Thesis (doctoral)--Göteborgs Universitet, 1982. / Extra t.p. with thesis statement inserted. Includes the author's published papers. Includes bibliographical references.

Thesis (Ph. D. in Mathematics)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

Streptococcus pyogenes infections can give rise to a diverse array of long-term secondary sequelae, including those in the brain characterized by both motor and neuropsychiatric disorders: Sydenham’s chorea and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS). These conditions are thought to be mediated by neuroinflammatory responses and autoantibody entry into the brain, but the mechanisms are not well understood.Previous work by our laboratory has demonstrated that recurrent intranasal S. pyogenes (Group A Streptococcus, or GAS) inoculations in mice cause infiltration of CD4 T cells into the anterior brain, disruption of the blood-brain barrier (BBB), increased numbers of activated myeloid cells and degradation of excitatory synapses leading to neural circuitry deficits. However, the molecular mechanisms underlying these phenotypes have not been fully explored. To understand how the neurovasculature and myeloid cells respond to recurrent GAS infections at the transcriptome level, I profiled cells from mouse olfactory bulb (OB) and nasal lymphoid tissue by single-cell RNA sequencing (scRNAseq). I found marked shifts in both endothelial cell and microglia populations at the transcriptome level after GAS infections, including downregulation of BBB-associated transcripts by endothelial cells (ECs), and increased production of inflammatory cytokines and chemokines, type I interferon response, and antigen presentation genes by microglia (Chapter 3). I validated several differentially expressed genes using flow cytometry, immunosorbant assays, RNA fluorescence in situ hybridization (FISH), and multiplexed error-robust FISH (MERFISH). Single-cell spatial transcriptomics of the OB revealed regional heterogeneity among microglial responses to GAS, possibly driven by proximity to infiltrating T cells. Analysis of transgenic CX3CR1/TMEM119 dual myeloid reporter mice confirmed that perivascular and meningeal macrophage numbers increase in response to GAS, but, unlike in other neuroinflammatory diseases, few macrophages infiltrate the brain parenchyma. Our laboratory has previously shown that Th17 cells are critical for BBB damage and activated microglia in response to repeated intranasal GAS infections, but the contribution of T helper (Th) 17 cell-derived cytokines in this process, as well as the transcriptional effects of Th17 cells on endothelial cells and microglia are unknown. To expand on these findings, I performed scRNAseq on retinoic acid-related orphan receptor γt (RORγt) mutant mice (Chapter 4) which showed a significant rescue in BBB-associated genes (e.g. Mfsd2a, Itm2a and Itih5) in endothelial cells. Chemokine production and type I interferon gene expression by microglia was also significantly rescued in RORγt mutants; surprisingly antigen presentation by microglia in response to GAS was exacerbated, at both the gene and protein level. Interleukin (IL)-17A is a major cytokine produced by Th17 cells. To examine the role of IL-17A in disease pathogenesis, I treated wild-type mice with an IL-17A neutralizing antibody during the course of GAS infections (Chapter 4). This treatment was sufficient to recapitulate the transcriptional effects on microglia and endothelial cells, as well as rescue BBB permeability previously found in RORγt mutants, indicating that IL-17A may play a critical role in transcriptional responses of endothelial cells and microglia to recurrent GAS infections in vivo. However, IL-17A did not disrupt tight junctions or induce transcytosis on ECs in vitro, suggesting that its effects on ECs in vivo are indirect. Th17 cells are capable of considerable phenotypic plasticity in response to chronic inflammation. To understand this process during recurrent GAS infections, I performed a time course analysis of CD4 T cell subsets after two, three, four and five infections (Chapter 5). This analysis revealed that proportions of “pathogenic” interferon γ-expressing Th17 cells increased over time, as did the number of CD4 T cells expressing granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with pleiotropic effects on autoimmunity. Moreover, I determined that RORγt mutants have decreased proportions of GM-CSF+ CD4 T cells in their nasal mucosa, raising the question of whether GM-CSF may also contribute to CNS pathology (BBB permeability or microglial activation) in addition to IL-17A. To address this question, I generated mice deficient for GM-CSF in T cells and found that conditional deletion of GM-CSF in CD4+ cells partly rescued type I interferon and antigen presentation responses in microglia by scRNAseq, but did not rescue BBB leakage, suggesting that GM-CSF and IL-17A have distinct roles in the neurovascular and neuroinflammatory responses to GAS. To relate the findings in mice to the human disease, in Chapter 6 we performed cytokine profiling in sera from PANDAS/PANS patients at the acute phase of the disease using a multiplex bead-based immunoassay. We found that many chemokines and cytokines produced by activated microglia or macrophages in the mouse model were also highly elevated in the sera of PANDAS/PANS patients. These findings suggest an important link to the human disorder both to understand disease mechanisms in humans and to use them as future clinical biomarkers for diagnosis and treatment monitoring.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Pneumonia is considered to be one of the leading causes of death worldwide. The outcome depends on both, proper antibiotic treatment and the effectivity of the immune response of the host. However, due to the complexity of the immunologic cascade initiated during infection, the latter cannot be predicted easily. We construct a biomathematical model of the murine immune response during infection with pneumococcus aiming at predicting the outcome of antibiotic treatment. The model consists of a number of non-linear ordinary differential equations describing dynamics of pneumococcal population, the inflammatory cytokine IL-6, neutrophils and macrophages fighting the infection and destruction of alveolar tissue due to pneumococcus. Equations were derived by translating known biological mechanisms and assuming certain response kinetics. Antibiotic therapy is modelled by a transient depletion of bacteria. Unknown model parameters were determined by fitting the predictions of the model to data sets derived from mice experiments of pneumococcal lung infection with and without antibiotic treatment. Time series of pneumococcal population, debris, neutrophils, activated epithelial cells, macrophages, monocytes and IL-6 serum concentrations were available for this purpose. The antibiotics Ampicillin and Moxifloxacin were considered. Parameter fittings resulted in a good agreement of model and data for all experimental scenarios. Identifiability of parameters is also estimated. The model can be used to predict the performance of alternative schedules of antibiotic treatment. We conclude that we established a biomathematical model of pneumococcal lung infection in mice allowing predictions regarding the outcome of different schedules of antibiotic treatment. We aim at translating the model to the human situation in the near future.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

A collection of 29 clinical streptococcal isolates obtained from the University of KwaZulu-Natal, Medical School, Durban Metro area (South Africa) were studied to establish their penicillin G susceptibility patterns often refered to as minimal inhibitory concentration (MIC) and to determine the genetic diversity among them using two genotyping methods, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) analysis. All isolates with MIC less than or equal to 0.12 µg/ml were considered susceptible, intermediate resistant if MIC was between 0.25 µg/ml and 4 µg/ml and resistant if greater than 4 µg/ml, The percentage of isolates with resistance was relatively high (75.9%), only 10.3% of isolates showed intermediate resistance and 13.8% of the isolates were completely susceptible to penicillin G. Some of the resistant isolates were highly resistant reaching penicillin G MIC levels of 5000 µ/ml. They were speculated to contain Path altered penicillin binding proteins and high level of crosslinking cell wall induced by the gene products of the MurMN operon. RAPD analysis was performed using three primers, MBPZ-1, MBPZ-2, and MBPZ-3, respectively. RAPD analysis allowed for the identification of 27 RAPD types with MBPZ-1 and MBPZ-3 and 26 RAPD types with MBPZ-2. Ninety-eight percent of these isolates were clustered into two groups, group I and group II, with 90% to 100% dissimilarity among them. Fifty two percent of the isolates of MBPZ-1 group I were in MBPZ-2 group I, 72% isolates of MBPZ-1 group I were in MBPZ-3 group I, and 72% of the isolates of MBPZ-2 group I were in MBPZ-3 group 1. This shows the discriminatory ability of the primers used in this study. Despite clustering of isolates, relatively high diversity was seen. PFGE analysis of macrorestriction fragments obtained after digestion of chromosomal DNA by restriction enzyme, SmaI showed 24 PFGE patterns. The 24 PFGE patterns were divided into three groups (I, II and III) of isolates, with an average of 85% dissimilarity (15% homology) among them. At 25% homology, four clusters, A (13 isolates), B (9 isolates), C (4 isolates), and D (4 isolates) were observed. Two pairs of isolates in group I, cluster A, showed 100% homology. This suggested that each represent the same strain. Four isolates of group I, cluster B, also exhibited 100% homology. This study showed that most of streptococcal isolates with the same penicillin G susceptibility patterns grouped together in a phylogenetic tree by both RAPD and PFGE analysis. There was also some similarity between the results obtained by RAPD analysis and PFGE analysis. Seventeen and nine of the 29 isolates grouped into group I and group II, respectively, two pairs of isolates were indistinguishable, and two pairs of islates were closely related by both RAPD (using MBPZ-3) and PFGE analysis. Although, RAPD analysis is sensitive, specific, faster and cost effective, the ease with which PFGE analysis can be performed, high discriminatory power, reproducibility of the results, and the polymorphism seen in the patterns, suggests that PFGE method has the potential to be very useful for epidemiological evaluations of nosocomial streptococcal infections in KwaZulu-Natal. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Group A streptococcal disorders can result from autoantibodies generated against M proteins. These autoantibodies cross react with the basal ganglia resulting in movement disorders. Previously, we demonstrated binding of streptococcal mAb10F5, with CPu and phospholipids. To determine if mAb10F5 binding to basal ganglia and phospholipids is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hours. Brains were harvested and immunofluorescence was used to analyze brain slices. Control IgG2 rats showed significantly less fluorescence in the CPu than mAb10F5 injected rats at every time point. These findings reaffirm 10F5 is an anti-basal ganglia antibody. To evaluate mechanism of antibody entry, mAb10F5 was examined for anti-phospholipid activity. MAb10F5 displayed greater affinity to phospholipids when compared to IgG2 controls. Our findings support mAb10F5 is an anti-basal ganglia and anti-phospholipid antibody due to its own virulence. / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

Autoantibodies generated against M proteins can cause post-streptococcal disorders such as Rheumatic Fever. A severe complication of rheumatic fever is rheumatic heart disease which may involve both cardiomyopathy and valvulitis. Rheumatic fever has been associated with the class I M protein epitope of Group A streptococcus (GAS). This epitope can be recognized by monoclonal antibodies (mAbs) 10B6 and 10F5. Previously, we demonstrated binding of streptococcal mAb10F5 in the heart tissue (apex, atria, and valves) of Lewis rats as compared to anti-myosin binding. To determine if mAb10F5 binding in the heart is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hrs. Hearts were harvested and immunofluorescence was used to analyze the hearts. The immunofluorescence intensities for IgG2b were compared to mAb10F5 using previously acquired data. Control IgG2b rats showed significantly less immunofluorescence intensities in the heart regions than mAb10F5 injected rats at the 48 and 72 hr time points. These findings reaffirm mAb10F5 as an anti-cardiac antibody thatbinds heart tissue due its own virulence. To differentiate between the two IgG subtypes, binding intensities of IgG2a were compared to the binding intensities of IgG2b. The binding intensities of IgG2a increased with time. This finding was supported by previous work in our laboratory suggesting IgG2a remained in the bloodstream longer than the IgG2b. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science