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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Merced Electronic Theses and Dissertations (2021)

Neglected parasitic diseases, which overlap with known diseases of poverty,cost over 30 million disability-adjusted life years (DALY) and are a massive economic burden for developing countries (Hotez et al., 2014). Toxoplasma gondii is the causative agent of toxoplasmosis, termed a "neglected parasitic diseases" and is a major source of foodborne mortality according to the Center for Disease Control and Prevention (CDC). T. gondii is a globally distributed parasite that infects 30% of the entire human population and no protective vaccine exists for this pathogen. T. gondii has a wide range of virulence; from non-virulent strains endemic to western Europe and North America to highly virulent and genetically diverse strains in South America which are known to evade immunological memory. The overarching goal of my thesis is to find host immunological mechanisms responsible for immunity to virulent T. gondii strains. First, attempts were made to reverse T cell exhaustion that we demonstrated occurs in chronically infected mice given a virulent secondary challenge, but a variety of checkpoint blockade therapies failed to provide immunity to challenge, suggesting alternative immunological mechanisms are required for immunity to secondary infection. We then used an unbiased forward genetic screen to test for survival against virulent secondary infections, and discovered 4 genomic loci associated with immunity. The immunity locus on chromosome 7 encompassed the gene Nfkbid which encodes the protein IkBNS, an atypical regulator of NF-kB signaling required for B-1 cell development. We found that mice without Nfkbid (bumble) could survive primary infection with a low virulent strain, but were susceptible to secondary infections with virulent strains. T. gondii-specific T cell responses for both CD4s and CD8s were intact in bumble mice. However, antibody responses to T. gondii were found to be deficient in bumble mice, specifically parasite-specific IgM was completely abrogated and all parasite-specific IgG isotypes were reduced. Using a series of adoptive transfer and mouse chimera strategies, we were able to make two major observations: (1) B-2 cells are responsible for the majority of high avidity T. gondii-specific antibody response and (2) B-1 cells are required for full immunity to T. gondii infection. In addition, humoral responses were found to be largely enhanced in resistant mice, with greater activation and class-switch recombination found in both the B-1 and B-2 B cell compartments. Finally, we present further evidence that the GPI-anchor of T. gondii may be a favored moiety by antibodies to recognize T. gondii parasite antigen. Together, our data suggests that, in addition to T cells, the anti-T. gondii immune response must be "layered" by both B-1 and B-2 B cells, to generate protective immunity and that Nfkbid is central to the immunological memory responses against T. gondii.

    Cover page: Nfkbid-driven B cell and antibody responses against T. gondiiCreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2013)

    Objective

    We evaluated regional brain volumes and cerebral metabolite levels as correlates of HIV DNA in peripheral blood mononuclear cells (PBMCs).

    Methods

    In this cross-sectional study, 35 HIV+ subjects aged ≥40 years (25 with detectable PBMC HIV DNA; 10 with HIV DNA <10 copies/10(6) cells, the threshold of detection) and 12 seronegative controls underwent structural brain MRI and magnetic resonance spectroscopy at 3 T. HIV+ subjects were on combination antiretroviral therapy ≥1 year; all but 1 had plasma HIV RNA <50 copies/mL. We used logistic regression to evaluate relationships of likely predictor variables to the outcome of PBMC HIV DNA detectability in the HIV+ subjects. Effects of serostatus and HIV DNA on regional brain volumes (normalized to intracranial volume) and on metabolite ratios over creatine were evaluated by analyses of covariance, controlling for age.

    Results

    Relative to the HIV+ group with undetectable HIV DNA, subjects with detectable HIV DNA demonstrated decreased volumes of cerebellar (-14%, p = 0.020) and total subcortical (-10%, p = 0.024) gray matter. Compared to healthy controls, only the detectable HIV DNA group showed significant (p < 0.05) enlargement of lateral ventricles and volumetric reductions of caudate, putamen, thalamus, hippocampus, nucleus accumbens, brainstem, total cortical gray matter, and cerebral white matter. Detectable HIV DNA was not associated with significantly altered cerebral metabolite levels.

    Conclusion

    Inability to clear peripheral blood of HIV DNA is associated with regional brain atrophy in well-controlled HIV infection, supporting the involvement of peripheral viral reservoirs in the neuropathogenesis of persistent HIV-related neurocognitive disorders.
      • Article
      • Peer Reviewed
      UC Merced Previously Published Works (2018)
      T cell exhaustion is a state of hyporesponsiveness that develops during many chronic infections and cancer. Neutralization of inhibitory receptors, or "checkpoint blockade," can reverse T cell exhaustion and lead to beneficial prognoses in experimental and clinical settings. Whether checkpoint blockade can resolve lethal acute infections is less understood but may be beneficial in vaccination protocols that fail to elicit sterilizing immunity. Since a fully protective vaccine for any human parasite has yet to be developed, we explored the efficacy of checkpoint inhibitors in a mouse model of Toxoplasma gondii reinfection. Mice chronically infected with an avirulent type III strain survive reinfection with the type I RH strain but not the MAS, GUY-DOS, and GT1 parasite strains. We report here that mouse susceptibility to secondary infection correlates with the initial parasite burden and that protection against the RH strain is dependent on CD8 but not CD4 T cells in this model. When given a lethal secondary infection, CD8 and CD4 T cells upregulate several coinhibitory receptors, including PD-1, TIM-3, 4-1bb, and CTLA-4. Moreover, the gamma interferon (IFN-γ) response of CD8 but not CD4 T cells is significantly reduced during secondary infection with virulent strains, suggesting that checkpoint blockade may reduce disease severity. However, single and combination therapies targeting TIM-3, CTLA-4, and/or PD-L1 failed to reverse susceptibility to secondary infection. These results suggest that additional host responses, which are refractory to checkpoint blockade, are likely required for immunity to this pathogen.
        Cover page: PD-L1, TIM-3, and CTLA-4 Blockade Fails To Promote Resistance to Secondary Infection with Virulent Strains of Toxoplasma gondii
        • Article
        • Peer Reviewed
        UC Merced Previously Published Works (2021)
        Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice ("bumble") did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection.
          Cover page: Genetic mapping reveals Nfkbid as a central regulator of humoral immunity to Toxoplasma gondii
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2016)

          Background

          Persistent inflammation and immune activation has been hypothesized to contribute to increased prevalence of subclinical atherosclerosis and cardiovascular disease (CVD) risk in patients with chronic HIV infection. In this study, we examined the correlation of peripheral monocyte subsets and soluble biomarkers of inflammation to coronary artery calcium (CAC) progression, as measured by cardiac computed tomography scan.

          Methods

          We conducted a longitudinal analysis utilizing baseline data of 78 participants with HIV infection on stable antiretroviral therapy (ART) in the Hawaii Aging with HIV-Cardiovascular study who had available baseline monocyte subset analysis as well as CAC measurement at baseline and at 2-year follow up. Monocyte phenotypes were assessed from cryopreserved blood by flow cytometry and plasma was assayed for soluble biomarkers using antibody-coated beads in a high sensitivity Milliplex Luminex platform. Change in CAC over 2 years was analyzed as the primary outcome variable.

          Results

          Of all monocyte subsets and biomarkers tested, higher non-classical monocyte percentage (ρ = 0.259, p = 0.022), interleukin (IL)-6 (ρ = 0.311, p = 0.012), and monocyte chemoattractant protein (MCP)-1 (ρ = 0.524, p = <0.001) were significantly correlated to higher 2-year CAC progression in unadjusted Spearman's correlation. Non-classical monocyte percentage (ρ = 0.247, p = 0.039), and MCP-1 (ρ = 0.487, p = <0.001), remained significantly correlated to 2-year CAC progression, while IL-6 was not (ρ = 0.209, p = 0.120) after adjustment for age, hypertension, diabetes mellitus, total/HDL cholesterol ratio, smoking history, and BMI.

          Conclusion

          The percentage of non-classical monocytes and plasma MCP-1 levels were independently associated with CAC progression and may be related to the progression of atherosclerosis and increased CVD risk associated with chronic HIV infection on stable ART.
            Cover page: Non-Classical Monocytes and Monocyte Chemoattractant Protein-1 (MCP-1) Correlate with Coronary Artery Calcium Progression in Chronically HIV-1 Infected Adults on Stable Antiretroviral Therapy.
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2016)
            Inflammation associated with low 25-hydroxyvitamin D (25(OH)D) is associated with increased morbidity and mortality among HIV-infected patients with vitamin D deficiency. We investigated the association between 25(OH)D and soluble biomarkers among HIV-infected patients on stable antiretroviral therapy. This is a cross-sectional study. This study focuses on assessment in subjects 40 years or older on stable antiretroviral therapy (ART) for >3 months. Chemiluminescent immunoassay was used to determine plasma 25(OH)D levels. Plasma soluble biomarkers were measured by Luminex technology. Multivariable linear regression analysis was used to assess the associations between log10-25(OH)D and soluble biomarkers.Of 138 patients, median age was 50.5 (45, 57) years and 25(OH)D was 34.0 (25.0, 42.3) ng/mL. The majority were males (88%) and had undetectable HIV RNA (84.8%); 19 (13.8%) had 25(OH)D ≥50 ng/mL. Spline regression analyses suggested a J-shaped relationship between various plasma biomarkers and 25(OH)D. Among subjects with 25(OH)D ≥20 ng/mL, multivariable linear regression showed positive association between 25(OH)D and interleukin (IL)-10 (β = 1.84, P < 0.001), IL-6 (β = 0.72, P = 0.02), MPO (β = 0.47, P = 0.02), serum amyloid A (β = 1.20, P = 0.04), and tumor necrosis factor (TNF)-α (β = 0.51, P = 0.04). High 25(OH)D (≥50 ng/mL) was associated with higher IL-6 (β = 0.30, P = 0.009), IL-8 (β = 0.14, = 0.005), IL-10 (β = 0.43, P = 0.02), and TNF-α (β = 0.20, P = 0.04), independent of age, sex, ethnicity, body mass index, hepatitis C co-infection, current smoking status, CD4%, and HIV RNA.In older HIV-infected patients, high 25(OH)D was associated with higher (not lower) levels of proinflammatory cytokines. Higher-than-optimal 25(OH)D may be associated with immune dysregulation and may pose a potential health risk among HIV-infected patients.
              Cover page: High 25-hydroxyvitamin D is associated with unexpectedly high plasma inflammatory markers in HIV patients on antiretroviral therapy
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2017)
              Plasminogen activator inhibitor type 1 (PAI-1), a key negative regulator of fibrinolysis, has been investigated to be one of the potential mechanisms of the development of impaired insulin sensitivity, insulin resistance, and diabetes mellitus. Because chronically stable HIV-infected individuals frequently develop abnormal glucose metabolism, including insulin resistance and diabetes mellitus, we postulated that PAI-1 could be one of the multifactorial pathogenic roles in the development of impaired insulin sensitivity and insulin resistance among chronic HIV-infected individuals. From our longitudinal cohort study, we selectively recruited chronically stable HIV-infected individuals without diagnosis of diabetes mellitus at baseline (N = 62) to analyze the correlation of baseline inflammatory cytokines, including PAI-1 and whole-body insulin sensitivity, with 2-year follow-up, as measured by Matsuda Index. We found a negative correlation between baseline PAI-1 and Matsuda Index (r = -0.435, p = .001) and a negative correlation between baseline PAI-1 and Matsuda Index at 2 years (r = -0.377, p = .005). In a linear regression model that included age, total body fat mass percentage, serum amyloid A, and family history of diabetes mellitus, PAI-1 still remained significantly associated with Matsuda Index at 2-year follow-up (β = -.397, p = .002). Our longitudinal study suggests that PAI-1 is an independent predictor of impaired insulin sensitivity among chronic HIV-infected individuals.
                Cover page: Plasminogen Activator Inhibitor-1 Predicts Negative Alterations in Whole-Body Insulin Sensitivity in Chronic HIV Infection
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2015)

                Objective

                This study aimed to evaluate the relationship between inflammatory biomarkers and endothelial dysfunction (ED), as measured by brachial artery flow-mediated dilation (FMD).

                Methods

                We conducted a cross-sectional analysis utilizing baseline data of 135 participants with HIV infection on stable antiretroviral therapy (ART) in the Hawaii Aging with HIV-Cardiovascular (HAHC-CVD) study who had available baseline inflammatory biomarkers and brachial artery FMD measurements.

                Results

                We observed significant associations between brachial artery FMD and baseline brachial artery diameter, age, male gender, traditional cardiovascular disease (CVD) risk factors such as BMI, waist to hip ratio, hypertension, systolic blood pressure (BP), diastolic BP, and LDL cholesterol, and 10-year coronary heart disease (CHD) risk estimated by Framingham risk score (FRS). Of all biomarkers tested, higher level of C-reactive protein (CRP) (beta =  - 0.695, P = 0.030) and serum amyloid P (SAP) (beta =  - 1.318, P = 0.021) were significantly associated with lower brachial artery FMD in univariable regression analysis. After adjusting for baseline brachial artery diameter, age, and selected traditional CVD risk factors in multivariable model, SAP remained significantly associated with brachial artery FMD (beta =  - 1.094, P = 0.030), while CRP was not (beta =  - 0.391, P = 0.181).

                Discussion

                Serum amyloid P was independently associated with impaired brachial artery FMD and may potentially relate to ED and increased CVD risk in HIV-infected patients on stable ART.
                  Cover page: Serum amyloid P (SAP) is associated with impaired brachial artery flow-mediated dilation in chronically HIV-1 infected adults on stable antiretroviral therapy
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2016)

                  Background

                  Inflammation may contribute to cardiovascular disease (CVD) among antiretrovirally suppressed HIV-infected individuals. We assessed relationships of monocyte, CD8 T-cell activation and plasma biomarkers to changes in carotid artery intima-media thickness (CIMT).

                  Methods

                  Longitudinal study of HIV-infected subjects ≥40 years and on stable antiretroviral therapy (ART) ≥3 months. Peripheral blood mononuclear cells were immunophenotyped by multiparameteric flow cytometry to quantify classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)), non-classical (CD14(low/+)CD16(++)) and transitional (CD14(+)CD16(-)) monocyte subsets and activated (CD38(+)HLA-DR(+)) CD8(+) T-cells at baseline. Plasma biomarkers were assessed by multiplex Luminex assay. High-resolution B-mode ultrasounds of right carotid arteries were obtained. Changes in CIMT over two years at the right common carotid artery (CIMTCCA) and right bifurcation (CIMTBIF) were outcome variables.

                  Results

                  We studied 50 subjects: 84% male, median age 49 (Q1, Q3; 46, 56) years, median CD4 count 461 (317, 578) cells/mm(3), and with HIV RNA ≤ 50 copies/mL in 84%. Change in CIMTBIF correlated with log values of baseline absolute count of non-classical monocytes (r = 0.37, p = 0.020), and with MCP-1 (r = 0.42, p = 0.0024) and TNF-α (r = 0.30, p = 0.036) levels. In multivariable linear regression, only non-classical monocytes and MCP-1 predicted the change in CIMTBIF, independent of Framingham Risk Score and baseline CIMTBIF. No correlation was noted between CD8 T-cell activation and CIMTBIF change. Monocyte subsets, CD8 T-cell activation, and biomarker concentrations were not correlated with changes in CIMTCCA.

                  Conclusions

                  Our findings highlight the role of non-classical monocytes and MCP-1 in the progression of CIMTBIF in HIV-infected individuals on stable ART independent of traditional cardio-metabolic risk factors.
                    Cover page: Non-classical monocytes predict progression of carotid artery bifurcation intima-media thickness in HIV-infected individuals on stable antiretroviral therapy
                    • Article
                    • Peer Reviewed
                    UC Merced Previously Published Works (2024)
                    Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δpigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δpigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δpigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions.
                      Cover page: The GPI sidechain of Toxoplasma gondii inhibits parasite pathogenesis.
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