published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Human Immunodeficiency Virus (HIV) infection is characterized by immune dysfunction that predisposes infected individuals to opportunistic infections such as Mycobacterium Tuberculosis (MTB). The result of this is an exacerbation of HIV-TB related deaths annually. Therefore there is an imperative need for HIV-TB focused research that aims to identify immunological factors that are involved in the control of MTB and HIV in both mono- and co-infected individuals. The CD161+ CD4+ T cell subset is linked to a distinct phenotypic and functional profile. Importantly, these CD161+ T cells may act as an important component of immunological defense and provide protection in infected tissues. CD161+ CD4+ T cells have also been identified as the precursor population of Th17 cells and it has been previously reported that reduction of CD161+ CD4+ T cells during HIV infection may limit Th17 reconstitution (Prendergast et al., 2010). This may ultimately contribute to impairment of mucosal immunity leading to the acquisition of opportunistic infections such as MTB and disease progression in HIV infected individuals. Our study aimed to comprehensively characterise the impact of HIV and MTB infection on the CD161+ CD4+ T cell subset and to assess the frequency, phenotype and function of these cells. The study also aimed to correlate the longitudinal variation in frequency, phenotype and function with markers of HIV disease progression. Methods The frequency, phenotype and function of the CD161+ CD4+ T cell subset was measured by flow cytometry. For the frequency and phenotypic assessment, whole blood was collected from HIV negative and HIV/MTB mono and co-infected subjects (n = 17 per patient group). Whole blood was surface stained with antibodies specific to CD3, CD4, CD8, CD161 and chemokine receptors CD103, CCR6, CXCR4, CCR5 and CXCR6. The percentage positive expression of CD161 on CD4+ T cells and chemokine receptor expression was measured. The functional assessment of CD161+ CD4+ T cells involved PBMC stimulation with antigenic stimulant, phorbol 12-myristate 13-acetate (PMA) and ionomycin or ESAT-6/CFP-10, GAG, TB10.4 and Ag85a followed by intracellular cytokine staining for IFN-γ, IL-17A, IL-22 and TNF-α. A subgroup of HIV negative (frequency and phenotype, n = 10, function n = 7) and HIV mono-infected subjects (frequency and phenotype, n = 10, function n = 7) were longitudinally followed to assess variations in the frequency, phenotype and function of CD161+ CD4+ T cells over time. Results The CD161+ CD4+ T cell subset demonstrated high-level expression of chemokine receptors CCR5, CCR6, CXCR4 and low-level expression of CD103 and CXCR6. The subset also demonstrated the ability to produce cytokines IFN-γ, IL17A, IL-22 and TNF-α in healthy subjects. Analysis of HIV infected samples revealed a significant reduction in the frequency of the CD161+ CD4+ subset (median = 06.86%, p < 0.0001) compared to that of healthy individuals (median = 14.75%). Correlation of the subset frequency to markers of disease progression revealed a positive trend to CD4 count (r = 0.2590, p = 0.0787) and a significant negative correlation to viral load (r = -0.3522, p = 0.0152). Unlike with HIV infection, no significant changes in CD161+ CD4+ T cell frequency was observed in individuals with LTBI (mono- or HIV co-infected) or active TB disease compared to that of the healthy patient group. However, the exception to this was HIV infected individuals with active TB disease (co-infected) (median = 03.80%, p < 0.0001). Decreased CCR6 expression on CD161+ CD4+ T cells was observed in HIV monoinfected (p = 0.0065) and HIV infected individuals with active TB disease (p = 0.007). No functional changes were observed in both the HIV and MTB mono- and co-infected cohorts following non-specific stimulation. An interesting positive trend in correlation between IFN-γ production and CD4 count (r = 0.2727, p = 0.0733) was demonstrated with a significant negative correlation between IFN-γ production and viral load observed following non-specific antigenic stimulation (r = -0.3705, p = 0.0133). CD161+ CD4+ T cells demonstrated antigen-specific T cell responses to peptides ESAT-6/CFP-10, TB10.4, Ag85a and GAG in a small proportion of 69 study participants with variable ranges in magnitude of the responses observed. The longitudinal assessment of CD161+ CD4+ T cell frequency and phenotype demonstrated low-level proportion of CD4+ T cells expressing CD161 and CCR6 expression longitudinally maintained in HIV mono-infected compared to that of healthy individuals. Conclusion The phenotypic and functional profile of the CD161+ CD4+ T cell population indicates that it may be an important component of immunological defense that may provide mucosal defense and protection at epithelial sites and tissues e.g. expression of tissue homing markers like CCR6 and the production of cytokines such as IL-17A and IL-22 (important in mucosal immunity). HIV infection is associated with a reduced frequency of CD161+ CD4+ T cells. The correlation between CD161+ CD4+ T cell frequency and markers of disease progression suggests that the observed low-level frequency in HIV infected individuals may in part be a result of non-specific HIV-mediated depletion of CD4+ T cells. However, lower levels of CD161+ CD4+ T cells in HIV infected individuals could also be a result of naturally lower levels being present in individuals prior to infection, thereby making these individuals more susceptible to HIV infection. The significantly reduced levels of CCR6 expression on CD161+ CD4+ T cells in HIV monoinfected individuals may also be an indication of cell subset migration to gut associated lymphoid tissue (GALT, target site of HIV replication) during HIV infection. Given their potential role in mediating signals that are essential for immune responses to microbes and microbial products, migration of CCR6+ CD161+ CD4+ T cells to target sites of HIV infection could serve as a protective measure in the fight against HIV infection. Although there were no observable changes in the functional capacity of the CD161+ CD4+ T subset in HIV infection, we believe that the reduction in frequency may contribute to HIV disease progression and susceptibility to opportunistic infections such as MTB or active TB disease. Unlike with HIV infection, infection with MTB appeared to have no significant impact on CD161+ CD4+ T cells as there were no observable differences in frequency or the functional capacity of the cell subset following PMA stimulation. However, MTB and HIV antigen-specific responses were observed in a small proportion of the total 69 subjects tested. This therefore indicates that a subset of CD161+ CD4+ T cells may act in an HIV and MTB-specific manner. Additional MTB and HIV-specific responses may be present in this CD161+ CD4+ population and may only be identified through stimulation with additional antigenic targets. Further investigation of CD161+ CD4+ T cells should be performed at the actual sites of infection to investigate if CD161+ CD4+ T cells are concentrated at sites of disease. Also it may be important to investigate the polyfunctionality of CD161+ CD4+ T cells to understand the multifunctional capacity of the cell subset in providing immunological defense to pathogens such as HIV and MTB. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

Indiana University-Purdue University Indianapolis (IUPUI) / Dicalcium phosphate dihydrate (DCPD) cements have been used for bone repair due to its excellent biocompatibility and resorbability. However, DCPD cements are typically weak and brittle. To overcome these limitations, the sodium citrate used as a setting regulator and the coating of poly-L-lactide acid (PLLA) technique have been proposed in this study. The first purpose of this thesis is to develop composite PLLA/DCPD scaffolds with enhanced toughness by PLLA coating. The second purpose is to examine the biocompatibility of the scaffolds. The final purpose is to investigate the degradation behaviors of DCPD and PLLA/DCPD scaffolds. In this experiment, DCPD cements were synthesized from monocalcium phosphate monohydrate (MCPM) and 𝛽-tricalcium phosphate (𝛽 –TCP) by using deionized water and sodium citrate as liquid components. The samples were prepared with powder to liquid ratio (P/L) at 1.00, 1.25 and 1.50. To fabricate the PLLA/DCPD composite samples, DCPD samples were coated with 5 % PLLA. The samples were characterized mechanical properties, such as porosity, diametral tensile strength, and fracture energy. The mechanical properties of DCPD scaffolds with and without PLLA coating after the in vitro static degradation (day 1, week1, 4, and 6) and in vitro dynamic degradation (day 1, week 1, 2, 4, 6, and 8) were investigated by measuring their weight loss, fracture energy, and pH of phosphate buffer solution. In addition, the dog bone marrow stromal stem cells (dBMSCs) adhesion on DCPD and PLLA/DCPD composite samples were examined by scanning electron microscopy. The cell proliferation and differentiation in the medium conditioned with DCPD and PLLA/DCPD composite samples were studied by XTT (2,3-Bis(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt), and alkaline phosphatase (ALP) assay, respectively. The addition of sodium citrate and PLLA coating played a crucial role in improving the mechanical properties of the samples by increasing the diametral tensile strength from 0.50 ± 0.15 MPa to 2.70 ± 0.54 MPa and increasing the fracture energy from 0.76 ± 0.18 N-mm to 12.67 ± 4.97 N-mm. The DCPD and PLLA/DCPD composite samples were compatible with dBMSCs and the cells were able to proliferate and differentiate in the conditioned medium. The degradation rate of DCPD and PLLA/DCPD samples were not significant different (p > 0.05). However, the DCPD and PLLA/DCPD composite samples those used sodium citrate as a liquid component was found to degrade faster than the groups that use deionized water as liquid component

Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.