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Escherichia coli enteropatogênica (EPEC) atípica sorotipo O55:H7: descrição da antifagocitose a partir de um fator secretado. / Atypical enteropathogenic Escherichia coli (aEPEC) serotype O55:H7: description of anti-phagocytosis from a secreted factor.Keyde Cristina Martins de Melo 02 February 2011 (has links)
Escherichia coli enteropatogênica atípica (EPECa) é causadora de diarréia infantil e apresenta alta heterogeneidade quanto aos fatores de virulência. O objetivo deste trabalho foi estudar o comportamento de EPECa na interação com fagócitos profissionais. Duas amostras de EPECa sorotipo O55:H7 mostraram-se capazes de reduzir a fagocitose. Os sobrenadantes dos cultivos foram submetidos a SPE e HPLC e as frações com efeito antifagocítico foram submetidas a espectrometria de massas. A fração capaz de reduzir a fagocitose de bactérias reduziu também a fagocitose de Saccharomyces cerevisiae. Além de mostrar que EPECa é capaz de induzir a antifagocitose, mostrou-se também que o fator antifagocitico é secretado, solúvel em meio aquoso, termoestável, apresenta baixo peso molecular, não é microbicida ou citotóxico e, por último, há indicativos de que possa apresentar uma região glicosídica. Estes achados sugerem que o fator antifagocítico pode, embora não sozinho, exercer um papel importante na adaptabilidade e patogenicidade das EPECa. / Atypical enteropathogenic Escherichia coli (aEPEC) causes diarrhea mainly in children and presents a high heterogeneity of virulence factors. The objective of this work was to study the behavior of aEPEC regarding its interaction with professional phagocytes. Two samples of aEPEC serotype O55:H7 were able to reduce phagocytosis, The culture supernatants were submitted to SPE and HPLC and the active fractions were tested and analyzed by mass spectrometry. The results show that the fraction with bacterial antiphagocytic activity also reduces phagocytosis of Saccharomyces cerevisiae. In addition to demonstrating that aEPEC can induce antiphagocytosis, this work shows that it is due to a secreted antiphagocytic factor that is soluble in aqueous medium, is thermo-stable, has a low molecular weight, is not bactericide or cytotoxic and, finally, possibly presents a glycosidic region. These findings suggest that the antiphagocytic factor may, though maybe not alone, play an important role in the adaptability and pathogenicity of aEPEC.
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Using the polymerase chain reaction to determine the prevalence of Lyme Disease bacteria, Borrelia burgdorferi, in ixodes pacificus ticks from San Bernardino County in Southern CaliforniaAllen, Richard 01 January 2001 (has links)
The purpose of this study was to determine the prevalence of Lyme Disease (LD) bacteria in adult Ixodes pacificus ticks collected from the mountains of San Bernardino County in Southern California. Seven hundred fifty four I. pacificus adults were collected from the Pacific Crest Trail and adjacent areas. The Polymerase Chain Reaction (PCR) was used to screen ticks for Borrelia burgdorferi infection by targeting two different DNA loci. Oligonucleotide primers targeting both the ospA and fla genes were used in the assay. Ticks were processed in pools of three, and genomic DNA from the ticks was extracted with a commercial mini-kit utilizing silica matrix spin-columns. All ticks tested negative for B. burgdorferi infection regardless of primer pair used. In addition, ticks were negative following examination by dark-field microscopy. This study confirms previous reports that the prevalence of LD in Southern California is quite low.
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Genital Chlamydia Infection is Influenced by the Female Sex Hormones Estrogen and Progesterone in VivoGravitte, Amy Gail 01 December 2021 (has links)
Chlamydia is the most common bacterial sexually transmitted infection in the United States and worldwide. It often goes unnoticed due to lack of symptoms and left untreated it can ascend the female genital tract to cause sequelae like pelvic inflammatory disease and irreversible tubal infertility. In reproductive-aged women, female sex hormones estrogen (E2) and progesterone (P4) concentrations fluctuate during the menstrual cycle and are influenced by hormonal contraceptives and hormone replacement therapy. E2 and P4 influence genital Chlamydia infection in women and mice, but these multifactorial interactions are not entirely mapped out. The complex interplay of E2 and P4 with Chlamydia and the host response demand further study to determine the effect of hormonal environment and host susceptibility to Chlamydia.
E2 primarily signals through estrogen receptors (ER) ERα and ERβ. We used ERα or ERβ knockout (KO) mice to study the role of E2 and ERs in chlamydial progression and examined the host immune response at day 9 post-infection, when we expected the immune response to be the most robust. ERαKO, but not ERβKO mice had significant differences in the progression of Chlamydia and the host immune response. Future studies should test the immune response at additional timepoints, and a model should be utilized wherein ERα and ERβ are simultaneously silenced by chemical knockdown of ERβ in ERα knockout mice using ER agonist ICI 182, 680. 3 Mice are widely used in Chlamydia research, but due to its short estrus cycle, infection cannot be established naturally before infected cells are shed. To overcome this, mice are pretreated with depot medroxyprogesterone acetate (DMPA), an exogenous progesterone that halts the estrus cycle. However, a mouse model not reliant on DMPA pretreatment is needed because 1.) DMPA can affect the immune response and 2.) the hormonal environment in women is not static. Our model uses mice that are ovariectomized to stop the production of endogenous E2 and P4, then treated with physiologically relevant levels of E2 and P4 via implantation of a hormone-filled capsule. We observed that E2 protected mice from Chlamydia, making our model a good alternative for in vivo Chlamydia studies.
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The Role of CD4 T Cell Help in Effective CD8 T Cell Responses during Mycobacterium Tuberculosis InfectionLu, Yu-Jung 29 April 2021 (has links)
Tuberculosis (TB), a transmissible disease caused by Mycobacterium tuberculosis (Mtb), is a global health threat. To design an effective vaccine, we need to better understand how different elements of our immune system collaborate to fight against Mtb. CD4 T cells are crucial in protective immunity to Mtb because they produce cytokines including interferon-γ. In contrast, CD8 T cells are thought to play a modest role. Whether CD4 T cells act as “helper” cells to promote optimal CD8 T cell responses during TB is unknown. We argue CD8 T cells’ role are likely underestimated because CD8 T cell functions are compromised without CD4 T cells. Here, using two independent models, I show that CD4 T cell help promotes CD8 T cell effector functions and prevents CD8 T cell exhaustion. I demonstrate CD4 and CD8 T cells synergistically enhance the survival of infected mice. Purified helped, but not helpless, CD8 T cells effectively restrict intracellular Mtb growth. Thus, CD4 T cell help is indispensable for generating protective CD8 T cell responses. In addition, I investigate the mechanisms of CD4 T cell help. Signals from CD4 T cells, and signals relayed by antigen presenting cells collectively shape CD8 T cell responses. We infer that vaccines aimed for eliciting both CD4 and CD8 T cells, in which CD8 T cells are properly helped by CD4 T cells, are more likely to be successful.
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Quantification of Progesterone and 17-β Estradiol in Mouse Serum by Liquid Chromatography-Tandem Mass SpectrometryKennard, Benjamin, Cobble, Allison, Gravitte, Amy, Galloway, Kaleigh, Kintner, Jen, Hall, Jennifer, Brown, Stacy C 05 May 2020 (has links)
Quantification of progesterone and 17-β estradiol in mouse serum by liquid chromatography-tandem mass spectrometry
Authors:
Benjamin Kennard, Allison Cobble, Amy Gravitte, Keleigh Galloway, Jen Kintner, Jennifer Hall, Stacy Brown
Introduction: In the United States, Chlamydia trachomatis is a commonly appearing sexually transmitted infection1. It affects the U.S. healthcare system to a tune of about $500 million dollars annually2. In women, it generally appears asymptomatic and can lead to severe secondary complications such as pelvic inflammatory diseases or infertility1. Female sex hormones, estrogen and progesterone, are being identified to have a role in chlamydial infection. Specifically, this study aims to create quantification methods to detect levels of estrogen and progesterone in mice, infected with Chlamydia muridarum, plasma samples.
Methods: Progesterone samples were prepared using solid-liquid extraction (SLE+) cartridges with ethyl acetate as the elution solvent. Estradiol samples were prepared using liquid-liquid extraction (LLE) with methyl tert-butyl ether and subsequent derivatization with DMIS. Following sample preparation, hormones were quantified in samples using LC-MS/MS with a gradient elution of 1 mM ammonium fluoride in water and acetonitrile. The separation was achieved using a UCT C18 column (100 x 21.mm, 1.8 μm particle size) maintained at 50oC. The mass spectrometer was set up to isolate molecular ions for progesterone (m/z 315.0910) and derivatized estradiol (m/z 431.1835). Quantification was facilitated by the use of deuterium-labeled internal standards and their corresponding molecular ions in the mass spectrometer (d9-progesterone; m/z 324.1230 and d5-estradiol; m/z 436.2922).
Results: Several aspects of the assay presented have been optimized for maximum analyte recovery and analytical sensitivity, including column choice, mobile phase, derivatizing agents for estradiol, and extraction protocols for progesterone. The LC-MS/MS method was investigated for precision and accuracy over three separate days. The dynamic range of the progesterone assay was 5 – 100 ng/mL, with a limit of detection of 1 ng/mL. Likewise, the estradiol assay was linear in the range of 5 – 100 ng/mL, with a limit of detection of 0.5 ng/mL. The average precision, represented by % RSD was 0.74 – 8.5% and 6.3 – 13.4% for progesterone and estradiol, respectively. The accuracy of the method, represented by % error was 1.6 – 14.4% and 4.0 – 10.5% for progesterone and estradiol, respectively. Successful validation was defined as < 15% RSD and error (< 20% at the limit of quantification), per current FDA Guidelines.
Conclusions: The developed LC-MS/MS method is specific for progesterone and estradiol, and the extraction is suitable for preparation of mouse serum samples. This assay could be successfully applied to hormone quantification in mouse samples to support the investigation of the link between chlamydia infection and hormone levels in female animals.
References
1. Chlamydia - 2017 Sexually Transmitted Diseases Surveillance. https://www.cdc.gov/std/stats17/chlamydia.htm. Accessed October 23, 2018.
2. Owusu-Edusei K, Chesson HW, Gift TL, et al. The Estimated Direct Medical Cost of Selected Sexually Transmitted Infections in the United States, 2008. Sex Transm Dis. 2013;40(3):197-201. doi:10.1097/OLQ.0b013e318285c6d2
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The Effects of Farnesol, a Quorum Sensing Molecule from Candida albicans, on Alcaligenes faecalisHutson, Savannah 01 May 2020 (has links)
Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.
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Molecular and Functional Properties of Transmitted HIV-1 Envelope Variants: A DissertationKishko, Michael G. 17 February 2011 (has links)
In 2008 the Nobel Prize in Physiology or Medicine was awarded to the co-discoverers of the Human Immunodeficiency Virus Type 1 (HIV-1), the causative agent of Acquired Immunodeficiency Syndrome (AIDS). This award acknowledged the enormous worldwide impact of the HIV-1/AIDS pandemic and the importance of research aimed at halting its spread. Since the syndrome was first recognized, 25 million people have succumbed to AIDS and over 33 million are currently infected with HIV-1 (www.unaids.org). The most effective strategy for ending the pandemic is the creation of a prophylactic vaccine. Yet, to date, all efforts at HIV-1 vaccine design have met with very limited success. The consistent failures of vaccine candidates stem in large part from the unprecedented diversity of HIV-1.
Among the novel theories of vaccine design put forward to address this diversity is the targeted vaccine approach. This proposal is based on the finding that mucosal transmission of HIV-1, the most prevalent form, occurs across a selective bottleneck such that typically only a single (or a few) variants of the viral swarm present in a donor are passed to the recipient. While the mechanisms controlling the selection are largely unknown, the targeted vaccine approach postulates that once they are identified, we can utilize this understanding to design vaccines specifically targeted to the characteristics shared by the rare, mucosally transmissible HIV-1 variants.
The studies described in this work were conducted to improve our understanding of the factors influencing viral variant selection during mother-to-child-transmission of HIV-1, a route of mucosal transmission which has globally become the leading cause of child infection. A unique panel was generated, consisting of nearly 300 HIV-1 envelope genes cloned from infected mother-infant pairs. Extensive characterization of the genotypes, phenotypes and phylogeny of these clones was then done to identify attributes differentiating early infant from maternal variants. Low genetic diversity of HIV-1 envelope variants was detected in early infant samples, suggesting a bottleneck and active selection of variants for transmission. Transmitted variants did not differ from non-transmitted variants in CD4 and CCR5 use. Infant isolates replicated poorly in macrophages; a cell subtype hypothesized to be important in the establishment of infection. The sensitivity of infant envelope variants to neutralization by a panel of monoclonal antibodies, heterologous and autologous plasmas and HIV-1 entry inhibitors varied. Most intriguingly, envelopes cloned from infants infected during delivery exhibited a faster entry phenotype than maternal isolates. Together, these findings provide further insight into viral variant selection during mother-to-child transmission. Identification of properties shared by mucosally transmitted viral variants may allow them to be selectively targeted, resulting in improved methods for preventing HIV-1 transmission.
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Characterization of Drug Resistance in Mycobacterium Tuberculosis via Saturating Mutagenesis of Drug Targets: A Master’s ThesisHarris, Michelle J. 15 June 2012 (has links)
Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.
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Sensitization of CD8 T Cells During Acute Viral Infections Impacts Bystander and Latecomer CD8 T Cell Responses : A DissertationMarshall, Heather D. 19 October 2009 (has links)
Many virus infections induce a transient state of immune suppression in the infected host. Virus-induced T cell suppression can be caused by T cell activation-induced cell death (AICD), dendritic cell (DC) apoptosis, DC dysfunction, and/or the enhanced expression of immune-suppressive cytokines. It has been previously demonstrated that naïve bystander CD8 T cells derived from hosts experiencing an acute virus-specific T cell response underwent AICD when polyclonally activated by anti-CD3 in vitro (Zarozinski et al., 2000). Susceptibility of naïve bystander T cells to AICD could prevent the development of a new T cell response during an ongoing immune response, and thus render infected hosts immune suppressed. Although immune suppression could result in an enhanced susceptibility to superinfections, virus-infected individuals are more commonly resistant to superinfecting pathogens. Because of these seemingly contradictory conditions, we sought to investigate how acute viral infections impact naïve bystander CD8 T cells in vivo. More specifically, we asked whether bystander CD8 T cells are susceptible to immune suppression or whether they can contribute to the resistance to superinfections. In order to address this, we examined the responses of bystander CD8 T cells activated with cognate antigen during acute viral infections in vivo. We generated several in vivomodels using P14 (LCMV glycoprotein-specific), HY (male antigen-specific), and OT-I (ovalbumin-specific) transgenic CD8 T cells, which we defined as bystander during acute infections with lymphocytic choriomeningitis virus (LCMV), Pichinde virus (PV), vaccinia virus (VV), and murine cytomegalovirus (MCMV).
Consistent with the enhanced susceptibility to cell death noted in vitro, we found that bystander CD8 T cells activated with cognate antigen in vivo during acute viral infections underwent markedly reduced proliferation. Virus-induced transient T cell suppression in vivo was not exclusively mediated by Fas-FasL- or TNF-induced AICD or due to an enhanced susceptibility to apoptosis. Instead, immune suppression in vivowas associated with a delayed onset of division, which we found not to be due to a defect in antigen presentation, but rather due to a T cell intrinsic defect.
Despite the suppressed proliferation of TCR-stimulated bystander CD8 T cells in vivo, we found an enhancement of the effector functions exerted by bystander CD8 T cells activated during acute viral infections. During acute viral infections or after stimulation with type 1 IFN (IFN-αβ) inducers, some bystander CD8 T cells were sensitized to immediately exert effector functions such as IFN-γ production and degranulation upon stimulation with high affinity cognate antigen. Sensitization of naïve CD8 T cells required self-MHC I and indirect effects of IFN-αβ, while IL-12, IL-18, and IFN-γ were not individually required. IL-15 was not required for the rapid expression of IFN-γ, but was required for up-regulation of granzyme B (GrzB). P14 and OT-I CD8 T cells, which are capable of homeostatic proliferation, could be sensitized by poly(I:C), but HY CD8 T cells, which are poor at homeostatic proliferation, could not, suggesting that the requirement for MHC I may be to present low affinity cryptically cross-reactive self antigens. Sensitized naive CD8 T cells up-regulated the t-box transcription factor Eomesodermin (Eomes), which can regulate these rapid effector functions.
In conclusion, we demonstrate in this thesis that acute viral infections impact naïve bystander CD8 T cells such that their response to cognate antigen is altered. Prior to cognate antigen engagement, bystander CD8 T cells up-regulated Eomes, CD122, and GrzB. Following cognate antigen engagement, bystander CD8 T cells rapidly degranulated and expressed the effector cytokine IFN-γ. The ability of bystander CD8 T cells to rapidly exert effector functions may contribute to the resistance of virus-infected individuals to superinfections. Despite these rapid effector functions, the proliferation of TCR-stimulated bystander CD8 T cells was markedly inhibited. This reduced proliferation was found not to be a defect in antigen presentation, but was a T cell intrinsic defect in initiating division. Thus, bystander CD8 T cells were also susceptible to virus-induced immune suppression.
It is also likely that virus-specific CD8 T cells that are not activated until later in the response, so-called latecomer CD8 T cells, may also be susceptible to immune enhancement and suppression. Thus, latecomer CD8 T cells would be able to rapidly exert effector functions at the expense of proliferation. Taken together, we propose that during an immune response, due to spatial and temporal gradients of antigen and inflammation, it is likely that a combination of heterogeneous T cells with different signal strengths and sequences of exposure from cytokines and peptide-MHC constitute the total T cell response to pathogens.
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M.tb Killing by Macrophage Innate Immune Mechanisms: A DissertationHartman, Michelle L 07 September 2011 (has links)
Macrophages infected with a heavy burden of M.tb Erdman undergo a cell death that initially resembles apoptosis but quickly transitions to necrosis. Unlike the previously reported TNF dependent apoptosis induced by avirulent Mycobacterium [1], this form of macrophage cell death is not microbicidal [2]. Microbicidal effects are observed however, when the heavily infected macrophage encounters an uninfected naïve macrophage. My studies describe in part, the crosstalk between the uninfected and infected macrophage that results in the killing of the intracellular M.tb Cell contact between the two cell populations is not necessary for this killing of bacilli to occur and the soluble “signal” of communication between the two cell populations is transferrable, without naïve macrophages present, to newly infected cells also resulting in the reduced viability of the bacilli. We have found that when the IL-1 receptor is absent in the naïve macrophage population that the co-culture antimycobacterial effect is abrogated, suggesting that IL-1 released by the infected dying macrophage is critical for naïve macrophages to respond in a way that results in the decrease in mycobacterial viability. The signaling between the two cell population ultimately converges on activation of iNOS in the infected cell however ROS appears not to be involved.
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