Joseph Delano Robles

BACKGROUND: Mesenchymal stromal cells (MSCs) are proven to have immunosuppressive functions via various mechanisms. These mechanisms were demonstrated by administering bone marrow derived human MSCs (hMSCs) to graft versus host disease (GVHD) murine models. METHODS: BALB/c host mice were irradiated prior to receiving C57BL/6 donor T cell depleted bone marrow (TCDBM) cells (negative control) and donor CD4+ T lymphocyte with (treatment group) or without hMSCs (positive control). The presence of hMSCs in target tissues and lymphoid organs was documented by using in vivo imaging and measuring the expression of EphB2 and ephrin-B2 by RTqPCR. Survival rate and GVHD score were also monitored. Tissue sections were obtained for histopathologic analysis. Flow cytometry was used to document donor T cell alloreactivity and expression of CCR5, CXCR3 and CCR7. ELISA was utilized to determine levels of proinflammatory cytokines, RANTES (CCL5) and phosphorylated STAT 5A/B. RTqPCR was performed to quantify expression of CCL3 and CXCL9. Western blotting was performed to qualitatively measure iNOS expression. RESULTS: Survival rate and GVHD score improved with hMSC treatment. Pathologic changes of GVHD were abrogated. Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs. Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed. CONCLUSIONS: These mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.

Norovirus infections are increasingly being recognized as important causes of diarrhea in hematopoietic stem cell transplantation (HSCT) recipients. This retrospective study aimed to evaluate the cumulative incidence, risk factors, and outcomes of norovirus infection in pediatric HSCT recipients. Among 55 patients age <21 years who underwent first HSCT between July 2007 and June 2011, 49 patients developed diarrhea and had stool tested for norovirus. Eight of these patients were found to be infected with norovirus. All were sporadic cases and manifested with nausea, vomiting, and diarrhea. The median age of these patients was 5.2 years (range, 0.5-18.5 years). Six were males. Seven patients underwent unrelated donor HSCT, and 1 patient underwent autologous cord blood HSCT. Two patients had norovirus infection before HSCT that persisted after transplantation. In the remaining 6 patients, norovirus developed at a median of 36.5 days posttransplantation (range, 5-517 days). The cumulative incidence of norovirus infection was 12.9% at 2 years posttransplantation. Risk factors for norovirus infection included the use of peripheral blood or cord blood as the stem cell source (P = .043) and administration of fludarabine (P = .002) and alemtuzumab (P = .011). The median time to viral clearance was 145 days (range, 13-263 days). Four-year survival was similar in norovirus-infected patients and noninfected patients (56.3% versus 58.3%).

This thesis is comprised of two parts, both exploring complications of hematopoietic stem cell transplantation. The first part investigated the immunosuppressive mechanisms of human mesenchymal stromal cells (MSCs) in graft versus host disease (GVHD) murine models. MSCs have been documented to possess the capability to suppress proliferation of T lymphocytes which is implicated in acute GVHD, which accounts for around 20% mortality amongst matched sibling and matched unrelated transplants (CIBMTR, 2013). A number of mechanisms have been described using in vitro or in vivo studies either by human MSCs administered to humanized murine models of GVHD or murine MSCs given to GVHD mice. This part involved exploration of the various mechanisms of immunosuppression using human MSCs administered to murine GVHD models and monitoring GVHD clinical score, survival, donor T cell alloreactivity, homing of MSCs to lymphoid organs and target organs of GVHD, and their corresponding histopathologic changes. Elucidation of the role of chemokine receptors and ligands, members of the ephrin family of receptors and ligands, inducible nitric oxide synthase and phosphorylated STAT 5 proteins in the immunosuppressive capacity of MSCs were also studied. This study attempted to form a coherent explanation of these mechanisms spanning clinicopathologically and in terms of the immune response of lymphoid organs and target tissues of acute GVHD. Apart from these, investigation of some of the receptors, ligands and soluble mediators reported to be produced by MSCs and use lymphoid organs and target tissues of GVHD to test these forming a coherent picture of how MSCs exert immunosuppression in GVHD in an in vivo setting were performed.&#13;\n&#13;\nThe second part of this thesis involved retrospective studies on Human Herpes Virus 6 and 7 (HHV-6, HHV-7) and Norovirus infections in paediatric hematopoietic stem cell transplant recipients. Their incidence in a tertiary hospital in Hong Kong, profile of patients affected, predisposing or reactivation risk factors, manifestations and management options were analysed and discussed. These data may provide clinicians recommendations when their patients become infected with these viruses post bone marrow transplantation in the future.