*p<0.05, PBMCs from BCG+ donors compared to PBMCs from BCG- donors. The concentrations of cytokines detected in the cell culture supernatants also varied depending upon whether PBMCs were from BCG+ or BCG- donors. on the immune responses to infection. Aggregates of PBMCs were initially observed on day 3 and the size of aggregates continued to increase on day 8 post-infection, where macrophages and T cell subsets were identified to be present. Similarly, mycobacterial load progressively increased in infected PBMCs during the 8 days of culture but were significantly lower in infected PBMCs from BCG vaccinated (BCG+) donors compared to unvaccinated (BCG-) donors. The levels of INF-, TNF-, IL-4, IL-6, IL-10 and IL-17 in the supernatants of infection. Introduction Tuberculosis (TB) infection affects approximately one in Pirarubicin Hydrochloride three people in the world and causes approximately 1.5 million deaths worldwide each year . The disease is caused by complex (bacillus is phagocytosed by dendritic cells (DCs) and monocyte-derived macrophages [3C6] where the bacillus survives within these cells . The host cellular immune response to infection includes the recruitment of new macrophages [8C11] and T cells from the circulation to the site of infection within the parenchyma of the lung. These recruited immune cells interact with the pre-existing macrophages and DCs in the lung in support of the immune response against infection . This series of events leads to the formation of a mature granuloma, a multicellular structure composed of infected and uninfected macrophages, epithelioid cells, giant cells (multinucleated cells derived from fused macrophages), T cells and B cells to contain the bacilli Pirarubicin Hydrochloride and to prevent spread of the infection [13C15]. We have previously reviewed a variety of approaches to better understand the development of a granuloma and to control the pathophysiology of . Due to the limited access to human biopsy samples of granulomas, several three-dimensional models have Igf1 been used to study the structure and function of granulomas. In particular, the three-dimensional granuloma model of infection which consists of donor PBMCs in a collagen matrix  has allowed the study of host factors that drive the formation of a granuloma [15,18,19]. Human PBMCs infected with members of the complex formed aggregates of bacteria and monocyte-derived macrophages as well as T cells, which may represent an early granuloma formation [16,17,19C23]. In this study, we used the three-dimensional granuloma model of infection and characterized the human immune response to attenuated H37Ra. Although use of attenuated strains in infection models may not mirror infection with wild-type virulent strains, use of attenuated strains allow us to assess the impact of potential confounders on experimental models using tools that are outside of a Biosafety Level 3 laboratory . We postulated that one key confounder of Pirarubicin Hydrochloride infection models that should be monitored in experiments is Bacille Calmette-Gurin (BCG) vaccination history of PBMC donors. Some evidence suggests that a history of BCG vaccination may influence results of studies using granuloma models by generating protection, albeit variable, against infection, and would be a significant confounder of studies . Given the sparse literature in the field, this study was initiated with two aims. The first was to characterize the early host immune responses in human PBMCs infected with an attenuated H37Ra strain, as well as the growth of this strain during infection. The second aim was Pirarubicin Hydrochloride to determine the impact of BCG vaccination history of PBMC donors on the immune and bacterial responses in three dimensional granuloma model of infection. Materials and methods Materials were obtained from Fisher Scientific, Ottawa, Ontario unless stated otherwise. Ethics statement This study was approved by the University of Alberta Health Research Ethics Board (Pro00057636) and all methods were performed in accordance with institutional guidelines and regulations. Informed written consent was obtained from all study participants. PBMC donor enrollment A questionnaire approved by the institutional ethics board was used to assess potential confounders in blood donors. Potential donors were asked 1) what is Pirarubicin Hydrochloride their age and country.