Dissertations / Theses: 'Hemic and Lymphatic Diseases'

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Luo, Alice, Joseph Maguire, Manisha Nukavarapu, and Ashokkumar Gaba. "Peeling away the layers to anemia." Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/asrf/2018/schedule/95.

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Anemia is a significant public health issue that affects a great number of people in developed and developing countries. The World Health Organization states when the Hb value is/dL in an adult male and/dL in a nonpregnant female, the individual is considered as anemic. Iron deficiency is one of the most common causes of anemia. Inadequate intake of iron, chronic blood loss, and/or a combination of both factors typically lead to iron deficiency anemia. In developed countries, chronic blood loss from gastrointestinal, genitourinary, and gynecological sources are the most common etiologies of iron deficiency anemia. Although there are reports of iron deficiency anemia leading to self-inflicted skin excoriation, there are few cases of chronic blood loss from skin excoriation resulting in severe iron deficiency anemia. We present a 49 year old female with significant past medical history of depression, anxiety and chronic back pain who presented after she was found to have profound anemia with hemoglobin of 4.1g/dl. During interviewing, she denied hematuria, hemoptysis, hematochezia, and had been without menstrual cycles for the past year. Urinalysis was negative for blood as well as two documented negative fecal occult blood tests. Iron studies completed showed severely reduced iron levels. Upon further interviewing, the patient reported a supposedly self-diagnosed keratin disorder. For the past ten years she has been self-treating the keratin disorder by applying topical tretinoin and then wrapping it in saran wrap. She would then peel off areas of skin over multiple areas of her body including all extremities and her face. Multiple pictures of bloody piles of tissue were shown. The patient required 3 units of packed red blood cells and was started on iron supplementation. Gastroenterology was consulted and agreed there was no GI source of her blood loss. Psychiatry further evaluated the patient and diagnosed her with obsessive-compulsive disorder with somatic delusions. The prevalence of anemia among chronic psychiatric patients is more frequent than general population. This coexistence deteriorates the quality of life of the patients, prolongs the psychiatric treatment period, and could even cause an increase in morbidity and mortality. Treatment-related factors, drugs taken, physical conditions, negative lifestyle habits, and nutritional disorders are the reasons for anemia among chronic psychiatric patients. Even though iron deficiency anemia in developed country is most often caused by chronic blood loss from gastrointestinal, genitourinary, and gynecological causes, it is important to evaluate for other factors when none of these are present. Psychiatric causes when warranted from history including somatic delusions from obsessive-compulsive disorder should be considered on the differential when other etiologies are less clear.

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Cromwell, Mary A. "The Role of T Lymphocytes in the hu-PBMC-SCID Mouse Model of Epstein-Barr Virus-Associated Lymphoproliferative Disease." eScholarship@UMMS, 1995. https://escholarship.umassmed.edu/gsbs_diss/158.

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Epstein-Barr virus (EBV) is associated with a spectrum of benign and malignant lymphoproliferative disorders, including acute infectious mononucleosis (IM), Burkitt's lymphoma (BL) and immunosuppression-associated B cell lymphoproliferative disease (LPD). Immunosurveillance mediated by virus-specific cytotoxic T lymphocytes is believed to protect immunocompetent hosts from EBV-associated lymphoma and LPD. Due to the lack of an adequate animal model, however, the precise immunologic mechanisms which provide this protection have not been directly demonstrated in vivo. Human peripheral blood mononuclear cell-reconstituted C.B.-17-scid/scid mice (hu-PBMC-SCID mice) develop EBV-positive LPD following intraperitoneal injection of PBMC from EBV-seropositive donors. The SCID mouse disease mirrors human EBV-associated LPD in morphology, presence of the EBV genome, clonality, and patterns of expression of latent viral cellular differentiation antigens. The hu-PBMC-SCID mouse provides a unique small animal model of EBV+ LPD, and it was used in this study to examine the role of CD8+ CTL in controlling LPD. Survival time increase significantly when EBV-specific cytotoxic T-cell lines (CTL) are adoptive transferred into hu-PBMC-SCID mice, demonstrating suppression of LPD in vivoby a CTL-mediated virus-specific mechanism. Survival time also increases significantly with administration of alloreactive CTL lines, suggesting that a non-virus-specific mechanism also contributes to control of EBV-associated LPD by CTL. NOD-SCID mice reconstituted with PBMC from donors with latent EBV infection develop EBV+ LPD with significantly less frequency than do C.B.17-SCID mice reconstituted with PBMC from the same donors. Administration of anti-CD8 mAb to these mice depletes human CD8+ cells and increases the incidence of LPD to 100%, demonstrating that CD8+ T cells are neccessary for protection from EBV-associated LPD. Adoptive transfer of human CD8+ T cells, but not CD4+ T cells, prevents LPD in CD8-depleted NOD-SCID mice. In vivo depletion of CD4+ T cells prevents engraftment of human T cells, and LPD does not develop in most mice after CD4+ cell depletion. These studies are the first to directly demonstrate both the protective role of CD8+ T cells and a requirement for CD4+ T cells in EBV -associated LPD in an in vivo model.

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Castaneda-Avila, Maira A. "The Role of a Monoclonal Gammopathy of Undetermined Significance Diagnosis in Healthcare Utilization." eScholarship@UMMS, 2021. https://escholarship.umassmed.edu/gsbs_diss/1135.

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Background Monoclonal Gammopathy of Undetermined Significance (MGUS) is an understudied precursor of multiple myeloma (MM), the second most prevalent hematologic malignancy in the United States. This dissertation was designed to: (1) Describe the trajectories of serum biomarkers over time in patients with an MGUS diagnosis, (2) Determine if an MGUS diagnosis is associated with changes in healthcare service utilization, and (3) explore the patient- and provider-level drivers of healthcare utilization in patients with MGUS. Methods Data sources include health claims and electronic health records from a community-based population of patients seeking care in central Massachusetts and primary qualitative data collected from providers and patients’ interviews. The analyses included descriptive statistics, group-based trajectory modeling, conditional Poisson regression, and qualitative data analyses. Results (1) Three distinct multi-trajectory groups of creatinine and hemoglobin were identified. (2) The rates of emergency room, hospital, and outpatient visits were higher for patients with MGUS than patients without MGUS. (3) Patients have a basic understanding of MGUS; however, some patients feel anxiety, which may affect other aspects of their lives. Patients primarily see hematologists for follow-up care; other providers have less knowledge about MGUS. Conclusions Biomarker trajectories characterize specific subpopulations of patients with MGUS over time. We found that an MGUS diagnosis is associated with higher healthcare utilization, especially during the months surrounding the diagnosis date. Finally, our study suggests that some patients with MGUS may need psychosocial support services and identifies a gap in knowledge around caring for MGUS patients among primary care providers.

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Cullion, Kathleen J. "Mechanisms of NOTCH1 Mediated Leukemogenesis: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/537.

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Gain of function NOTCH1 mutations are common in both patients with T-ALL and in mouse models of the disease. Inhibiting the Notch pathway in T-ALL cell lines results in growth arrest and/or apoptosis in vitro, suggesting a requirement for Notch signaling in T-ALL. Therefore, we sought to examine the role of Notch1 signaling in both premalignancy and in the maintenance of leukemic growth. Using a murine model of T-ALL, in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development, our preleukemic studies reveal that Notch1 mutations are early events that contribute to the clonal expansion of DN3 and DN4 progenitors. We also demonstrate that progenitors are maintained within the tumor and are enriched in leukemia-initiating cell (L-IC) activity, suggesting Notch1 may contribute to L-IC self-renewal. By studying the effects of Notch signaling in murine T-ALL cell lines, we also demonstrate that Notch1 promotes the proliferation and survival of leukemic blasts through regulation of Lef1 and the Akt/mTOR pathways. Given that T-ALL cell lines are dependent on Notch signaling in vitro, we investigated the effects of Notch inhibition in vivo. We provide evidence that Notch1 can be successfully targeted in vivo and that Notch inhibition, with γ-secretase inhibitors (GSIs), significantly extends the survival of leukemic mice. We also demonstrate that administration of GSIs in combination with rapamycin inhibits human T-ALL growth and extends survival in a mouse xenograft model. Given that NOTCH1 may be required to maintain both L-IC and bulk leukemic growth, targeting NOTCH1 may prove to be an efficacious targeted therapy for T-ALL patients with aberrant NOTCH1 activation.

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Nie, Siwei. "Role of TNF in Heterologous Immunity between Lymphocytic Choriomeningitis Virus and Vaccinia Virus: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/394.

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Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.

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Precopio, Melissa Lynn. "EBV-Specific CD4+ T Cell Responses in Acute Infectious Mononucleosis: a Dissertation." eScholarship@UMMS, 2004. https://escholarship.umassmed.edu/gsbs_diss/113.

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Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes a life-long latent infection of B cells. It is usually asymptomatic in healthy individuals; however, individuals with compromised immunity often develop EBV-induced lymphoma. EBV also encodes potential oncogenes that can contribute to tumorigenesis. Therefore, vaccine and immunotherapeutic strategies targeting EBV are desirable. Recent studies have shown that infusion of EBV-specific CD8+T cells can elicit remission of lymphomas arising after administration of immunosuppressive drugs during transplantation, suggesting an important role for T cells in the prevention of EBV-induced malignancy. A better understanding of the cellular immune components involved in the control of EBV will aid in the development of methods to prevent infection and/or treat EBV-associated disease. While EBV infection is usually acquired asymptomatically during childhood, primary infection of adolescents and young adults can result in an illness termed acute infectious mononucleosis (AIM). Because of the characteristic symptoms of the illness, individuals with AIM can be readily identified and diagnosed with acute EBV infection. Thus, primary CD4+ and CD8+ T cell responses against the virus can be evaluated. It has been previously found that there is a marked expansion of lytic EBV protein-specific CD8+ T cells early during AIM, with delayed detection of lower frequencies of latent EBV protein-specific CD8+ T cells. The magnitude and specificity of CD4+T cell responses during AIM has been less well characterized. This thesis dissertation presents data from both functional assays and direct staining experiments documenting the timing, magnitude, and antigen-specificity of CD4+ T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4+ T cells were readily detected by intracellular IFN-γ production at presentation with AIM and declined rapidly thereafter. Blood EBV load was also quantitated and found to decrease over time following AIM. By contrast, CD8+T cell IFN-y responses remained high for several weeks following presentation with AIM. Direct staining of lytic epitope-specific CD4+ T cells during AIM revealed high frequencies of virus-specific cells with low proliferative and IFN-γ-producing potential. Blood EBV load in these patients was persistently high through 6 wk following AIM. These data suggest a relationship between high EBV load during acute infection and impaired EBV-specific CD4+ T cell responses, which are compatible with impaired CD4+ T cell responses reported during high viremia associated with other viral infections. This may represent a mechanism by which persistent viruses, such as EBV, are able to establish a life-long infection in their hosts.

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Pan, Feng. "Understanding Ten-Eleven Translocation-2 in Hematological and Nervous Systems." FIU Digital Commons, 2014. http://digitalcommons.fiu.edu/etd/1925.

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I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

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Shockett, Penny E. "Regulation of IgA Class Switch Recombination in the I.29μ B Cell Lymphoma by Cytokines and Inhibitors of Poly(ADP-ribose) Polymerase: A Thesis." eScholarship@UMMS, 1993. https://escholarship.umassmed.edu/gsbs_diss/133.

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Heavy chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region which will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and the degree to which the regulation of germline transcripts correlates with the regulation of switching, we studied this process in the murine B-lymphoma cell line I.29μ, which in the presence of bacterial lipopolysaccharide (LPS) switches primarily to IgA and less frequently to IgE. Levels of α-germline transcripts initiating upstream of α switch (Sα) sequences are elevated in clones of this line which switch well as compared to clones which switch less frequently. TGFβ1 has been shown to increase α-germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B-cells. We now demonstrate in I.29μ cells that TGFβ also increases switching to IgA and increases the level of α-germline transcripts 5 to 9 fold. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGFβ appears to direct switching to IgA by inducing transcription from the unrearranged Sα- CαDNA segment. Germline α RNA is quite stable in I.29μ cells, having a half life of about 3 to 5 hours, and we find only slight stabilization in the presence of TGFβ. Levels of ε-germline transcripts are not increased by TGFβ . IL-4, which modestly increases switching to IgA in I.29μ cells, slightly increases trancription of α-germline RNA. However, we present evidence suggesting that endogenously produced IL-4 may also act at additional levels to increase switching to IgA. IFNγ, which reduces IgA expression in these cells, also reduces the level of α-germline transcripts. IFNγ also reduces the level of ε-germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines in turn regulates the specificity of recombination. In studies aimed at identifying other signalling pathways that promote class switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase lipopolysaccharide (LPS)-induced switching to IgA in the B cell lymphoma I.29μ and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In I.29μ cells, PARP inhibitors increase IgA switching by day 2 and cause a 5-fold average increase in switching on day 3 as assayed by immunofluorescence microscopy. The PARP inhibitor, nicotinamide, also causes a reduced intensity of hybridization of Cμ and Cα specific probes to genomic DNA fragments containing the expressed VDJ-Cμ and the unrearranged Sα - Cα segments, respectively, indicating that PARP inhibition increases rearrangment of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogs or reduced by inhibitors of protein kinase A (PKA). Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged Cα gene, although TGFβ is required for optimal induction by PARP inhibitors, consistent with a requirement for transcription of the unrearranged CH gene. PARP inhibitors do not overcome the requirement for endogenously produced IL-4.

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Litman, Rachel. "Characterization of the BACH1 Helicase in the DNA Damage Response Pathway: a Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/329.

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DNA damage response pathways are a complicated network of proteins that function to remove and/or reverse DNA damage. Following genetic insult, a signal cascade is generated, which alerts the cell to the presence of damaged DNA. Once recognized, the damage is either removed or the damaged region is excised, and the original genetic sequence is restored. However, when these pathways are defective the cell is unable to effectively mediate the DNA damage response and the damage persists unrepaired. Thus, the proteins that maintain the DNA damage response pathway are critical in preserving genomic stability. One essential DNA repair protein is the Breast Cancer Associated gene, BRCA1. BRCA1 is essential for mediating the DNA damage response, facilitating DNA damage repair, and activating key cell cycle checkpoints. Moreover, mutations in BRCA1 lead to a higher incidence of breast and ovarian cancer, highlighting the importance of BRCA1 as a tumor suppressor. In an effort to better understand how BRCA1 carried out these functions, researchers sought to identify additional BRCA1 interacting proteins. This led to the identification of several proteins including the BRCA1 Associated C-terminal Helicase, BACH1. Due to the direct interaction of BACH1 with a region of BRCA1 essential for DNA repair and tumor suppression, it was speculated that BACH1 may help support these BRCA1 function(s). In fact, initial genetic screenings confirmed that mutations in BACH1 correlated not only with hereditary breast cancer, but also with defects in DNA damage repair processes. The initial correlation between BACH1 and cancer predisposition was further confirmed when mutations in BACH1 were identified in the cancer syndrome Fanconi anemia (FA) (complementation group FA-J), thus giving BACH1 its new name FANCJ. These findings supported a previously established link between the FA and BRCA pathways and between FA and DNA repair. In particular, we demonstrated that similar to other FA/BRCA proteins, suppression of FANCJ lead to a substantial decrease in homologous recombination and enhanced both the cellular sensitivity to DNA interstrand cross-linking agents and chromosomal instability. What remained unknown was specifically how FANCJ functioned and whether these functions were dependent on its interaction with BRCA1 or other associated partners. In fact, we identified that FANCJ interacted directly with the MMR protein MLH1. Moreover, we found that the FANCJ/BRCA1 interaction was not required to correct the cellular defects in FA-J cells, but rather that the FANCJ/MLH1 interaction was required. Although both the FA/BRCA and MMR pathways undoubtedly mediate the DNA damage response, there was no evidence to suggest that these pathways were linked, until recently. Our findings not only indicate a physical link between these pathways by protein-protein interaction, but also demonstrated a functional link.

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Zhang, Haojian. "The Molecular Mechanisms for Maintenance of Cancer Stem Cells in Chronic Myeloid Leukemia: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/614.

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Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the Philadelphia chromosome (Ph) that arises from a reciprocal translocation between chromosomes 9 and 22, thereby resulting in the formation of the chimeric BCR-ABL oncogene encoding a constitutively activated tyrosine kinase. BCR-ABL tyrosine kinase inhibitors (TKIs) induce a complete hematologic and cytogenetic response in the majority of chronic phrase CML patients. However, TKIs cannot efficiently eradicate leukemia stem cells (LSCs) because of the insensitivity of LSCs to TKIs. Therefore, developing new strategies to target LSCs is necessary and critical for curing CML, and success of this approach depends on further understanding the molecular mechanisms by which LSCs survive and are maintained. In Chapter I, I briefly introduce CML disease, BCR-ABL oncoprotein, and TKIs. I also describe the identification and features of LSCs. Several key pathways in LSCs including Wnt/ß-catenin, hedgehog, FoxO, Bcl6 and HIF1, are discussed. I also propose our strategy to identify unique molecular pathways that are important for LSCs but not their normal stem cell counterparts. In Chapter II, I describe our finding about the function of the positive regulator, HIF1α, in CML development and LSC survival. I show that loss of HIF1α impairs the maintenance of CML through impairing cell cycle progression and inducing apoptosis of LSCs, and I also report that p16Ink4a and p19Arf mediate the effect of HIF1α on LSCs, as knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α-/- LSCs. As detailed in Chapter III and IV, through comparing the global gene expression profiles of LSCs and HSCs, I find two novel regulators, Blk and Scd1, which act as tumor suppressors in CML development. In Chapter III, I show that Blk is markedly down-regulated by BCR-ABL in LSCs, and that c-Myc and Pax5 mediate this down-regulation. Deletion of Blk accelerates CML development; conversely, Blk overexpression significantly delays the development of CML and impairs the function of LSCs. I also demonstrate that p27, as a downstream effector, is involved in the function of Blk in LSCs. Blk also functions as a tumor suppressor in human CML stem cells, and inhibits the colony-forming ability of human CML cells. In Chapter IV, I investigate the function of another negative regulator, Scd1, in CML LSCs, and find that expression of Scd1 is down-regulated in mouse LSCs and human CML cells. We report that Scd1 acts as a tumor suppressor in CML, as loss of Scd1 causes acceleration of CML development and overexpression of Scd1 delays CML development. Using a colony-forming assay, I demonstrate that Scd1 impairs the maintenance of LSCs due to the change of expression of Pten, p53 and Bcl2. Importantly, I find that both Blk and Scd1 do not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Taken together, our findings demonstrate that HIF1α is required for the maintenance of CML LSCs, and conversely that Blk and Scd1 suppress the function of LSCs, suggesting that combining TKI treatment with specific targeting of LSCs will be necessary for curing CML.

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Mallick, Emily M. "A New Murine Model For Enterohemorrhagic Escherichia coli Infection Reveals That Actin Pedestal Formation Facilitates Mucosal Colonization and Lethal Disease: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/601.

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Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.

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Peng, Cong. "Novel Therapeutic Targets for Ph+ Chromosome Leukemia and Its Leukemia Stem Cells: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/473.

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The human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. The resulting chimeric BCR-ABLoncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of CML patients, but is unable to completely eradicate BCR-ABL–expressing leukemic cells, suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. Two major reasons cause the imatinib resistance. The first one is the BCR-ABL kinase domain mutations which inhibit the interaction of BCR-ABL kinase domain with imatinib; the second one is the residual leukemia stem cells (LSCs) in the patients who are administrated with imatinib. To overcome these two major obstacles in CML treatment, new strategies need further investigation. As detailed in Chapter II, we evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble Hsp90 inhibitor, IPI-504, in our BCR-ABL retroviral transplantation mouse model. We found that BCR-ABL mutants relied more on the HSP90 function than WT BCR-ABL in CML. More interestingly, inhibition of HSP90 in CML leukemia stem cells with IPI-504 significantly decreases the survival and proliferation of CML leukemia stem cells in vitro and in vivo. Consistent with these findings, IPI-504 treatment achieved significant prolonged survival of CML and B-ALL mice. IPI-504 represents a novel therapeutic approach whereby inhibition of Hsp90 in CML patients and Ph+ ALL may significantly advance efforts to develop a cure for these diseases. The rationale underlying the use of IPI-504 for kinase inhibitor–resistant CML has implications for other cancers that display oncogene addiction to kinases that are Hsp90 client proteins. Although we proved that inhibition of Hsp90 could restrain LSCs in vitro and in vivo, it is still unclear how to define specific targets in LSCs and eradicate LSCs. In Chapter III, we took advantage of our CML mouse model and compared the global gene expression signature between normal HSCs and LSCs to identify the downregulation of Pten in CML LSCs. CML develops faster when Pten is deleted in Ptenfl/fl mice. On the other hand, Pten overexpression significantly delays the CML development and impairs leukemia stem cell function. mTOR is a major downstream of Pten-Akt pathway and it is always activated or overepxressed when Pten is mutated or deleted in human cancers. In our study, we found that inhibition of mTOR by rapamycin inhibited proliferation and induced apoptosis of LSCs. Notably, our study also confirmed a recent clinical report that Pten has been downregulated in human CML patient LSCs. In summary, our results proved the tumor suppressor role of Pten in CML mouse model. Although the mechanisms of Pten in leukemia stem cells still need further study, Pten and its downstream, such as Akt and mTOR, should be more attractive in LSCs study.

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Almasmoum, Hibah. "Biochemical analysis of the BTG1 variants associated with Non-Hodgkin's lymphoma." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/47720/.

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Non-Hodgkin’s lymphoma (NHL) is a group of lympho-proliferative disorders characterised by genetic mutations resulting in the selection of a malignant clone. Recently, mutations in the anti-proliferative B-cell translocation 1 gene (BTG1) and B-cell translocation 2 gene (BTG2) have been identified in in NHL cases, which suggests a direct involvement of BTG1 and BTG2 in malignant transformation. BTG1 and BTG2 are members of the human BTG/TOB family. They are characterised by the conserved amino-terminal BTG domain, which mediates interactions with the human Caf1(hCaf1) catalytic subunit of the Ccr4-Not deadenylase complex .In addition, the BTG domain binds to the cytoplasmic poly (A)-binding protein (PABPC1). This complex plays a critical role in mRNA deadenylation and degradation as well as translational repression. It is currently unclear how, or indeed whether, mutations in BTG1 and BTG2 affect the function of the gene products. Therefore, a combination of sequence analysis and molecular modelling was used to predict the functional consequences of mutations previously identified in NHL. Sorting intolerant from tolerant (SIFT) and Suspect (Disease-Susceptibility-based SAV Phenotype Prediction) prediction tools enabled the identification of amino acid residues that would potentially interfere with the protein function, and hence may be associated with disease. In total 45 mutations in BTG1 and BTG2 were assessed. These mutations were derived from NHL samples, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma and Burkitt's lymphoma. Of the variants analysed, 15 were predicted to interfere with the function of BTG1 using SIFT analysis (Score ≤0.05). Only seven of these variants were predicted to be likely associated with disease using Suspect algorithm (Score ≥50), and an additional variant, BTG1 C149del, was predicted to interfere with protein function using PROVEN (Protein Variation Effect Analyzer). The ability of these protein variants to interact with known partners was established using yeast two hybrid assays. In addition, functionally assessment of the role of the mutated proteins in cell cycle progression, translational repression and mRNA degradation was also performed. Using a yeast two-hybrid system, ten BTG1 variants were shown to affect the interaction of BTG1 with the hCaf1 (CNOT7/CNOT8) catalytic subunit of the Ccr4-Not deadenylase complex. In addition, when BTG1 variants were transfected into mammalian cells, these BTG1 variants (M11I, F25C, R27H, F40C, P58L, G66V, N73K and I115V), unlike the wild-type proteins, were not able to inhibit cell cycle progression. These results suggest that anti-proliferative BTG1 is required for hCaf1 (CNOT7/CNOT8) deadenylase activity. The remaining BTG1 variants (L37M, L94V, L104H and E117D) were not consistent in the correlation of BTG1 interaction with hCaf1 (CNOT7/CNOT8) and inhibition of cell growth which led to the suggestion that BTG1 may require an additional factor such as PABPC1. Interestingly, several BTG1 variants (M11I, F25C, R27H, P58L, N73K I115V and E117D) did not require interaction with the hCaf1 (CNOT7/CNOT8) deadenylase enzyme to reduce reporter activity as established using 3’ UTR tethering assays. This suggests that BTG1 may also have a role in regulating cell cycle progression and RNA degradation via Ccr4-Not deadenylase complex independent mechanisms. The data show that variants in BTG1 commonly found in DLBCL, are functionally significant and are likely to contribute to malignant transformation and tumour cell grow.

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Ahankari, A. S. "Maharashtra Anaemia Study : an investigation of factors associated with adolescent health and pregnancy-related outcomes in women from Maharashtra State, India." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/47078/.

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Maharashtra Anaemia study (MAS) was conducted as a part of the PhD programme of Dr Anand Ahankari through a joint collaboration of the University of Nottingham, UK and Halo Medical Foundation, India. The main goal of the study was to establish baseline epidemiological data for anaemia research in pregnant women and adolescent girls in Maharashtra state of India. Iron deficiency anaemia is the most common form of anaemia observed in India, and assessed based on haemoglobin (Hb) levels in blood. Clinically, anaemia is categorised in mild, moderate and severe form based on Hb levels. The project had three main sections, a) Adolescent girls cross sectional survey, b) Pregnant women prospective study, and c) Maharashtra state birth registry analysis. The study aimed to investigate individual and village level risk factors of anaemia in adolescent girls (13 to 17 years), and pregnant women (3 to 5 months) living in rural Maharashtra. Data from pregnant women were also used to examine risk factors associated with low birth weight (LBW). A recently introduced non-invasive haemoglobin (Hb) technology (known as NBM 200) was validated in this Indian setting by comparing Hb measurements obtained from the NBM 200 with reference blood measurements. In the adolescent survey, Sahli’s hemometer (finger prick technique) was used to estimate reference Hb values, while in pregnant women venous blood samples were obtained to measure Hb using an automated analyser. Anaemia was defined using Hb levels based on the following cut offs, (a) Hb < 12.0 g/dl in adolescent girls, and in (b) Hb < 11.0 g/dl in pregnant women. Multivariable regression technique was used to identity risk factors associated with anaemia and LBW. The Maharashtra state birth registry records covering a 32-year period (1980 to 2011) were investigated to assess temporal changes in the sex ratio at birth to investigate impacts of sex determination prevention legislations (known as PNDT 1994 and PCPNDT 2003). The adolescent girls’ survey showed a very high prevalence of anaemia (87%). Of 45 factors assessed in the survey, four were associated with adolescent anaemia. Anaemia likelihood increased significantly with age (Odds Ratio [OR] 1.41 per year, 95% CI: 1.17 to 1.70). Factors associated with decreased risk of anaemia were higher mid upper arm circumference (> 22 cm) (OR 0.51, 95% CI: 0.31 to 0.82), and ≥3 days/week consumption of fruit (OR 0.35, 95% CI: 0.23 to 0.54). At village level piped water supply was associated with higher Hb levels (β coefficient 0.61 g/dl, 95% CI: 0.39 to 0.82). Results from the NBM 200 reported wide agreement levels in the Bland-Altman analysis (mean difference of -2.70 g/dl, 95% CI: -2.84 to -2.55) demonstrating an overestimation of Hb by the NBM 200 compared to Sahli’s hemometer. The NBM 200 showed low sensitivity (23.6%) and moderate specificity (61.8%) for the diagnosis of anaemia in the adolescent population. Findings from pregnant women showed high anaemia prevalence (77%). Of 51 factors assessed in the study, three were associated with maternal anaemia. Increased risk of anaemia was seen in women with consanguineous marriages (OR 2.41, 95% CI: 1.16 to 5.01). Post-delivery data from full-term singleton live births showed the prevalence of LBW babies was 7%. Consanguineous marriage was a major risk of LBW babies in our study population (OR 5.68, 95% CI: 1.58 to 20.32). Village level risk factors showed lower likelihood of maternal anaemia with regular access to government nurses (OR 0.48, 95% CI: 0.25 to 0.93). The NBM 200 validation showed overestimation of Hb levels and underestimation of anaemia. Bland-Altman analysis showed a mean difference of -1.8 g/dl (95% CI: -2.06 to -1.71) indicating a systematic overestimation by the NBM 200 compared to venous Hb measurements. The device showed low sensitivity (33.7%) but high specificity (91.8%) for the diagnosis of anaemia in the pregnant woman population. The 32 years of longitudinal birth registry data showed a significant increase in the sex ratio at live birth from 1980 to 2004, and then a subsequent decrease in sex ratio. The annual state male:female sex ratio of Maharashtra increased from a baseline of 1.11 in 1980 to a maximum value of 1.23 in 2003, before decreasing to 1.16 in 2011. This represented an increase in the annual sex ratio at live birth from 1980 to 2004 of 0.005 units per year (p < 0.001), and a decrease of 0.009 units per year after 2004 (p < 0.01). The increase in the sex ratio was consistent with the hypothesis of both increasing availability and acceptability of ultrasound scanning during this period, enabling foeticide of females in utero. The probable cause for the decrease in sex ratio after 2004 is likely to be due to the strengthening of the legislation banning sex-specific foeticide.

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Aldosari, Sahar. "Assessment of DNA damage and DNA damage response and repair in dormancy-enriched leukemia cells." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/47257/.

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Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by clonal expansion of abnormal/immature hematopoietic precursor cells in the bone marrow. A side compartment in the BM niche consists of abnormal, quiescent cells, which are called dormant leukemic initiating cells (DLICs). Patients with AML tend to respond well to remission induction chemotherapy, but relapse is common because current therapies cannot completely eradicate leukemic cells. It is widely accepted that CD34+CD38− DLICs are more resistant to chemotherapy and that they contribute to drug resistance and relapse of AML to a greater extent than progenitor CD34+CD38+ cells. DLICs have been extensively characterised, but they remain a critical area of investigation for clinical research because of the low prevalence of DLICs and similarity to normal HSCs. A model of dormancy in vitro that shows most of the features of DLICs had previously been established in the Nottingham Haematology Group. This study used this model and aimed to investigate whether the response to DNA damage was different in dormancy-enriched cells compared to cycling leukemic cells following chemotherapy. The amount of DNA damage was assessed up to 24 hours pre- and post- drug treatment using the neutral Comet assay. Lower levels of damage were observed in dormancy-enriched cells following etoposide (ETO) treatment at 4 hours (p = 0.04), although this switched at the 24 hour time point where accumulated DNA double-stranded breaks (DSBs), in dormancy-enriched KG1a cells were associated with a higher percentage of viable cells. DNA damage response cascade markers in both dormancy-enriched and cycling cells showed phosphorylation by flow cytometry (phospho-H2AX139, pATM-S1981, H2AX142, and pChk-Thr68) in response to conventional AML chemotherapy. Significantly lower levels of cleaved PARP-Asp214 and active caspase 3 were observed in dormancy-enriched cells treated with ara-c (p = 0.0001) or ETO (p = 0.0001) at 24 hours, strongly indicating that survival responses are activated in dormancy-enriched cells. Induction of 53BP1 foci, the hallmark of non-homologous end joining (NHEJ) was observed following treatment with ara-c (p = 0.038) and ETO (p = 0.049) in dormancy-enriched cells, indicating the NHEJ repair pathway is the preferred mechanism for DSB repair. At the molecular level, BTG2 expression was involved in the DNA damage response. Significant induction of BTG2 was detected in cycling treated cells with ETO for 24 hours. In conclusion, this study provides evidence that phosphorylation of H2AX139 and H2AX142 is a key response marker that may explain the mechanism underlying the drug resistance of DLICs and induction of repair. Therefore, results of this study may help in devising novel treatment strategies for AML that target H2AX142 of DLICs to permanently eradicate all leukemic cells and improve overall survival.

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Aram, Jehan Jalal. "Granulocyte-macrophage colony-stimulating factor : expression and regulation in human immune responses with relevance to multiple sclerosis." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/48853/.

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Background: Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a haematopoietic growth factor and a pro-inflammatory cytokine produced by T cells and other immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. Few recent studies have detected GM-CSF expression by immune cells in MS. In this thesis, the expression of GM-CSF and its receptor by different subtypes of peripheral blood mononuclear cells (PBMCs) in MS was investigated. In addition, GM-CSF regulation was studied in the above-mentioned cells in MS. Finally, GM-CSF neutralization was performed in a phase Ib clinical trial, and some immune-related effects were investigated. Aims: To evaluate the expression of GM-CSF and its receptor by PBMC subsets in MS; to determine the key factors regulating their expression by PBMC subsets in MS; to detect the differentiation of helper T cells producing GM-CSF (Th-GM) in MS patients, and to detect the frequency of immune cells after GM-CSF neutralization in MS in vivo. Subjects and Methods: Patients were mainly untreated relapsing-remitting MS (RRMS) during remission stage, and some were MS patients during a relapse. Healthy controls were also enrolled. All subjects consented to participation in the study before donating peripheral blood. PBMCs were isolated using Ficoll density gradient centrifugation. Flow cytometry and q-PCR were used to detect the expression of GM-CSF and its receptor. Multiplex bead assay was used to quantify GM-CSF with other pro-inflammatory and anti-inflammatory cytokines. Results: The frequency of stimulated GM-CSF-expressing cells (helper T (Th), cytotoxic T (Tc), monocytes, NK cells, and B cells) is significantly higher in the mixed PBMC population of untreated RRMS patients when compared to healthy volunteers. The frequency of Th1 cells expressing GM-CSF was higher in MS patients than healthy controls. The expression of GM-CSF by isolated and stimulated NK cells was not different in MS patients and controls. PBMC culture supernatants were shown to contain significantly higher concentrations of IL-2, IL-12, IL-1β, and GM-CSF in MS patients than controls. Blocking IL-2 and IL-12 significantly reduced GM-CSF expression by Tc, NK, and B cells in MS patients, but not in healthy controls. MS patients during relapse had significantly higher frequency of Th-GM (CD3+CD8-IL-17-IFN-γ-IL-3+GM-CSF+) cells than healthy controls. EBV infected B cells expressed GM-CSF receptor in less frequency than non-infected B cells. In vivo GM-CSF neutralization in MS patients resulted in significant reduction in the frequency of CD8+ T cells and CD4+CD45RA+CD25++ (naïve) Tregs and an increase in CD4+CD35+foxp3 (total) Tregs. Conclusions: Th1 (and Th in general), Tc, monocytes, NK and B cells are all high producers of GM-CSF in MS. IL-2 and IL-12 are the main regulators of GM-CSF expression by Tc, NK, and B cells in MS patients. GM-CSF and its receptor may not be major survival or proliferation factors for EBV infected B cells. The newly identified Th-GM cells were detected in higher frequency in MS patients during relapse, which may suggest a new source for GM-CSF production in MS. The recent safety, tolerability, and immune-related results of GM-CSF neutralization in MS are encouraging. Therefore, GM-CSF is a potential therapeutic target in MS.

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Ma, Leyuan. "Targeting Drug Resistance in Chronic Myeloid Leukemia: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/870.

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Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive. First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells. Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model. Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients. Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.

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Ma, Leyuan. "Targeting Drug Resistance in Chronic Myeloid Leukemia: A Dissertation." eScholarship@UMMS, 2011. http://escholarship.umassmed.edu/gsbs_diss/870.

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Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive. First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells. Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model. Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients. Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.

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Bird, Lydia. "A randomised controlled trial of a rehabilitation programme to assist physical and psychosocial recovery after stem cell transplantation." Thesis, University of Nottingham, 2008. http://eprints.nottingham.ac.uk/13857/.

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Background Stem cell transplantation is routinely used in the treatment of haematological malignancy. However, it is an intensive treatment frequently associated with a considerable deterioration in patients' wellbeing and prolonged recovery. Research into the amelioration of the negative biopsychosocial factors associated with stem cell transplantation is essential, facilitating nurses and other health professionals to provide the best possible care to individuals who have been treated with a stem cell transplant. Study Design 58 patients who had been treated with a stem cell transplant were recruited and randomly allocated to either a health profession led rehabilitation programme or a self managed rehabilitation programme. Follow-up measures (SF-36, QHQ, Graham and Longman QoL Scale and SWT) were taken at three and six months. Qualitative interviews were conducted with 15 of the 58 participants and with five members of staff. Results In all dimensions of the SF-36 the scores of patients recovering after stem cell transplantation indicated poorer health status in comparison to UK population norms supporting the need for rehabilitation services for this patient group. No evidence of a difference between the two modes of rehabilitation was observed for any of the trial outcomes. The qualitative interview data indicated that from patients' and staff's perspectives there was scope for improvement in the rehabilitation programmes. The interview data also highlighted that staff were concerned that the trial conditions had negatively impacted the provision of rehabilitation, drawing attention to the difficulties inherent in the evaluation of complex interventions. Conclusions Existing literature, the SF-36 data collected in this study and the experiences of both patients and health professionals expressed in the qualitative component of this study all indicate that rehabilitation is an important component of health care following stem cell transplantation. However, the rehabilitation needs and desires of this patient group are complex and therefore any rehabilitation programme must reflect this complexity. Enabling patients to work collaboratively with health professionals in determining the most appropriate provision of rehabilitation may result in enhanced levels of patient satisfaction with rehabilitation services. However, the efficacy of rehabilitation following stem cell transplantation remains unproven and the provision and evaluation of patient centred rehabilitation raises numerous practical and methodological challenges.

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Bhayat, Fatima. "The epidemiology of leukaemia in the UK." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11726/.

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INTRODUCTION Approximately 9% of all new malignant diagnoses in the UK are due to haematological malignancies. The acute and chronic leukaemias constitute 2.5 % of all cancers and leukaemia is the 12th most common cancer registered in the UK. Approximately 7 000 people are diagnosed with the disease and more than 4 300 people die from leukaemia in the UK each year. As such, they have an important impact on the health of the public and represent a significant cost to the health care budget. AIMS AND OBJECTIVES The research presented in this thesis firstly aimed to quantify the incidence of and mortality from the acute and chronic leukaemias in the UK, and to define their associations with gender, age, socioeconomic class, calendar time, and geographic region of residence. A further aim was to determine whether the use of non-steroidal anti-inflammatory drugs (NSAIDs) had a protective effect on the incidence of and mortality from these leukaemias, as has been shown to be the case for a number of other cancers. Finally, the impact of alcohol consumption on leukaemia incidence and mortality was investigated. A surprising result from the incidence and mortality studies was that survival in AML, but not other leukaemias, was worse with increasing socioeconomic deprivation. This generated an additional hypothesis surrounding potential class bias in bone marrow transplantation in these patients, a new area that was also investigated, in addition to the original aims and objectives of the research. METHODS Both general practice and hospital data were used to conduct these population-based studies. 'The Health Improvement Network' (THIN) general practice dataset was used to conduct the cohort studies of incidence and mortality, as well the case-control studies investigating non-steroidal anti-inflammatory drug use and alcohol consumption, as potential risk factors for leukaemia. Hospital Episode Statistics (HES) data were used to investigate the additional hypothesis generated by results of the incidence and mortality studies, which showed that mortality in AML patients worsens with increasing socioeconomic deprivation. RESULTS A total of 4162 cases of leukaemia were identified, 2314 (56%) of whom were male. The overall incidence of leukaemia is 11.25 per 100 000 person-years and is independent of socioeconomic class. Median survival from leukaemia is 6.58 years and mortality increases with increasing age at diagnosis. The prognosis in AML is dismal and worsens with increasing socioeconomic deprivation, a phenomenon not seen in other leukaemias. Bone marrow transplantation declines with increasing socioeconomic deprivation (p for trend <0.01). Patients with AML in the most deprived socioeconomic quintile are 40% less likely to have a bone marrow transplantation than those in the most advantaged socioeconomic class (OR 0.60, p<0.01, 95% C.I. 0.49 - 0.73), even after adjusting for gender, age at diagnosis, year of bone marrow transplantation and co-morbidity. The risk of leukaemia overall appears to increase marginally with increased use of NSAIDs prior to diagnosis. This is not seen when individual leukaemia subtypes are examined, however, except perhaps in CLL where patients who had received 2-5 prescriptions/year were 29% more likely to be diagnosed with CLL than those who had not had any NSAID prescriptions (O.R. 1.29, p=0.05, 95% C.I. 1.00 - 1.67). There is no statistically significant association between exposure to NSAIDs prior to leukaemia diagnosis, and survival. There is no statistically significant association between alcohol consumptionand risk of developing leukaemia overall, nor with any of the leukaemia subtypes studied here. Alcohol consumption is associated with a lower risk of death in leukaemia overall (HR 0.83, p=0.04, 95% C.I. 0.69 - 0.99), as well as in All (HR 0.14, p<0.01, 95% C.I. 0.04 - 0.44) and CLL (HR 0.71, p=0.02, 95% C.1. 0.53 - 0.96), when compared to those who had not consumed any alcohol.

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Al-Asadi, Mazin Gh. "Investigation of dormancy in acute myeloid leukaemia cells and the induction of dormancy in their response to genotoxic stress." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/43320/.

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Cancer cells can exist in a reversible state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely due to dormant cells escape frontline treatment. Dormant AML cells have been identified in the bone marrow (BM) endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. As the first step in this project, we developed an in vitro model of AML cell dormancy by exploiting these features. Following a preliminary investigation of four AML cell lines, the CD34+CD38- line TF1-a was selected for in-depth investigation. TF1-a showed significant inhibition of proliferation, with features of dormancy and stemness, in response to 72 hours of TGFB1+mTOR inhibitor treatment (mTOR pathway inhibition mimics major effects of nutrient scarcity) without affecting cell viability or inducing differentiation of these cells. Secondly, whole human genome gene expression profiling using Affymetrix microarray strips (HuGene2.1ST) was conducted to molecularly characterise the dormancy-induced TF1-a cells. As a result, we identified 240 genes which were significantly up-regulated by at least twofold, including genes involved in adhesion, stemness, chemoresistance, tumor suppression and genes involved in canonical cell cycle regulation. The most upregulated gene was osteopontin (17.1fold). Immunocytochemistry of BM biopsies from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular BM. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABCC3 and CDKN2B) as well as little-known ones (e.g. ITGB3, BTG2 and PTPRU), are potential therapeutic targets in AML. AML cells which are identified in the bone marrow (BM) endosteal region are likely to survive chemotherapy for several reasons including the low concentration of the drug delivered to this poorly perfused area of the BM. We hypothesised that these cells might induce dormancy features that help them escaping chemotherapy. Moreover, little is known regarding the molecular changes in those AML cells surviving genotoxic stress. The third aim of this study was to investigate the AML cells surviving genotoxic stress. Therefore, we developed and characterised an in vitro model of prolonged sub-lethal genotoxicity in AML cells by utilising the TF1-a cells treated with etoposide for 6 days. TF1-a cells survived this conditioning with significant inhibition of proliferation and limited damage and apoptosis. The molecular signature of these cells was characterized using GEP of the whole human genome and was compared to that of the dormancy-induced TF1-a cells in an aim to identify genes commonly up-regulated in both scenarios that might act as possible drivers of dormancy in AML cells residing in a sub-lethal genotoxic environment. In this context, 31 genes were significantly commonly upregulated in both scenarios including ITGB3, SLFN5, C15orf26 and GRAP2 which are candidates for in-depth investigation in AML. To sum up, in this study we developed and molecularly characterised in vitro models of dormancy and sub-lethal genotoxicity in AML cells with stemness characteristics through novel approaches that took bone marrow microenvironmental features into consideration. These features likely contribute to the resistance of the residual sub-population of AML cells that causing disease relapse. The current models helped to overcome the obstacles facing the in-depth studying of these rare AML cells due to the difficulty in obtaining them from clinical samples. Finally, the molecular findings of this study paved the way to potentially important future directions of research that could help to achieve the ultimate goal of eradicating AML cells and preventing relapse.

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Grundy, Martin. "Activity of the aurora kinase B inhibitor AZD1152 in acute myeloid leukaemia." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12758/.

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Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies including those of leukaemic origin. Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells whose prognosis is particularly poor and where standard induction therapy has changed little over the past thirty years. This thesis evaluated the effects of AZD1152-hQPA (barasertib-hQPA), a highly selective inhibitor of aurora-B kinase, in AML cell lines and primary samples. Inhibition of phospho-Histone H3 (pHH3) on serine 10 can be used as a biomarker for AZD1152-hQPA activity and an assay was optimized to measure pHH3 in our cell lines and primary samples. AZD1152-hQPA inhibited pHH3 in our cell lines resulting in polyploid cells, apoptosis, and cell death, irrespective of cellular p53 status. Over-expression of the ATP-binding cassette (ABC) drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in AML. A cell line which over-expresses Pgp was developed by selecting for daunorubicin (DNR) resistance in OCI-AML3 cells. Pgp and also BCRP expressing AML cell lines were found to be resistant to AZD1152-hQPA and it was found that AZD1152-hQPA is effluxed by these transporters. pHH3 inhibition by low dose AZD1152-hQPA was seen in all of the primary samples tested with Pgp and BCRP positive samples being less sensitive. However, 50% inhibition of pHH3 by AZD1152-hQPA was achieved in 94.6% of these samples. The FLT3-ITD-expressing MOLM-13 and MV4-11 cell lines were particularly sensitive to AZD1152-hQPA. Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor are found in approximately 25% of AML patients and are associated with a poor prognosis. It was demonstrated that AZD1152-hQPA directly targets phosphorylated FLT3 in the FLT3-ITD cell lines along with inhibiting its downstream target pSTAT5. FLT3-ITD primary samples were particularly sensitive to clonogenic inhibition and pSTAT5 down-regulation after treatment with AZD1152-hQPA compared with FLT3 wild-type (WT) samples.

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Baliousis, Michail. "Psychological intervention to alleviate distress in haematopoietic stem cell transplantation : a phase II study." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/32779/.

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Background. Haematopoietic stem cell transplantation (HSCT) is an intensive procedure associated with psychological distress particularly during the first weeks (acute phase). Based on the self-regulatory model of adjustment to illness, a preparatory group intervention was developed aiming at alleviating distress by reducing negative perceptions of HSCT and fostering helpful coping. Aims. The present study aimed to evaluate the feasibility of delivering the intervention and of conducting a trial to assess its efficacy. It also aimed to explore the applicability of the self-regulatory model in HSCT. Methods. Participants were adults from consecutive referrals at two transplant centres. Half were randomised to the intervention and half to treatment as usual at each site. Psychological distress, HSCT perceptions, and coping were assessed at baseline (following consent), on transplant day, two weeks, and four weeks after transplantation. Results. Of 99 eligible patients, 45 consented. Main barriers included inability to consent prior to transplantation, competing priorities, being unwell, and long travel distance. Of 21 participants randomised to intervention, five attended. Main barriers included being unable to attend prior to transplantation and having competing priorities. Groups could not be held sufficiently frequently to enable attendance prior to transplantation, as randomising participants to the control group prevented sufficient accrual at each site. Anxiety peaked two weeks following transplantation but depression increased throughout the acute phase. Intervention effects were small but sample sizes for a full trial appeared feasible. Negative perceptions of HSCT and use of a range of coping styles (including styles considered helpful) predicted higher distress throughout the period. Conclusions. The findings revealed considerable barriers to delivering a group-based intervention and conducting a trial to assess its effectiveness. This highlighted a need for better integration with routine care and alternative trial procedures. However, the findings illustrated complex psychological needs during the acute phase of HSCT and the role of negative HSCT perceptions and unhelpful coping in underpinning distress.

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Yu, N. "Identifying and targeting dormant cells in acute myeloid leukaemia." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33679/.

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Relapse in AML is thought to arise from dormant leukaemic cells that are characterised by low RNA synthesis activity, protected by the bone marrow (BM) niche, and may evade the effects of chemotherapeutic drugs. Our aim was to investigate agents which might be able to overcome chemoprotection by targeting the intrinsic apoptosis pathway. We developed in vitro assays to identify and characterise the dormant AML cells using combinations of markers, including the cell-division marker PKH26, leukaemia-associated phenotypes (LAPs), and dormancy markers. In a dormancy model based on 12-day AML/stroma co-culture, we have shown that the expression of some aberrant phenotypes can persist for several days. Also, after 12 days, some of the CD34+, PKH26high (dormant), and LAP+ (leukaemic) cells maintained their primitiveness and were still clonogenic. Furthermore, our chemosensitivity data showed that novel agents TG02, and BH3 mimetics ABT-737 and ABT-199, which inhibit the B-cell lymphoma 2 (BCL-2) family of anti-apoptotic molecules, could efficiently target BM niche-mediated chemoresistance, which is thought to be one of the main obstacles to traditional chemotherapy. We explored various candidate dormancy markers based on the low RNA, non-proliferative profile of dormant cells. Among those tested, the RNA synthesis marker Pyronin Y (PY), and an antibody to the transferrin receptor CD71 were the most reproducible in terms of marker expression and stability. We endeavoured to characterise cell dormancy on the molecular level by investigating gene expression in the PYlow (dormant) and PYhigh (proliferating) subsets and have obtained limited results. In summary, this study has identified and partly characterised dormant AML cells by development of in vitro assays, and has shown chemosensitivity to novel agents TG02, ABT-737 and ABT-199 in dormant leukaemia cells.

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Afolabi, Bosede. "Plasma volume in normal and sickle cell pregnancy." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12073/.

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Plasma volume (PV) rises by up to 50% in normal pregnancy, a phenomenon associated with a favourable pregnancy outcome. A previous study of pregnant women with sickle cell (haemoglobin SS) disorder found that PV paradoxically contracts in late pregnancy. A cross-sectional study was performed to determine PV (Evans blue method) and volume regulatory hormones and electrolytes in pregnant women with haemoglobin (Hb) SS and in non-pregnant and Hb AA controls. PV rose in pregnant HbAA and was significantly correlated with plasma angiotensinogen. Non-pregnant Hb SS women had supranormal PV measurements and reduced glomerular filtration rate (GFR). Their PV did not rise in pregnancy and was not correlated with angiotensinogen. Their plasma renin concentration also failed to rise significantly by 36 weeks gestation and was significantly less than in Hb AA pregnancy although aldosterone concentration was raised as expected. A general vasoconstriction in pregnancy can cause inactivation of the renin-angiotensin system and could explain this, with aldosterone being elevated by non Angiotensin II dependent stimulation such as plasma potassium, which was significantly higher in the pregnant Hb SS women. Further studies demonstrating a deficiency of vasodilator substances in pregnant Hb SS women will strengthen this hypothesis.

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Zgair, Atheer. "Intestinal lymphatic transport of cannabinoids : implications for people with autoimmune diseases and immunocompromised individuals." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/44494/.

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There has been an escalating interest in the medicinal use of Cannabis sativa in recent years. Cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), the main constituents of Cannabis sativa, have well documented immunomodulatory effects in vitro and following administration of high doses to animals. However, these effects have not been clearly evident in humans following oral administration of cannabinoids, probably due to low systemic bioavailability. To note, cannabis and cannabis-containing medicines are currently used for symptomatic relief in autoimmune diseases, such as multiple sclerosis (MS), and in cases of immunodeficiency, such as in cancer patients on chemotherapy regimens. In this thesis, we aimed to elucidate the impact of enhancing the transport of orally administered cannabinoids to the intestinal lymphatic system, the major host of immune cells, on the immunomodulatory effects of cannabinoids. Oral administration of lipophilic cannabinoids with long-chain triglycerides (LCT) was investigated as a simple approach to enhance the intestinal lymphatic transport. The effect of LCT on the intraluminal processing of orally administered cannabinoids was assessed by means of in vitro lipolysis model. The results of in vitro lipolysis demonstrated that at least one-third of CBD dose would be solubilised and readily available for absorption to the enterocytes when orally administered in LCT-formulation. The association of CBD with chylomicrons (CM) in the enterocytes and subsequent intestinal lymphatic transport was estimated using an in silico model, in vitro association by artificial CM-like lipid particles, and ex vivo uptake by plasma-derived CM from rats and humans. The results of CM association studies revealed high intestinal lymphatic transport potential for CBD in rats and humans. Similar high lymphatic transport potential was also reported for THC in our laboratory. Oral co-administration of CBD and THC with LCT to rats increased the systemic exposure by 3-fold and 2.5-fold, respectively, compared to lipid-free formulations. The underlying mechanism of increased bioavailability is likely to enhanced intestinal lymphatic transport and decreased pre-systemic metabolism in the liver. The results of biodistribution experiments indicated that the intestinal lymphatic transport of CBD and THC was, indeed, enhanced following oral co-administration of lipids as denoted by the dramatic increase in the concentrations recovered in MLN and intestinal lymph. The concentrations of CBD and THC in intestinal lymph fluid were in the range of 120 and 60 µg/mL compared to 0.5 and 0.6 µg/mL in plasma, respectively. Moreover, CBD and THC showed dose-dependent immunosuppressive effect on lymphocytes isolated from rats and peripheral blood mononuclear cells (PBMC) isolated from humans as assessed by lymphocyte proliferation assay and flow cytometry analysis of inflammatory cytokines. These effects were only significant at concentrations achieved in the intestinal lymphatic system, but not in plasma, following oral co-administration of cannabinoids with LCT. CBD showed more immunosuppressive effects on lymphocyte proliferation and the expression of inflammatory cytokines comparing to THC. Also, PBMC from MS patients were more susceptible to the immunomodulatory effects of cannabinoids than PBMC from healthy volunteers and cancer patients on chemotherapy. In conclusion, oral administration of cannabinoids with lipids can enhance the intestinal micellar solubilisation and augment the systemic exposure to cannabinoids by enhancing intestinal lymphatic transport. The concentrations of lipophilic cannabinoids recovered in the intestinal lymphatic system were extremely high and exceeded the immunosuppressive threshold of CBD and THC. The increase in systemic exposure to cannabinoids in humans is of potentially high clinical importance as it could turn a barely effective dose of orally administered cannabis into highly effective one, or indeed a therapeutic dose into a toxic one. In addition, administering cannabinoids, in particular CBD, with a high-fat meal, as cannabis-containing food, or in lipid-based formulations could represent a valid therapeutic approach to improve the treatment of MS, or other T cell-mediated autoimmune disorders. However, in immunocompromised patients, administration of cannabinoids in this way may deepen the immunosuppressive effects.

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Partanen, Taina A. "Lymphatic vs blood vascular endothelial growth factors and receptors in human tissues and diseases." Helsinki : University of Helsinki, 2000. http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/partanen/.

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28

Eipper-Mains, Marcie A. 1979. "The role of the Bcl-2 family in proliferation and apoptosis and in mediating the development of lymphatic diseases." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28867.

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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.
Includes bibliographical references (leaves [54]-[66]).
The development of the immune system is a highly dynamic process, characterized by quickly and frequently changing cell types and numbers. The orchestration of cell growth and proliferation and also of cell death is a necessarily complex process, taking cues from a wide variety of sources. The nematode Caenorhabditus elegans has provided an elegant and simple model of the control of programmed cell death, or apoptosis, in metazoans. Apoptosis in mammals is regulated by pathways related to but more intricate than metazoans. Several key features define the onset of apoptosis in any given cell; these include DNA fragmentation and chromatin condensation, "blebbing" of the plasma membrane, and subsequent phagocytosis of the resulting cell fragments by adjacent cells (Kerr et al. 1972 and Wyllie et al. 1980).
by Marcie A. Eipper-Mains.
S.M.

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29

Villefranc, Jacques A. "Two Distinct Modes of Signaling by Vascular Endothelial Growth Factor C Guide Blood and Lymphatic Vessel Patterning in Zebrafish: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/557.

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Vascular Endothelial Growth Factor Receptor-3 (VEGFR3/Flt4) and its ligand Vegfc are necessary for development of both blood and lymphatic vasculature in vertebrates. In zebrafish, Vegfc/Flt4 signaling is essential for formation of arteries, veins, and lymphatic vessels. Interestingly, Flt4 appears to utilize distinct signaling pathways during the development of each of these vessels. To identify components of this pathway, we performed a transgenic haploid genetic screen in zebrafish that express EGFP under the control of a blood vessel specific promoter. As a result, we indentified a mutant allele of vascular endothelial growth factor c (vegfc), vegfcum18. vegfcum18 mutants display defects in vein and lymphatic vessel development but normal segmental artery (SeA) formation. Characterization of this allele led to the finding that the primary defect in vegfcum18 mutants was a general failure in vein and lymphatic vessel sprouting. Further genetic and biochemical analysis of this mutant revealed profound paracrine defects, which likely result in the observed loss of lymphatic and venous structures. Furthermore, double mutant analysis demonstrated that defects during SeA formation in vegfcum18 mutants were masked by inputs from the Vegfa signaling pathway. Endothelial cell autonomous expression of vegfcum18 induced angiogenic effects on blood vessels while endothelial cells lacking vegfc displayed defects in tip cell occupancy, suggesting a cell autonomous-autocrine role for Vegfc during developmental angiogenesis. Finally, we present genetic evidence that links processing of Vegfc by Furin during the formation of lymphatics in zebrafish. Together the data presented here suggest two discrete modes of signaling during blood and lymphatic vessel development, thus implying that regulation of Vegfc secretion and processing may play a pivotal role in the formation of these distinct vessel types in zebrafish.

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Schmidt, Madelyn R. "Virus-Lymphocyte Interactions: Virus Expression Is Differentially Modulated by B Cell Activation Signals: A Dissertation." eScholarship@UMMS, 1991. https://escholarship.umassmed.edu/gsbs_diss/51.

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It is shown here that the ability of B lymphocytes to act as supportive host cells for virus infections requires they be activated from the resting Gostage of the cell cycle. I have used a series of activation regimens, which allow B cells to progress to different stages in their activation/differentiation pathway toward antibody secretion, in order to evaluate the extent of activation required to support vesicular stomatitis or Newcastle disease virus infections. At least three distinct phases during B cell activation which affected VSV infection were defined. Freshly isolated resting murine splenic B cells in the Go phase of the cell cycle do not support VSV, assessed by protein synthesis, infectious center formation, and PFU production. Small B cells cultured for 48 hours without stimulation still do not support VSV. B cells stimulated with the lymphokines found in Con A activated supernatants from splenic T cells or cloned T cell lines transited into the G1 phase of the cell cycle but remain refractory to VSV. These VSV non-supportive B cell populations do take up virus particles and transcribe viral mRNAs which can be translated in vitro, suggesting a translational block to VSV. B cells stimulated into the S phase of the cell cycle with anti-immunoglobulin synthesize VSV proteins and increased numbers of infectious centers, but only low level PFU synthesis (center) is observed. Co-stimulation with anti-Ig and lymphokines, which supports differentiation to antibody secretion, enhanced PFU synthesis without further increasing the number of infected B cells. LPS, which activates B cells directly to antibody secretion by a pathway different from anti-Ig, induced infectious centers, and PFUs at levels comparable to those seen when stably transformed permissive cell lines are infected. Co-stimulation of LPS activated B cells with the same lymphokine populations that enhance PFU production when anti-Ig is used as a stimulator suppresses PFU production completely, suggesting that anti-Ig and LPS activated B cells are differentially responsive to lymphokines. NDV infection of murine B cells differed markedly from VSV infection, as all B cell populations examined gave a similar response pattern. NDV viral proteins were synthesized by B cells in each of the activation states previously described, even freshly isolated B cells. Infectious center formation increased up to 5-fold over the levels observed with unstimulated B cells after anti-Ig or LPS activation. However, PFU synthesis was low (center) for all B cell populations. These results suggest that these two similar viruses may be dependent on different host cell factors and that these factors are induced for VSV but not NDV by the B cell activators employed here or that the process of infection of B cell by these two viruses induces different cellular responses.

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Lambert, Julie. "Autoimmune Diabetes and Transplantation Tolerance Induced by Costimulation Blockade in NOD Mice: a Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/344.

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NOD mice model human type 1 diabetes and have been used to investigate tolerance induction protocols for islet transplantation in a setting of autoimmunity. Costimulation blockade-based tolerance protocols that induce prolonged skin and permanent islet allograft survival in non-autoimmune mice have failed in NOD mice. To investigate the underlying mechanisms, we generated NOD hematopoietic chimeras. We were able to show that dendritic cell maturation defects seen in NOD mice are partially corrected in mixed hematopoietic chimeras. Furthermore, skin allograft survival was dependent upon the phenotype of the bone marrow donor, demonstrating that in the NOD the resistance to tolerance induction resides in the hematopoietic compartment. In addition, we studied congenic NOD mice bearing insulin dependent diabetes (Idd) loci that reduce diabetes incidence. The incidence of diabetes is reduced in NOD.B6 Idd3 mice, and virtually absent in NOD.B6 Idd3Idd5 mice. Islet allograft survival in NOD.B6 Idd3 mice is prolonged as compared to NOD mice, and in NOD.B6 Idd3Idd5 mice islet allograft survival is similar to that achieved in C57BL/6 mice. Alloreactive CD8 T cell depletion in NOD mice treated with costimulation blockade is impaired, but is partially restored in NOD.B6 Idd3 mice, and completely restored in NOD.B6 Idd3Idd5 mice. Idd3 results from variations in Il2 gene transcription. We hypothesized insufficient levels of IL-2 in NOD mice contributes to impaired deletion of alloreactive CD8 T cells and shortened islet allograft survival. We observed using synchimeric mice that co-administration of exogenous IL-2 to NOD mice treated with costimulation blockade led to deletion of alloreactive CD8 T cells comparable to that in C57BL/6 mice and prolonged islet allograft survival. However, some Idd loci impaired the induction of transplantation tolerance. These data suggest that Idd loci can facilitate or impair induction of transplantation tolerance by costimulation blockade, and that Idd3 (IL-2) is critical component in this process.

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Marshall, Heather D. "Sensitization of CD8 T Cells During Acute Viral Infections Impacts Bystander and Latecomer CD8 T Cell Responses : A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/440.

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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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Todd, Derrick James. "Role of the Intestinal Immune System in the Pathogenesis of Autoimmune Diabetes in the BB Rat Model of Type 1 Diabetes Mellitus." eScholarship@UMMS, 2001. https://escholarship.umassmed.edu/gsbs_diss/138.

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The intestine is the largest lymphoid organ in the body, challenged constantly by an enonnous quantity and diversity of antigens. Distinct from peripheral lymphocytes, intestinal lymphocytes have evolved unique mechanisms of tolerance and appear to govern mucosal processes such as "chronic physiologic inflammation" and oral tolerance. Failure of mucosal tolerance has been implicated in the pathogenesis of several diseases, including inflammatory bowel disease, celiac disease, and even autoimmune diabetes. One population of intestinal lymphocytes, intraepithelial lymphocytes (IELs), exists within the intestinal epithelium itself and remains poorly characterized. IELs respond to unique activation signals and appear to be in part responsible for the maintenance of epithelial integrity and mucosal tolerance. Type 1 diabetes is one of the most common chronic childhood illnesses and causes significant morbidity and mortality. Type 1 diabetes mellitus is an autoimmune disease that results from immune-mediated destruction of insulin-producing pancreatic beta cells and is characterized by an absolute insulin deficiency. Several animal models are used to study the immunopathogenesis of type 1 diabetes, including the BB rat and NOD mouse. BBDP rats spontaneously develop autoimmune diabetes mellitus and are severely deficient in peripheral T cells. BBDR rats do not spontaneously develop autoimmune diabetes, have nonnal numbers of peripheral T cells, and can be induced to become diabetic by injections of a cytotoxic anti-ART2a mAb and low doses of poly I:C. The cause of autoimmune diabetes in BB rats and humans is still unknown, but both genetic and environmental factors appear to participate. I hypothesize that one important class of environmental factors--diet and enteromicrobial agents--participates in this pathogenic process through the mediation of the gut immune system. In this dissertation, I report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The yield of rat IELs using this method is 5-10 fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrate that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis reveals 5 major subsets of IELs, including populations of γδ T and natural killer (NK) cells present at levels not previously detected. I also report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, IENK cells differ from splenic NK cells phenotypically, and a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. I conclude that rat IELs harbor a large population of NKR-P1A+ CD3-cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive intraepithelial NK cells may participate in the regulation of mucosal immunity. I next demonstrate that, prior to diabetes, both BBDP and ART2a-depleted BBDR rats have a reduced total number of IELs and exhibit a selective deficiency of IENK cell number and function as compared to control BBDR rats. The deficiency of BBDP rat IELs can be corrected by engraftment of bone marrow from histocompatible WF donors. These results suggest 1) that the peripheral lymphopenia in BBDP rats extends to the IEL compartment, particularly to IENK cells, 2) that in BBDR rats the diabetes-inducing treatment depletes IELs, particularly IENK cells, and 3) that the defect in BBDP rat IELs is intrinsic to hematopoietic cells, not intestinal stromal cells. I also establish that, unlike BBDR and WF rats, BBDP rats are also deficient in γδTCR+IELs, a population of T cells that may play a role in normal mucosal tolerance. In addition, I report preliminary data supporting the hypothesis that systemic autoreactivity may be initiated in the intestine; peripheral autoreactive lymphocyte populations appear to emanate first from mesenteric lymph nodes that drain the intestine, and such cells may initiate a type 2 autoimmune phenomenon driven by IL-4. Collectively, my findings support the hypothesis that a failure of mucosal tolerance in BBDP rats, perhaps secondary to deficiencies in one or more IEL subpopulations, participates in the pathogenesis of autoimmune diabetes in these animals by activating peripheral autoreactive T cells. The nature of the autoimmune response in BB rats (driven by IL-4) appears to be distinct from that of NOD mice. Despite the differences between these two well-accepted animal models of autoimmune diabetes, until more is known about the pathogenesis of type 1 DM in humans, lessons learned from both the BB rat and NOD mouse continue to be of tremendous benefit to our understanding of human disease.

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McIntyre, Kim W. "Cytotoxic Lymphocytes in Viral Hepatitis: a Thesis." eScholarship@UMMS, 1987. https://escholarship.umassmed.edu/gsbs_diss/56.

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The immunological mechanisms involved in virus-induced hepatitis were examined by measuring the cytotoxic capabilities and the morphological and antigenic phenotypes of leukocytes isolated from the livers of virus-infected mice. Large granular lymphocytes (LGL) of both natural killer (NK) cell and cytotoxic T lymphocyte (CTL) phenoytpes [phenotypes] accumulated in livers of mice infected with lymphocytic choriomeningitis virus (LCMV) of either the nonhepatotropic Armstrong strain (LCMV-ARM) or the hepatotropic WE strain (LCMV-WE). NK cell activity and LGL number increased 3- to 4-fold between days 1 and 5 postinfection (p.i.). These LGL were characterized as NK cells on the basis of cell surface antigens, kinetics of appearance, target cell range, and morphology. By day 7 p.i., virus-specific, H-2-restricted, Thy-1+, Lyt-2+CTL activity was present in the liver, and its appearance correlated with a second wave of LGL accumulation. Total CTL activity, leukocyte numbers, and CTL/LGL numbers were at least 5-fold higher in the livers of LCMV-WE-infected mice than in the livers of LCMV-ARM-infected mice. Mice infected with the cytopathic viruses, mouse hepatitis virus and murine cytomegalovirus, experienced greater increases in NK/LGL by day 3 p.i. than did mice either infected with LCMV or injected with poly I:C. The early and late accumulations of LGL in the virus-infected liver were associated with the appearance of two waves of LGL with blast cell morphology expressing the phenotypes of NK cells and CTL, respectively. Thus, the organ-associated accumulation, blastogenesis, and in situ proliferation of cytotoxic LGL provide a means for the localization and site-specific augmentation of a host's cell-mediated antiviral defenses. The mechanism of inhibition of virus synthesis in vivo by immune splenocytes containing virus-specific CTL was examined in mice dually infected with two different viruses and then adoptively immunized with spleen cells immune to one of the two viruses. Only the titer of the virus to which the splenocytes were immune was reduced in titer, and no nonspecific antiviral effect was seen on the titer of the 'bystander' heterologous virus. These data are consistent with an in vivo mechanism of CTL-mediated antiviral resistance involving direct cytotoxicity rather than release and dissemination of antigen-nonspecific antiviral factors, such as interferon, following recognition of appropriate viral antigen.

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Pozzi, Lu-Ann M. "Macrophages Directly Prime Naïve CD8+ T Cells: a Dissertation." eScholarship@UMMS, 2004. https://escholarship.umassmed.edu/gsbs_diss/117.

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Professional antigen presenting cells (APCs) represent an important link between the innate and adaptive immune system. Macrophages (MΦs) and dendritic cells (DCs) serve as sentinels in the periphery collecting samples from their environment and processing this information. These cells then present antigenic fragments to T cells in the context of self-MHC molecules. Although a clear role for both of these APCs in the stimulation of already activated or memory T cells has been established, the ability of MΦs to activate naive T cells is still unknown. In this thesis the ability of bone marrow-derived MΦs and DCs to prime naive CD8+ and CD4+ T cells was investigated. Using adoptively transferred transgenic CFSE-Iabeled P-14 T cells, specific for gp33 from lymphocytic choriomeningitis virus in the context of Db, we were able to demonstrate the ability of both MΦs and DCs to induce naive CD8+ T cells proliferation. Once primed by MΦs these T cells gained effector function as shown by interferon- γ (IFN-γ) production and in vivo cytolysis. In addition, immunization of wild type animals with gp33-pulsed MΦs, as well as DCs, led to greater than a 95% reduction in lymphocytic choriomeningitis virus titers. To rule out the role of cross-presentation in the observed priming, two models were used. In the first model, lethally irradiated F1 bxs chimeras reconstituted with either H-2s or H-2b bone marrow were used as host for the adoptive transfer experiments. Since the gp33 peptide binds to Db, the H-2s reconstituted animals should be unable to cross-present the peptide to the P-14 T cells. Using this model, we were able to clearly demonstrate the ability of MΦs to activate naive P-14 T cells to undergo division. Additional experiments, demonstrated that these MΦ primed T cells went on to develop into effector cells. Finally, the ability of the MΦ primed T cells to develop into functional memory cells was demonstrated. To confirm the chimera results, these experiments were repeated using β2 microglobulin deficient animals (whose cells don't express MHC I) as host in adoptive experiments. MΦs were able to stimulate the naive P-14 T cells to divide and gain effector function as demonstrated by the ability to produce IFN-γ. In contrast to the CD8 system, MΦ were poor stimulators of D011.10 CD4+ T cell proliferation. Additionally, D011.10 T cells stimulated by DCs were able to produce interleukin-2 (IL-2), IL-4, tumor necrosis factor and granulocyte-macrophage colony stimulating factor where as MΦ stimulated D011.10 T cells were only able to produce IL-2. In conclusion this body of work clearly demonstrates the in vivo ability of MΦ to stimulate CD8+ T cell proliferation, effector function, as well as the formation of functional CD8+ T cell memory. Whether or not the nature of the memory pools stimulated by the two APCs is exactly the same is still unknown and needs further investigation. The ability of APCs other than DCs to stimulate functional protective memory needs to be considered in the quest to design vaccines that offer broad-spectrum protection.

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Morrison, Alan R. "Poly(ADP)-Ribose Polymerase Activity in the Eukaryotic Mono-ADP-Ribosyl Transferase, ART2: a Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/126.

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The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface.

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Mathew, Anuja. "Human T Cell Responses to Dengue Virus Infections: CD8+CTL and Acute Immunosuppression: a Dissertation." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/18.

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There are four serotypes of dengue virus designated dengue 1, 2, 3 and 4 (D1, D2, D3 and D4) and epidemiological studies indicate that a severe complication of dengue virus infection - dengue hemorrhagic fever (DHF) is more likely to occur following a secondary infection. DHF is hypothesized to be immunologically mediated and may be triggered by virus-specific T cells. It is also likely that dengue virus-specific cytotoxic T lymphocytes (CTLs) are important for recovery from dengue virus infections. An analysis of the immune response during acute illness and when the patient has recovered from the infection (immune state) is therefore important as it will provide insights into the immunopathological nature of the disease. This thesis initially examines the CD8+CTL responses in volunteers who have received live attenuated dengue vaccines and then investigates acute and immune T cell responses in children following natural infection with dengue. When this project was initiated, there was little available information on the human CD8+ T cell responses to dengue viruses. PBMC from one donor had generated memory CD8+CTL to the nonstructural protein NS3 of dengue virus. Memory CD8+CTL responses were therefore analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult American volunteers who had received monovalent live-attenuated candidate vaccines of the 4 dengue serotypes. All the donors had specific T cell proliferation to dengue viruses and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural proteins NS3 and NS1.2a and the structural protein E were recognized by CD8+CTLs from six, five and three donors respectively. All donors recognized either NS3 or NS 1.2a. In a donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using PBMC of two donors were serotype-specific whereas all other donors had serotype-cross reactive responses. For one donor, CTLs specific for E, NSl.2a and NS3 proteins were all HLA-B44 restricted. For the three other donors tested the potential restricting alleles for recognition of NS3 were HLA-B38, A24 and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with a single serotype of dengue virus are diverse and directed against a variety of proteins. The nonstructural proteins NS3 and NSl.2a appear to be immunodominant and should be considered when designing subunit vaccines for dengue. Previously T cell responses had not been examined in people who have had natural infections with dengue. The HLA diversity between North American Caucasians and populations where dengue is a serious health problem, calls for the analysis of immune responses in people who have been infected with natural circulating strains of the virus. We examined the memory cytotoxic T lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue infections. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for the NSl.2a and NS3 proteins respectively. All CTL lines derived from both patients were crossreactive with other serotypes of dengue virus. The CD8+ NS1.2a specific lines from patient KPP94-037 were HLA-B57 restricted and the CD8+ NS3 specific lines from patient KPP94-024 were HLA-B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA-DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize a.a. 221-232 on NS3. These results demonstrate that after symptomatic secondary natural dengue infections in Thai patients, CTLs are mainly directed against nonstructural proteins and are broadly crossreactive. The data correlate with our observations that nonstructural proteins are immunodominant proteins in volunteers who received dengue vaccines. We were interested in examining CTL responses in children during their acute illness and comparing them to memory CTLs obtained from the same children a year or more after the infection. A detailed analysis on samples from nine patients during their acute illness failed to generate any dengue virus-specific CTL responses. We therefore decided to determine if cell mediated responses are altered during acute dengue infection. Decreased proliferative responses to mitogens and recall antigens have been observed in PBMC obtained during several acute human viral infections. All responses of PBMC during acute illness were compared to the same patients PBMC obtained at least 6 months after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid and dengue antigens were significantly decreased in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous immune or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 antibodies restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12 also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation. The data generated from this project shed light on the nature of the immune responses during acute natural dengue infections. It strengthens the existing data on the human memory CD8+CTL responses to dengue viruses and validates the observations by examining memory CTL responses after natural dengue infection in patients from Thailand. In addition, we demonstrate a profound defect in lymphoproliferative responses during dengue illness.

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Bahl, Kapil. "Attrition of CD8 T Cells during the Early Stages of Viral Infections: a Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/351.

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Profound lymphopenia has been observed during many acute viral infections, and our laboratory has previously documented a type 1 IFN-dependent loss of most memory (CD44hi) and some naïve (CD44lo) CD8 T cells immediately preceding the development of the antiviral T cell response at days 2-4 following lymphocytic choriomeningitis virus (LCMV) infection. In this thesis, I will examine additional mechanisms involved in the early attrition of CD8 T cells and evaluate whether antigen-specific and non-specific CD8 T cells are equally susceptible. Lastly, I will examine whether the early attrition of CD8 T cells contributes to the generation of an effective immune response. Poly(I:C), a potent inducer of type 1 IFN, was previously shown to cause the attrition and apoptosis of CD8α+CD44hi cells in normal mice, but not in type 1 IFN receptor–deficient mice (IFN1-R KO). I questioned whether additional molecule(s) might contribute to the type 1 IFN-induced apoptosis of CD8α+CD44hi cells. I used a PCR array to determine the expression of 84 apoptosis-related genes at 6 hours post-poly(I:C) treatment, relative to an untreated control. There was an 11-fold increase in CD40 RNA expression in CD8α+CD44hi cells isolated from poly(I:C)-treated mice. CD40 protein expression was also increased on CD8α+CD44hi cells, peaking between 9 and 12 hours following poly(I:C) treatment, before declining thereafter. This increase in CD40 protein expression directly correlated with an increase in Annexin V reactivity, an indicator of early apoptosis. Nevertheless, CD40 was not required for the loss of CD8α+CD44hi cells, as both wildtype and CD40-deficient mice were equally susceptible to the poly(I:C)-induced attrition. Upon further characterization, I found this population of CD40+CD8α+CD44hi cells to be CD11c+B220-Thy1.2- MHCIIhi, which is consistent with a “lymphoid” CD8α+ DC phenotype. Kinetic analysis revealed a type 1 IFN-dependent increase in this CD8α+ DC population at 12 hours post-poly(I:C) treatment. This increase was only observed in the spleen, as no increase in percentage was observed in the peritoneal cavity (PEC), lungs, inguinal lymph nodes (iLN), or peripheral blood. Collectively, these results suggest that the type 1 IFN-dependent increase in splenic CD8α+DCs accounts for the observed increase in Annexin V reactive cells following poly(I:C) treatment. These findings required a re-evaluation of the type 1 IFN-induced attrition of CD8+CD44hi T cells with an anti-CD8β antibody, which is a more exclusive marker for T cells than the anti-CD8α antibody. Kinetic analysis revealed a significant decrease in splenic CD8β+CD44hi T cells at 12 hours post-poly(I:C) treatment. This reduction in splenic CD8β+CD44hi T cells was not due to trafficking to other organs, as the PECs, lungs, iLN, lungs, and peripheral blood all exhibited significant, although varying, decreases in the percentage of CD8β+CD44hi T cells at 12 hour following poly(I:C) treatment. These data support the notion that the type 1 IFN-induced attrition of CD8β+CD44hiT cells was a “global” phenomenon and could not be completely due to migration out of the spleen. The attrition of CD8β+CD44hi T cells was also dependent upon type 1 IFN at 3 days post-LCMV infection, as there was no significant reduction of this population in IFN1-R KO mice. The loss of wildtype CD8β+CD44hi T cells correlated with an increased activation of caspases 3 and 8, which are enzymes that play essential roles in apoptosis and inflammation. A significant loss of CD4+CD44hi T cells, which also correlated with an increased activation of caspases 3 and 8, was observed at 3 days post-LCMV infection. Collectively, these results suggest that attrition of both CD4+CD44hi and CD8β+CD44hiT cell populations is type 1 IFN-dependent and associated with the activation of caspases following LCMV infection. At 3 days post-LCMV infection, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had a higher frequency of cells with fragmented DNA, a hallmark characteristic of the late stages of apoptosis, as revealed by terminal transferase dUTP nick end labeling (TUNEL), relative to uninfected controls. This suggests that the loss of both populations was due to apoptosis. Therefore, I questioned whether the LCMV-induced apoptosis of both CD4+CD44hi and CD8β+CD44hi T cell populations occurred through a mitochondrial-induced pathway involving the pro-apoptotic molecule Bim. The attrition of both CD4+CD44hi and CD8β+CD44hi T cells was significantly higher in wildtype mice compared to Bim KO mice at 3 days post-LCMV infection. Moreover, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had higher frequency of TUNEL+ cells, relative to Bim KO populations. These results suggest that the apoptosis of CD8β+CD44hi and CD4+CD44hiT cells, following LCMV infection, might occur through a mitochondrial-induced pathway involving Bim. Studies have shown “lymphoid” CD8α+ DCs to be involved in the phagocytosis of apoptotic lymphocytes. Therefore, I evaluated whether host CD8α+ DCs are capable of phagocytosing apoptotic lymphocytes by adoptively transferring CFSE-labeled wildtype donor splenocytes (Ly5.1) into congenic wildtype hosts (Ly5.2), followed by inoculation with poly(I:C). There was an increased frequency of donor cells (Ly5.1, CFSE+) within the host CD8α+CD11c+ gate at 9 and 12 hours post-poly(I:C) treatment. The results suggest that type 1 IFN-activated CD8α+DCs might aid in the rapid clearance of apoptotic cells during the type 1 IFN-induced attrition associated with viral infections. I next questioned whether TCR engagement by antigen would render CD8 T cells resistant to attrition. I tested whether a high concentration of antigen (GP33 peptide) would protect LCMV-specific naïve TCR transgenic P14 cells specific for the GP33 epitope of LCMV and GP33-specific LCMV-immune cells from depletion. Both naïve P14 and memory GP33-specific donor CD8 T cells decreased substantially 16 hours after inoculation poly(I:C), regardless of whether a high concentration of GP33 peptide was administered to host mice beforehand. The increased activation status of naïve antigen-specific cells via peptide inoculation did not confer resistance to type 1 IFN-induced depletion. Donor naïve P14 and LCMV-specific memory cells were also depleted from day 2 LCMV-infected (Clone 13) hosts by 16 hours post-transfer. These results indicate that antigen engagement does not protect CD8 T cells from the type 1 IFN-induced attrition associated with viral infections. Computer models indicated that early depletion of memory T cells may allow for the generation for a more diverse T cell response to infection by reducing the immunodomination caused by cross-reactive T cells. To test this in a biological system, I questioned whether the reduced apoptosis of the crossreactive memory CD8 population (NP205), in aged LCMV-immune mice (18-22 months), following heterologous virus challenge (PV), would allow it to dominate the immune response. At day 8 post-PV infection, the cross-reactive memory CD8 T cell response (NP205) was more immunodominating in aged LCMV-immune mice relative to younger LCMV-immune mice. This was indicated by the increased ratio of the cross-reactive NP205 response to the newly arising noncross-reactive, PV-specific NP38 response in older LCMV-mice relative to younger LCMV immune-mice, at day 8 post-PV infection. These data suggest that the early attrition of T cells allows for the generation of a more diverse T cell response to infection by reducing the immunodomination caused by crossreactive T cells. Collectively, these findings offer further insight into the early attrition of T cells associated with viral infections.

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Pikora, Cheryl A. "Type-Specific Immunity in HIV-1 Vertically Infected Infants." eScholarship@UMMS, 1995. https://escholarship.umassmed.edu/gsbs_diss/83.

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High frequencies of CTL recognizing laboratory strains of HIV-1 are present in HIV-1 infected adults as early as preseroconversion. The presence of HIV-1 specific CTL during primary infection has been correlated with better control of early viremia and a more delayed onset of CD4 lymphocyte loss. Previous experiments in our laboratory have demonstrated that, unlike HIV-1 infected adults, the majority of vertically infected infants lack CTL which recognize laboratory strains of HIV-1 within the first year of life. ADCC antibody responses against laboratory strains of HIV-1 env gene products are also delayed until at least two years of age. As a possible correlate, disease progression is also more rapid in vertically infected infants. We hypothesized that HIV-1-specific CTL are type-specific in early infancy and that the use of target cells expressing laboratory strain gene products might limit the detection of HIV-1-specific CTL. To address this hypothesis, HIV-1 env genes from early isolates of four infants were PCR amplified, cloned, and used to generate recombinant vaccinia vectors (vv). The frequencies of CTL precursors (CTLp) recognizing env gene products from autologous isolates and the IIIB strain of HIV-1 were measured at time points from early infancy to 19 months using limiting dilution analysis (LDA). ADCC titers were also measured against autologous and IIIB env gene products at 4 time points spanning 2 months to 2 years of age. CTL precursors from 3 of 4 of these patients were specific only for autologous HIV-1 env gene products during the first 6 to 12 months of age. A pattern of CTL responsiveness was observed in these 3 patients in which type-specific CTL precursors observed in early infancy were replaced by cross-reactive, group-specific CTL by 6 to 12 months of age. CTL precursors from a fourth patient at 12 months of age recognized IIIB env and 1 out of 2 envs derived from 2 autologous viral isolates. High titers titers of ADCC antibodies against autologous env were detected in two infants prior to the detection of ADCC antibodies to IIIB. In two other infants, group specific ADCC antibody responses were detected in late infancy. Our results demonstrate that young infants can mount HIV-1 specific CTL and ADCC responses. The ability of young infants to mount cellular immune responses to HIV-1 also provides support for the concept of perinatal vaccination to prevent HIV-1 transmission. Furthermore. the lack of broadly-reactive CTL in early infancy suggests that the use of vaccines based on laboratory strains of HIV-1 may not afford protection from vertical infection.

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Van, Epps Heather Lin. "Long-Lived Memory T Lymphocyte Responses Following Hantavirus Infection: a Dissertation." eScholarship@UMMS, 2001. https://escholarship.umassmed.edu/gsbs_diss/112.

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Hantaviruses are members of the virus family Bunyaviridaethat cause two potentially life-threatening diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (BPS). HFRS is caused by Old World hantaviruses that are endemic in many Asian and European countries. Infections with Old World hantaviruses can range in severity from asymptomatic to moderate or severe, depending primarily on the infecting serotype of virus. HPS is caused by New World hantaviruses in North and South America. New World hantaviruses are rarely asymptomatic and are severe in the majority of cases. These syndromes are distinct from one another in the primary target organ of virus infection (kidney vs. lung), but have important clinical features in common, including fever, thrombocytopenia, and a capillary leak syndrome. These common clinical manifestations suggest that the underlying mechanisms of disease may be similar in the two syndromes. The precise mechanisms of pathogenesis of HFRS and HPS are poorly characterized, but may be mediated in part by immunopathology. Hantaviruses are able to establish infections in many human cell types, including primary human endothelial cells, without having any cytopathic effect on these cells. Human infections with hantavirus result in a robust activation of the humoral and cellular immune response, and we hypothesize that these immune responses contribute to the pathology of disease. Evidence for the activation of T lymphocytes, and their potential involvement in immunopathology, includes increases in the number of circulating, activated CD8+ T cells during HFRS, the presence of lymphocytic infiltrates (predominantly CD8+T cells) in kidney biopsies from patients with acute HFRS, and associations between certain HLA haplotype and disease severity following hantavirus infection. This thesis is the first examination of human T lymphocyte responses that are generated during HFRS. Initially, we studied memory T cell responses in scientists who were sub-clinically infected with Hantaan virus (HTNV), the prototype hantavirus. We later investigated memory T cell responses in healthy Finnish adults who had HFRS caused by Puumala virus (PUUV), a hantavirus endemic primarily in Scandinavia. At the onset of these studies, there was no available information on human T lymphocyte responses to Old World hantaviruses. Virus-specific CD8+ and CD4+human T cell lines had been isolated from patients with acute HPS caused by Sin Nombre virus (SNV) infection. In that study, conducted in our laboratory, several human T cell epitopes on the nucleocapsid (N) protein and G2 envelope glycoprotein of SNV were identified and characterized. We decided to perform similar analyses on PBMC from donors who had been infected with HTNV and PUUV, in order to determine the specificity and diversity of the T cell response to Old World hantaviruses. The initial study of three donors who had sub-clinical infections with HTNV demonstrated that virus-specific T cell responses could be detected in all the donors following in vitro stimulation of PBMC with inactivated virus. In two of the donors, the virus-specific cytolytic T cells (CTL) recognized the HTNV N protein, and in the third donor the virus-specific CTLs recognized the HTNV G1 glycoprotein. Isolation and characterization of virus-specific T cells from two donors resulted in the identification of two CD8+ T cell epitopes on the HTNV N protein, which were restricted by either HLA A1 or B51. These CTL lines included both HTNV-specific (HLA B51-restricted) and serotype-cross reactive (HLA A1 restricted) lines. In one subject, these virus-specific T cell responses were detectable in IFN-γ ELISPOT assays following peptide stimulation, and in bulk cultures after short-term stimulation with inactivated HTNV. These results indicated that the CD8+CTL responses of humans after sub-clinical infection with HTNV were readily detectable and were directed against a limited number of viral proteins and epitopes. In addition, sub-clinical infection resulted in the generation of both virus-specific and cross-reactive CTL responses. We reasoned that hantavirus infections that lead to clinical illness may result in the generation of more robust and/or diverse virus-specific T cell responses than in sub-clinical infections. To address this question, we studied the memory CD8+ T cell responses in a group of healthy adults from Finland who had HFRS caused by PUUV infection between the years 1984 and 1995. We detected virus-specific CTL in the bulk cultures of seven of eleven immune individuals tested following stimulation with infectious virus. The PUUV proteins N, G1 and G2 were recognized by CTLs in six, five, and two donors respectively. Extensive cloning of T cells from two donors resulted in the isolation of sixty-three virus-specific CTL lines, the majority of which (61/63) were specific for the PUUV N protein. Six novel CD8+ CTL epitopes and one CD4+ CTL epitope were identified on the N protein, all of which clustered in the center of the protein between amino acids 173 and 251. The CTL lines specific for these epitopes were restricted by a variety of HLA alleles including A2, A28, B7 and B8, and were primarily serotype specific when tested against target cells expressing HTNV or SNV N protein. IFN-γ ELISPOT analysis using the defined epitopes to stimulated PBMC, revealed high frequencies of circulating N-specific CD8+ T cells in eight of thirteen individuals tested. Finally, T cell receptor (TCR) Vβ analysis of CTL clones specific for one epitope (N204-12) demonstrated that cells in this population expressed up to five different Vβ chains. These results demonstrated that the PUUV N protein may be the dominant target of the CTL response, that the N-specific CD8+ CTL responses are diverse, heterogeneous, and primarily serotype specific, and that virus-specific memory CD8+T cells can persist at high levels for up to 15 years after the primary infection. In order to understand the pathology of HFRS and HPS, we must be able to assess the contribution of various factors that could potentially contribute to disease. The virus burden in the infected individual is likely to be an important factor in the severity of the resulting disease. Quantitative RT-PCR analysis of plasma samples from acute HPS patients demonstrated that a higher virus burden (as reflected by viral RNA copy number) is associated with more severe HPS. In order to perform similar analyses in patients with HFRS caused by PUUV, we established a quantitative RT-PCR assay for the detection of PUUV S segment RNA in patient plasma. The design and optimization of the PUUV-specific RT-PCR is described in this report. This assay will allow us to measure the virus burden in patients and compare these data with levels of T cell activation and with parameters of disease severity. In this way, we hope to gain an understanding of the kinetics and magnitude of both the virus burden and virus-specific T cell response during the acute illness. This thesis provides the first description of human virus-specific T cell responses to HTNV and PUUV. These data shed light on the nature of the CD8+ T cell responses that are generated following natural infections with PUUV and sub-clinical infections with HTNV. The studies of memory CD8+ T cell responses to PUUV, and the development of a PUUV-specific quantitative RT-PCR assay, establish the framework for future studies of the immunopathology of acute HFRS. Quantitative analysis of both virus burden and T cell responses during acute illness will provide insight into their relative contributions to the pathology of disease.

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Castro, Cláudia Irene Emilio de. "Alterações cariotípicas dos cromossomos 7 e 8 em Síndromes Mielodisplásicas de novo (SMD-p) e sua correlação com variáveis biológicas de significado prognóstico." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-24042015-150822/.

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As Síndromes Mielodisplásicas (SMD) são doenças clonais, que se originam em células hemopoéticas totipotentes, com distúrbio na proliferação e perda da maturação celular gerando hipercelularidade na medula óssea (MO) e citopenias no sangue periférico (SP). As SMD ocorrem predominantemente em pacientes mais vellhos e estão associadas com um alto risco de progressão para leucemia mielóide aguda (LMA). A etiologia das SMD é deconhecida na maioria dos casos (de novo), porém 10% das SMD estão associadas ao uso de agentes xenobioticos, como os agentes antineoplásicos e solventes orgânicos (SMD secundária). Os pacientes com SMD apresentam diferente evolução clínica, embora todos sejam incuráveis. Alguns pacientes podem viver por uma década com apenas uma anemia enquanto outros apresentam uma sobrevida curta devido à uma rápida evolução para LMA. Diferentes aspectos biológicos da doença têm sido usados para dividir os pacientes em grupos e auxiliado nas decisões clínicas a serem tomadas em cada caso. A maioria dessas classificações levam em conta os subtipos morfológicos (classificação FAB) e os dados citogenéticos do clone mielodisplásico. Anormalidades cromossômicas são importantes marcadores das SMD. A maioria dos pacientes com esta síndrome apresenta aberrações citogenéticas que podem auxiliar no diagnóstico das SMD. Entretanto, algumas dessas aberrações podem ser correlacionadas com um mau prognóstico, como a monossomia do 7 e a trissomia do 8. Essas aneuploidias têm sido investigadas por estudos citogenéticos convencionais (bandamento G) e moleculares (\"FISH\" - Hybridization in situ Fluorescente). No intuito de se comparar a eficiência de ambas as técnicas e de se definir a freqüência e o significado prognóstico da monossomia do 7 (-7) e trissomia do 8 (+8) em SMD, foram estudados 28 pacientes com SMD-p (17 homens e 11 mulheres) por citogenética convencional (banda G) e molecular (\"FISH\" em intérfase), previamente a tratamento quimioterápico. Para compararmos o estudo cariotípico por bandamento G com o de \"FISH\", 19 pacientes foram estudados e a -7 foi detectada por \"FISH\" em seis pacientes e somente um deles o foi pela citogenética convencional. Os pacientes com -7 apresentaram uma menor sobrevida e um maior risco de transformação leucêmica. Cinco dos seis pacientes com -7 foram previamente classificados (FAB) como subtipos de mau prognóstico. Dois pacientes apresentaram +8, detectada por ambas as técnicas. Não houve diferença na sobrevida destes pacientes. Nossos resultados demonstraram que o \"FISH\" é uma técnica mais eficiente para detectar anomalias cromossômicas numéricas (-7) com importância prognóstica, especialmente em amostras pobres em metáfases, uma vez que \"FISH\" pode ser realizado em núcleos interfásicos.
Myelodisplastic Syndromes (MDS) are clonal disordes of hematopoietic stem cells characterized by ineffective hematopoiesis leading to blood cytopenias. They predominate in elderly patients and are associated with a high risk of progression to acute myelogenous leukemia (AML). The etiology of MOS is unknown in most cases (de novo) but about 10% of MDS are secondary to the use of xenobiotics, such as antineoplastic agents and organic solvents (secondary MDS). Though incurable, MDS patients have different clinical courses. Some patients can live for a decade with anemia being the only complaint whilst others present a very short term survival due to a rapid development of a treatment-refractory AML. Scales taking in account different biological aspects of the disease have been used in order to allocate patients in discrete risk levels. This risk stratification might help clinical decisions in a patient-by-patient basis. Most of these scales take in account the morphological subtype (FAB classification) and the cytogenetic status of the myelodysplastic clone. Chromosome abnormalities are very important markers of MDS. Most patients with this syndrome present cytogenetic aberrations which can be helpful in the diagnosis of SMD. Moreover, some of these aberrations have been correlated to a poorer prognosis, such as monosomy 7 and trissomy 8. These aneuploidies have been investigated by conventional (G-Banding) and molecular cytogenetic studies (In Situ Hybridization - FISH). In order to compare the efficiency of both techniques and to define the frequency and significance of monosomy 7 and trisomy 8 in MDS in a single institution population, chromosome studies of 28 patients were performed in metaphases by G-banding and in interphases by FISH (α satellite probes), before chemotherapy treatment. Chromosome studies by both techniques were obtained for 19 patients; monosomy 7 was detected by FISH in 6 patients; only one of these has been detected by conventional cytogenetics. Clinical follow-up of this monosomy 7 group patients disclosed short survival and increased risk for developing AML. Five out of the six patients with monosomy 7 were previously classified as the FAB subtypes of poor prognostic. 2 patients presented trisomy 8 in this series and were detected by both techniques. No difference in survival could be seen in these patients. Our results demonstrate that FISH is much more efficient to investigate chromosome numerical anomaly (at least monossomy 7) with prognostic relevance. This is specially true mainly when bone marrow samples lack metaphases since the studies can be performed in interphases cells.

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Sirois, Cherilyn M. "Nucleic Acid Sensing by the Immune System: Roles For the Receptor For Advanced Glycation End Products (RAGE) and Intracellular Receptor Proteins: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/551.

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As humans, we inhabit an environment shared with many microorganisms, some of which are harmless or beneficial, and others which represent a threat to our health. A complex network of organs, cells and their protein products form our bodies’ immune system, tasked with detecting these potentially harmful agents and eliminating them. This same system also serves to detect changes in the healthy balance of normal functions in the body, and for repairing tissue damage caused by injury. Immune recognition of nucleic acids, DNA and RNA, is one way that the body detects invading pathogens and initiates tissue repair. A number of specialized receptor proteins have evolved to distinguish nucleic acids that represent “threats” from those involved in normal physiology. These proteins include members of the Toll-like receptor family and diverse types of cytosolic proteins, all of which reside within the confines of the cell. Few proteins on the cell surface have been clearly characterized to interact with nucleic acids in the extracellular environment. In this dissertation, I present collaborative work that identifies the receptor for advanced glycation end products (RAGE) as a cell surface receptor for nucleic acids and positions it as an important modulator of immune responses. Molecular dimers of RAGE interact with the sugar-phosphate backbones of nucleic acid ligands, allowing this receptor to recognize a variety of DNA and RNA molecules regardless of their nucleotide sequence. Expression of RAGE on cells promotes uptake of DNA and enhances subsequent responses that are dependent on the nucleic acid sensor Toll-like receptor 9. When mice deficient in RAGE are exposed to DNA in the lung, the predominant site of RAGE expression, they do not mount a typical early inflammatory response, suggesting that RAGE is important in generating immune responses to DNA in mammalian organisms. Further evidence suggests that RAGE interacts preferentially with multimolecular complexes that contain nucleic acids, and that these complexes may induce clustering of receptor dimers into larger multimeric structures. Taken together, the data reported here identify RAGE as an important cell surface receptor protein for nucleic acids, which is capable of modulating the intensity of immune responses to DNA and RNA. Understanding of and intervention in this recognition pathway hold therapeutic promise for diseases characterized by excessive responses to self nucleic acids, such as systemic lupus erythematosus, and for the pathology caused by chronic inflammatory responses to self and foreign nucleic acids.

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Chang, Serena Soyoung Yunmee. "Toll-Like Receptors: Target of Hepatitis C Virus: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/386.

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Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

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Babon, Jenny Aurielle B. "The Subtype Specific and Cross-Reactive T Cell Responses to Influenza Viruses in Humans: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/603.

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Human influenza is a contagious respiratory disease resulting in substantial morbidity and mortality worldwide. With the recent cases of avian influenza infections in humans and the heightened concern for an influenza pandemic arising from these infections, it is essential to understand host responses that would confer protective immunity to influenza. The cell-mediated immune responses to influenza virus play an important role during influenza infection. To analyze the specificity and diversity of memory T-cell responses, we performed a genome-wide screening of T cell epitopes to influenza A virus in healthy adult donors. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-(IFN-γ) enzyme-linked immunosorbent spot assays (ELISPOT) using peripheral blood mononuclear cells (PBMCs) from four healthy adult donors. We found that among 11 influenza viral proteins, hemagglutinin (HA) and matrix protein 1 (M1) had more T-cell epitopes than other viral proteins. The donors were not previously exposed to H5N1 subtype, but we detected H5 HA T cell responses in two of the four donors. To confirm that HA is a major target of T cell responses we also analyzed H1 and H3 HA-specific T-cell responses using PBMC of additional 30 adult donors. Fifteen out of thirty donors gave a positive response to H3 HA peptides, whereas five of thirty donors gave a positive response to H1 HA peptides. Because we detected T cell responses to the H5 HA peptides in donors without prior exposure to H5N1 subtype, we asked if cross-reactive T cells to H5 HA peptides can be attributed to a prior exposure to H2N2 subtype, the closest HA to the H5 based on their phylogeny. We compared younger donors who have no prior exposure to H2N2 subtype and older donors who were likely to be exposed to H2N2 subtype, and both groups responded H2N2 peptides at similar level, suggesting that memory T cells cross-reactive to H5 HA peptides can be generated by prior exposure to the H1N1 and H3N2 subtypes, and the exposure to H2N2 subtype is not necessary. We subsequently identified a CD4+ T cell epitope that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of HA of influenza A and the HA of the influenza B virus. A CD4+ T cell line specific to this epitope recognizes target cells infected with various influenza A viruses including seasonal H1N1 and H3N2, a reassortant H2N1, the 2009 pandemic H1N1, H5N1 and influenza B virus in cytotoxicity assays and intracellular cytokine staining assays. Individuals who have the HLA-DRB1*09 allele have ex vivo IFN-γ responses to this epitope peptide in ELISPOT. Although natural infection or standard vaccination may not induce strong T and B cell responses to this very conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4+ T cells which are cross-reactive to both influenza A and B viruses.

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Friberg-Robertson, Heather L. "CD8+ T Cell Serotype-Cross-Reactivity is a Predominant Feature of Dengue Virus Infections in Humans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/513.

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The four serotypes of dengue virus (DENV 1-4) have a significant and growing impact on global health. Dengue disease encompasses a wide range of clinical symptoms, usually presenting as an uncomplicated febrile illness lasting 5-7 days; however, a small percentage of infections are associated with plasma leakage and bleeding tendency (called dengue hemorrhagic fever, DHF), which can result in shock. Epidemiological studies indicate that severe dengue disease most often occurs during secondary heterotypic DENV infection. Additionally, plasma leakage (the hallmark of DHF) coincides with defervescence and viral clearance, suggesting that severe disease arises from the immune response to infection rather than a direct effect of the virus. A number of studies have found increased levels of markers of immune cell activation in patients with DHF compared to patients with the less severe form of disease (DF). These markers include IFNγ, TNFα, soluble CD8, soluble IL-2 receptor, soluble TNF receptor, and CD69, which support a role for T cells in mediating immunopathology. Because of the high homology of DENV 1-4, some degree of serotype-cross-reactivity is seen for most T cell epitopes. A high percentage of DENV-specific T cells recognize multiple DENV serotypes, as demonstrated by peptide-MHC (pMHC) tetramer binding and in vitro functional assays performed on PBMC from subjects vaccinated with an experimental DENV vaccine or naturally-infected subjects with secondary (>1) DENV infection. This thesis sought to address several gaps in the literature, specifically whether T cell responses differ in primary versus secondary (natural) infection. We studied the frequency, phenotype, and function of DENV-specific T cells. We demonstrated substantial serotype-cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary as well as secondary DENV infection. The frequency of A11-NS3133 epitope-specific T cells during acute infection did not correlate with disease severity. However, the peak frequency occurred earlier in primary infection while the frequency of CD45RA+ T cells declined quicker in secondary infection, suggesting the expansion of DENV-specific memory T cells. DENV-immune T cells exhibited different functional capabilities that were dependent on the particular serotype of infection. Specifically, DENV-1 or -3 stimulation of A11-NS3133 epitope-specific T cell lines resulted in robust function that included IFNγ production, whereas DENV-2 stimulation resulted in limited function that often included MIP-1β but not IFNγ production. These data support a role for T cells in DENV infection and offer new insights into their potential contribution to dengue pathology.

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Seedhom, Mina O. "Immunity, Pathogenesis, and Prevention of Poxvirus Infections: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/514.

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Vaccinia virus (VAC) is the prototypical member of the orthopoxvirus genus of the poxvirus family and the virus used for smallpox vaccinations. The following describes the testing of VAC variants designed to have similar immuno-protective profiles with decreased pathogenicity, examines the immune response to VAC after lethal infection in wild type and lupus-prone mice, and describes a method that allows for the enumeration of VAC-specific CD8+ T in naïve and VAC-immune mice. The first part describes work examining VAC Wyeth (VAC-Wy) variants engineered to be less pathogenic in vivo. VAC-Wy variants included genes that code for three immunomodulatory proteins, an interferon-γ (IFNγ) binding protein (B8R), an interleukin 18 (IL-18) binding protein (C12L), and a complement binding protein (C3L, or C21L) or various combinations of the three knockouts, and a triple knockout (VAC-Wy -/-/-) in which all three genes were knocked out of a variant virus. The immunomodulatory effects of other IFNγ binding proteins on VAC-Wy pathogenesis in the mouse were also examined. Virus recombinants where the B8R gene was replaced with a truncated mouse IFNγ receptor gene or a gene that encodes a B8R/IFNγ fusion that allows for dimerization of the secreted IFNγ receptor were studied. As the knockouts and variants were made in the current vaccine VAC-Wy strain, only high dose (1x107 PFU’s) intra nasal (I.N.) infection of mice reliably resulted in detectable virus in the lungs. Further testing revealed that all knockout and variant viruses grew to similar levels after high dose I.N. infections. Protection induced by vaccination with the VAC-Wy variants was studied in comparison to immunizations with the VAC-Wy parental strain. Mice were immunized by tail skin scarification to mimic human immunizations, and this was followed months later by I.N. challenge with 20 LD50’s of VAC-WR. All VAC-Wy recombinants tested, including the VAC-Wy -/-/-, provided similar levels of protection as the parental VAC-Wy strain from a lethal VAC-WR I.N. infection. Mice immunized with the VAC-Wy -/-/- induced similar amounts of neutralizing antibody and similar numbers of CD8+ T cells specific to a subdominant determinant as VAC-Wy. While examining high dose, normally lethal, VAC-WR I.N. infections, a profound splenic CD8+ T cell immune suppression was noted that might have been caused by Fas dependent activation induced cell death (AICD). Using high dose intra-peritoneal (I.P.) and I.N. models of VAC-WR infection, decreased weight loss, decreased virus titers, and increased T cell numbers were found in Fas mutant (B6.MRL-Faslpr/J) mice in comparison to B6 wild type mice on day 6. It would be expected that Fas-deficient CD8+ T cells from B6.MRL-Faslpr/J mice (B6-lpr) would survive a high dose VAC-WR infection better than CD8+ T cells that could express Fas if T cells were being eliminated by Fas-dependent AICD, but co-adoptive transfer experiments using splenocytes from B6-lpr and B6.Cg- IgHaThy-1aGPi-1a/J (IgHa) wild type counterparts found no difference in the numbers or proliferation of donor CD8+ T cells at day 6. As the B6-lpr mice were better protected from VAC-induced weight loss early after lethal VAC-WR infections, it was possible that B6-lpr mice might be protected early in infection. In fact, Fas mutant mice had decreased virus loads in the fat pads, livers, and spleens in comparison to B6 wild type mice at days 2 and 3. In addition to the decreased virus titers, the severe splenic lymphocyte deficiency noted in B6 wild type mice as early as day 2 after high dose I.P. infection was ameliorated in B6-lpr mice. Further experiments demonstrated that uninfected B6-lpr mice had increased numbers of memory phenotype (CD44+) CD4+, CD8+ and γδ+ T cells, with an increased number of γδ+ T cells and NK cells in splenic lymphocytes in comparison to wild type B6 mice. Uninfected B6-lpr mice also had increased numbers of IFNγ+ CD8+ T cells after polyclonal stimulation with an antibody against CD3ε. In lymphocyte depletion experiments performed at day 3, antibody depletion of CD4, CD8, or NK or treatment with an antibody that was specific to the γδ+ TCR did not significantly alter virus loads in B6-lpr mice. In co-adoptive transfer experiments, splenocytes from wild type or B6-lpr mice survived high dose VAC-WR challenge similarly suggesting that B6- lpr splenocytes were not intrinsically better protected from lymphocyte depletion by lack of the Fas protein. On day 2 after high dose I.P. VAC-WR infection, B6- lpr mice had increased numbers of IFNγ+ NK cells, IFNγ+ CD8+ T cells, and IFNγ+ CD4+ T cells. B6-lpr and B6 mice treated with an antibody against IFNγ had significantly increased virus titers in the spleens and livers. Interestingly, there was no significant difference in liver or spleen virus titers when comparing anti- IFNγ antibody treated B6 mice or anti-IFNγ antibody treated B6-lpr mice. These results suggest that multiple leukocyte populations co-operatively or redundantly provide B6-lpr mice with increased protection from high dose VAC-WR infections through increased production of IFNγ. The third part of this work describes the enumeration of total numbers of pathogen-specific CD8+ T cells in a mouse through use of an in vivo limiting dilution assay (LDA). The extensive proliferation of virus-specific CD8+ T cells that occurs after virus infection was used to enumerate numbers of virus-specific CD8+ T cells in a naïve mouse. By transferring limiting amounts of carboxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1+Ly5.2+ heterogeneous CD8+ T cells into Thy1.2+Ly5.1+ hosts, CD8+ T cell precursor frequencies to whole viruses can be calculated. The calculations are based on finding the number of donor CD8+ T cells that results in CFSElo (i.e. proliferated) donor CD8 T cells in 50% of the hosts. Using probit or Reed and Muench 50% endpoint calculations, CD8+ T cell precursor determinations were made for naïve and immune states to a virus challenge. It was found that in naïve B6 mice, 1 in 1444 CD8+ T cells proliferated in response to VAC-WR (~13,852 VAC-WR-specific CD8+ T cells per mouse) and 1 in 2956 proliferated in response to lymphocytic choriomeningitis virus (LCMV) (~6,761 LCMV-specific CD8+ T cells per mouse). In mice immune to VAC-WR, the number of VAC-WR-specific LDA precursors, not surprisingly, dramatically increased to 1 in 13 (~1,538,462 VAC-WR- specific CD8+ T cells per mouse) consistent with estimates of VAC-WR-specific memory T cells. In contrast, precursor numbers to LCMV did not increase in VAC-WR-immune mice (1 in 4562, ~4384 LCMV-specific CD8+ T cells in a VAC-WR-immune mouse) consistent with the fact that VAC-WR provides no heterologous immunity to LCMV. Using H-2Db-restricted LCMV GP33-specific P14 transgenic T cells it was found that, after accounting for take of donor T cells, approximately every T cell transferred underwent a full proliferative expansion in response to an LCMV infection and a high efficiency was also seen in memory populations. This suggests that most antigen-specific T cells will proliferate in response to infections at limiting dilution. These results, which are discussed in comparison to other methods, show that naïve and memory CD8+ T cell precursor frequencies to whole viruses can be remarkably high. In total this work further advances knowledge of the immunity, pathogenesis, and prevention of poxvirus infections. This was accomplished by studying VAC-Wy recombinants as improved vaccines, by examining the mechanisms and cell types important in early protection from high dose poxvirus infections in B6 and B6-lpr mice, and by describing a method to enumerate total numbers of virus-specific CD8+ T cells in a mouse.

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Hardy, Olga T. "Role of the Monocyte/Macrophage Cell Lineage in Obesity-Related Insulin Resistance." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/464.

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Background Obesity is an important risk factor for resistance to insulin-mediated glucose disposal, and is a precursor of type 2 diabetes and other disorders. Objectives To identify molecular pathways in adipose tissue and inflammatory cells that may result in obesity-associated insulin resistance, we exploited the fact that not all obese individuals are prone to insulin resistance. Thus the degree of obesity as a variable was removed by studying obese subjects of similar body mass index (BMI) who are insulin-sensitive (IS) versus insulin-resistant (IR). Methods Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from 10 obese-IR and 10 obese-IS patients undergoing gastric bypass surgery. In a secondary study, we isolated monocytes from 4 obese-IR, 3 obese-IS, and 4 nonobese-IS adolescent and young adult subjects for purposes of assessing differences in expression of inflammatory genes in monocytes using RT-PCR. Results Gene sets related to chemokine activity and chemokine receptor-binding were identified as most highly enriched in the omental tissue from obese-IR compared to obese-IS subjects, independent of BMI. Strikingly, insulin resistance, but not BMI, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size. In the adolescent and young adult cohort, expression of two cytokine signaling molecules (IL8, SOCS3) and two downstream products of the JNK pathway (JunB, c-Fos) showed increased expression in the obese-IR subjects compared to the obese-IS and nonobese-IS subjects, suggesting the presence of a proinflammatory phenotype in monocytes in obesity, which is exacerbated in the insulin resistant state. Conclusions Our findings demonstrate that inflammation of omental adipose tissue and activation of proinflammatory monocytes is strongly associated with insulin resistance in human obesity. Manipulation of these pathways may result in the prevention of or delay in the onset of obesity-related co-morbidities.

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Raval, Forum M. "Innate Signaling Pathways in the Maintenance of Serological Memory: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/635.

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Long-term antiviral antibody responses provide protection from re-infection and recurrence of persistent viruses. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88-deficient mice generate low levels of virus-specific IgG after the acute phase of infection and that these IgG responses have a skewed isotype distribution with low levels of IgG2a/c. Moreover MyD88-deficient mice have reduced numbers of long-lived plasma cells in the bone marrow. These studies suggest an important role of MyD88-mediated signaling in long-term antiviral responses. Our lab has shown that T cell-deficient mice can also maintain long-term virus-specific IgG responses following PyV infection. The goal of this thesis is to evaluate the role of innate signaling pathways in maintaining serological memory to persistent virus infection and to elaborate on how long-term antiviral responses can be maintained in an immunocompetent or partially immune compromised, T cell-deficient host. Regarding T cell-dependent B cell responses, I set out to investigate the upstream and downstream components of the MyD88-mediated pathways required for normal antibody isotype and long-term humoral responses. IgG2a is a predominant immunoglobulin isotype in most virus infections. Wild type mice, in response to PyV infection, primarily induce antiviral IgG2a with some IgG1. MyD88-deficient mice in response to PyV infection display attenuated levels of virus-specific IgG2a, but normal levels of IgG1. Using Unc93B1 mutant mice (3d mice), which are defective in TLRs 3, 7 and 9 signaling, I show that 3d mice also generated low levels of virus-specific IgG2a following PyV infection. Studies in individual TLR3-/-, TLR7-/- or TLR9-/- mice displayed PyV-specific IgG2a responses similar to wild type responses. TLR7 and TLR9 double deficient mice generated similar skewed antibody isotype responses, where virus-specific IgG2a was reduced compared to wild type mice. This shows that TLR7 and TLR9-MyD88 mediated pathways are important in regulating IgG2a responses during a PyV infection. To investigate what components downstream of MyD88 are involved in mediating IgG2a responses, I worked with IRF5-deficient mice. IRF5 is a transcription factor that is activated upon stimulation of TLR7 or TLR9-MyD88-mediated pathways. Moreover, IRF5-deficient mice cannot generate autoantibodies specifically of the IgG2a isotype in a mouse lupus model, suggesting that IRF5 plays an important function in mediating class switching to IgG2a. In vitro studies where IRF5-/- B cells were stimulated with TLR7 or TLR9 ligands also generated low levels of γ2a germ-line transcripts, suggesting a B cell-intrinsic role for IRF5 in regulating γ2a germ-line transcription. PyV infection of IRF5-deficient mice resulted in similar skewed isotypes as observed in MyD88-deficient and 3d mice. To investigate a B cell-intrinsic role for IRF5 in regulating IgG2a responses in vivo upon PyV infection, I transferred IRF5-/- B cells and WT T cells into RAG KO mice prior to infection and compared the responses of these mice with mice reconstituted with wild type B6 B and T cells. Diminished numbers of IgG2a+ B cells and reduced levels of virus-specific IgG in mice reconstituted with IRF5-/- B cells were seen compared to mice reconstituted with wild type B cells. Regarding the defect in long-term IgG production in MyD88-/- mice upon PyV infection, I conducted studies in IRF5-/-, 3d, single TLR3-/-, TLR7-/-, TLR9-/- and TLR7/9 double deficient mice. These studies reveal an important and redundant role for TLR7- and TLR9-MyD88 signaling in maintaining long-term anti-PyV IgG responses. To determine how MyD88 signaling affects the generation of long-lived plasma cells and memory B cells, I investigated germinal center (GC) responses in MyD88-deficient mice. A defect in GC B cell numbers is observed in MyD88-deficient mice after the acute phase of infection. The GC reaction is essential for the generation and maintenance of long-lived plasma cells and memory B cells. T follicular helper (TFH) cells are absolutely required to generate normal GC. l found reduced numbers of TFH cells in MyD88-deficient mice. Lower numbers of T FH cells suggests that poor T cell help may contribute to the diminished number of GC B cells. However, interaction with B cells is required for the formation of fully differentiated TFH cells. Along with B cell function, MyD88 signaling can affect T cell and dendritic cell function as well. Thus, it is not clear at this point whether the requirement for intact MyD88 signaling for the formation and maintenance of long-term B cell populations is completely B cell-intrinsic. Some viruses can induce T cell-independent B cell responses, perhaps due to their complex arrays of repetitive antigenic epitopes on virions, coupled with the induction of innate cytokines. Nevertheless, T cell help is usually necessary for generating long-term antibody responses in the form of long-lived plasma cells and memory B cells. In contrast, our lab has found that T cell-deficient mice infected with PyV develop long-lasting, protective antiviral IgG responses. I questioned whether these mice could generate TI B cell memory cells or long-lived plasma cells. I show that long-lasting anti-PyV antibody in T cell-deficient mice was not due to the presence of long-lived plasma cells or memory B cell responses. TCRβδ deficient mice, which lack both CD4 and CD8 T cells, had ~10 a times higher virus load persisting in various organs. Therefore, I hypothesized that the high level of persistent PyV antigen, in completely T cell-deficient mice, may activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses. Prior to PyV infection, T cell-deficient mice received wild type CD8 T cells, which reduced PyV loads, and this was associated with decreased levels of antiviral serum IgG over time. As in TCRβδ deficient mice, high PyV loads were detected in the bone marrow, which is the site for B cell lymphopoiesis, I questioned how B cells develop in the presence of PyV antigen and still stay responsive to PyV, generating long-term antiviral IgG responses in the periphery. Studies have shown that self-antigens that trigger both B cell receptor signaling and TLR-MyD88 signaling pathways in the bone marrow lead to the breaking of B cell tolerance and production of autoantibody in the periphery. Thus, we hypothesized that high PyV levels in the bone marrow signal through both B cell-receptors and TLRs, allowing continuous antiviral antibody production by B cells. Using mice that are deficient in T cells and MyD88 signaling, I found that PyV-specific TI IgG levels gradually decreased, supporting this hypothesis. Thus, high PyV loads and innate signaling together can break B cell tolerance. During a persistent virus infection this can result in sustaining long-term protective T cell-independent IgG responses.

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Trobaugh, Derek W. "Primary and Secondary Immune Responses During Sequential West Nile Virus and Japanese Encephalitis Virus Infections: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/581.

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Abstract:

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related Flaviviruses that are important arthropod-borne human pathogens. Both of these viruses can cause encephalitis with significant morbidity and mortality after infection. Flaviviruses co-circulate in many areas of the world, which raises the risk for sequential infection between heterologous viruses. Sequential infection between dengue virus serotypes can lead to cross-protection, but in some cases, it leads to a severe outcome, dengue hemorrhagic fever. Previous work in hamsters and non-human primates demonstrated that prior JEV immunity protects against a lethal WNV infection. However, the ability of prior WNV immunity to protect against a lethal JEV infection has been inconclusive. WNV-immune hamsters were fully protected from JEV viremia, but in non-human primates, prior WNV-immunity only reduced disease severity, with symptoms of encephalitis still observed. These differences in cross-protection led to further investigation on the directionality as well as the underlying mechanisms for this phenomenon. Previous work in our lab found that JEV-immune C57BL/6J (B6) mice were fully protected against a lethal WNV infection, and JEV-immune CD4+ and CD8+ T cells were required for this cross-protection. In other mouse models, memory cross-reactive CD4+ and CD8+ T cell responses may induce protection or immunopathology upon secondary heterologous viral challenge. We hypothesize that JEV/WNV cross-reactive CD4+and CD8+ T cells preferentially expand upon 2o infection and contribute to cross-protection. To elucidate the potential role of T cells in sequential flavivirus infection, we identified and characterized cross-reactive CD4+ and CD8+ T cell responses between JEV and WNV. A previously reported WNV NS4b CD8+ T cell epitope and its JEV variant elicited CD8+ T cell responses in both JEV- and WNV-infected mice. Despite similarities in viral burden for pathogenic JEV and WNV viruses, CD8+ T cells from pathogenic JEV-infected mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. We believe the differences in the CD8+ T cell responses during primary JEV and WNV infection are due at least in part to the low levels of peripheral replication seen in JEV-infected mice compared to WNV-infected mice. We also found that WNV-immune B6 mice were protected against a lethal JEV infection. Cross-reactive CD8+ T cells in JEV-immune mice rapidly expanded after WNV infection. Even though WNV-immune mice had higher frequencies of memory CD8+ T cells, cross-reactive CD8+ T cells did not expand after secondary JEV infection. Neutralizing antibodies to JEV were detected in WNV-immune mice; however, cross-reactive CD8+ T cells did not expand even in the absence of these cross-reactive neutralizing antibodies. We did not detect any differences in the CD8+ T cell repertoires between JEV- and WNV-infected mice nor were WNV-immune CD8+ T cells functionally exhausted. In fact, proliferation of memory CD8+ T cells did not correlate with the ability of WNV-immune CD8+ T cells to restrict recombinant vaccinia viruses expressing the cross-reactive epitope or lyse peptide-coated targets. These data suggest that the higher frequency of memory CD8+ T cells and cross-reactive antibodies in WNV-immune mice are better able to prevent neuroinvasion following 2o JEV infection.

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Mueller, Christian. "Lack of CFTR in CD3+ Lymphocytes Leads to Aberrant Cytokine Secretion and Hyper-Inflammatory Adaptive Immune Responses: A Master's Thesis." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/595.

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Abstract:

Background: Cystic fibrosis (CF) remains the most common fatal monogenic disease in the US, affecting 1 in 3,300 live births. CF is the result of mutations in CFTR, a chloride channel and regulator of other ion channels. The mechanisms by which CFTR mutations cause chronic lung disease in CF are not fully defined, but may include the combined effects of altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr-/- mice mount an exaggerated IgE response towards Aspergillus fumigatus (Af) when compared to Cftr+/+ mice. Along with the increased IgE levels, the Cftr-/- mice had higher levels of IL-13 and IL-4, mimicking both the Th-2 biased immune responses and predilection to mounting Af-specifc IgE seen in CF patients. Herein we hypothesize that these immune aberrations are primarily due to the lack of Cftr expression in lymphocytes rather than with Cftr deficiency in the epithelium. Results: Our results indicate that adoptive transfer experiments with Cf splenocytes confer higher IgE response to Af in host mice as compared to hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount Th2 responses was confirmed by in vitro antigen recall experiments, where higher levels of IL-13 and IL-4 where seen only in the presence of Cftr-deficient lymphocytes. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T-cell lineages developed the higher IgE titers as compared to their wild-type littermate controls. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca 2+ flux in response to T cell receptor activation as compared to normal lymphocytes. This was accompanied by a significant increase in nuclear localization of the calcium-sensitive transcription factor NFAT, which could contribute to the enhanced secretion of IL-13 and other cytokines. Conclusions: In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. This is the first instance that a CF related phenotype has been entirely modeled in vivo by selectively knocking out CFTR in the immune system. Specifically, Cftr deficient lymphocytes directed skewed responses to Aspergillus fumigatus , leading to a higher than normal IgE response. These findings implicate the lymphocyte population as a potentially important target for therapeutics directed to the treatment of CF lung disease.

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Dissertations / Theses on the topic 'Hemic and Lymphatic Diseases'

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