG until adulthood, with the same monitoring applied to a group of unvaccinated mini-piglets. Further, we challenged both vaccinated and non-vaccinated animals via the aerosol route with HN878 and we characterized important changes between the two groups in the course of immune responses following challenge. Based on comparison of immune responses to BCG in mini-pigs and infants, our findings suggest that mini-pigs have the potential to serve as an effective neonatal animal model for TB vaccine development.Item Open Access Novel therapeutics, associated adverse effects, and changes in immune responses during pulmonary infection with Mycobacterium tuberculosis(Colorado State University. Libraries, 2024) Ali, Malik Zohaib, author; Gonzalez-Juarrero, Mercedes, advisor; Basaraba, Randall J., committee member; Moreno, Julie A., committee member; Chen, Chaoping, committee memberPatients diagnosed with multidrug resistant (MDR) or extensively drug resistant (XDR) tuberculosis (TB) have limited treatment options. The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) collectively termed as BPaL for the treatment of TB. This regimen achieved remarkable results as almost 90% of the participants suffering from MDR- or XDR-TB had favorable outcomes. Despite the extraordinary outcomes, many patients also developed severe adverse effects (AEs) which were associated with the long-term administration of the oxazolidinone protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a potent protein synthesis inhibitor of Mycobacterium tuberculosis (Mtb) with an excellent safety profile, but which lacks oral bioavailability. In chapter 2, we hypothesized that inhaled spectinamide 1599, combined with BPa ––BPaS regimen ––, has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c (permissive resistant) and C3HeB/FeJ (permissive susceptible) murine chronic TB efficacy models. Both regimens promoted similar bactericidal effects in lung and spleen of both models after 4 weeks. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cell counts while increased the number of B and helper and regulatory T cells. This combined data suggests that inhaled spectinamide 1599 combined with BPa is an effective TB therapy that avoids L-associated AEs. The granuloma formation is the pathological hallmark of TB, and several studies suggest that there are temporal and spatial changes in their distinct immune responses. These changes differ not only from one granuloma to another in a single individual but also depend on the severity of the disease. In chapter 3, we attempted to understand longitudinal changes in immune cells, their relationships, and their spatial distribution in granulomas of Mtb infected BALB/c and C3HeB/FeJ mouse models using a novel technique of multispectral imaging microscopy. Multiplex fluorescence immunohistochemistry (mfIHC) is unique in its ability to provide both expression and location of several immune cells along with their co-localization in a single tissue section while preserving tissue architecture and spatial context. The results showed that as the infection progresses, there are also dynamic changes in the immune phenotypes forming the granulomas and those located within the parenchymal tissue. Moreover, the histologically similar granulomas manifested complexity in their immune cell composition mainly due to the presence of adaptive immune responses. The advanced cellular granulomas in BALB/c TB model were mainly predominated by CD4 and CD8 T cells, Ly6G stained neutrophils, B220 B cells and all these were surrounded by F4/80 macrophages. With time post infection, there was an increased uniform recruitment of CD4 and Foxp3 T cells, F4/80 macrophages and Ly6G neutrophils within granulomas compared to parenchymal tissue where IFNγ and IL-10 secreting cells were in abundance. Moreover, B220 B cells and CD8 T cells also showed increased but heterogeneous distribution among the advancing granulomas especially B220 B cells formed clusters. The spatial analysis showed an increased median distance for Ly6G neutrophils, whereas this distance was decreased for B220 B cells when measured from CD4 and CD8 cells. In summary, combining the spatial and temporal data in addition to the mere cell counts helps to uncover interactions and relationships between different immune cells within the granuloma.Item Open Access The effects of mannose capped lipoarabinomannan on dendritic cell function(Colorado State University. Libraries, 2009) Lee, Eric John, author; Gonzalez-Juarrero, Mercedes, advisor; Schenkel, Alan, committee member; Chen, Chaoping, committee memberM. tuberculosis is one of the leading causes of death due to infectious disease in the world. While the majority of people are capable of controlling the initial infection, many progress to a latent stage of disease where the M. tuberculosis bacilli persist for long periods of time within the host. The M. tuberculosis cell wall lipoglycan mannose capped lipoarabinomannan (ManLAM) has been characterized as one of the immunomodulatory factors associated with the bacteria [3-5]. ManLAM interacts with dendritic cells (DCs) via DC-SIGN, mannose receptors and to a lesser extent TLR-2 [6-8]. Thus we set out to examine the effects that ManLAM has on DCs both in vitro and in vivo. ManLAM treatment of bone marrow derived DCs (BMDCs) prevents their phenotypic maturation reduces the expression of MHC class II and CD1d. BMDCs stimulated with ManLAM also exhibit altered phagocytic capacity and the inability to stimulate naïve CD4+ T-cell proliferation.