published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The human prolactin-inducible protein (PIP) or gross cystic disease fluid protein -15 (GCDFP-15) is a 15 kD protein secreted by human breast cancer cells and is abundant in fluids from gross cystic breast disease. Previous results from our laboratory showed that PIP KO mice had significantly lower numbers of CD4+ T cells in their secondary lymphoid organs, and these cells are impaired in their ability to differentiate into Th1 cells in vitro and in vivo leading to failure to control Leishmania major infection. In the present study, we further assessed the role of PIP in adaptive immunity by comparing cytokine production and intracellular signaling events in macrophages from WT and PIP KO mice following IFN-γ and lipopolysaccharide (LPS) stimulation. We show that although the expressions of IFN-γR and TLR4 on macrophages from KO and WT mice were comparable, PIP KO macrophages were significantly impaired in producing proinflammatory cytokines following IFN-γ and LPS stimulation. This was associated with impaired phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducers of activation of transcription (STATs) proteins in IFN-γ and LPS-stimulated macrophages from PIP KO mice. Interestingly, the expression of suppressors of cytokine signaling (SOCS) 1 and 3 proteins, known to suppress IFN-γ and LPS signaling, was higher in PIP KO macrophages compared to those from WT mice. Collectively, our studies clearly show that deficiency of PIP significantly affects intracellular signaling events leading to proinflammatory cytokine production in macrophages, and further confirm a role for PIP as important immunoregulatory protein involved in host defense. / October 2015

Immunological mediators are thought to be responsible for many of the pathophysiologic changes observed in human glomerulonephritis. Previous studies have shown the importance of immunological mediators such as immune-complexes, complement and neutrophils. Recently, a significant role for the monocyte/macrophage system in glomerular injury has been emphasized by several investigators. The present study was designed to investigate the effect of macrophage supernatant factor(s) on glomerular cell metabolism in an in vitro tissue culture system. Human glomerular cells are grown in vitro and were characterized by their phagocytic capacity and their morphologic characteristics. Light microscopy, scanning and transmission electron microscopy, and observation of their growth pattern revealed two basic cell types: an epithelial and a mesangial cell. Epithelial cells were represented by large (100-200 μ in length) and small (50-70 μ in length) flat polygonal cells. The larger epithelial cell was primarily seen in the initial outgrowth and was not easily maintained in culture. Therefore they were not used for the metabolic experiments. On the other hand, the smaller epithelial cell was maintained in culture for an average of 5 to 8 passages. The mesangial cells were medium-sized (75-120 μ in length), of variable morphology but mostly spindle-shaped and grew in a characteristic storiform pattern. Both cell types kept their morphologic appearance with subculturing and cryopreservation. Cultured glomerular cells were treated with dialyzed macrophage supernatants obtained from mouse or human peripheral blood monocytes. Undiluted or diluted macrophage supernatants were over-layed on glomerular cells cultured in 96-well flat-bottom microtiter plates. DNA, RNA and protein synthesis were evaluated by incorporation of radio-labelled precursors. Macrophage supernatants failed to stimulate DNA synthesis in epithelial cells as measured by incorporation of 3HTdR. The same macrophage supernatant did, however, significantly increase the uptake of 3HUdR and a 14-C amino acid mixture, indicating an increase in RNA and protein synthesis. The results with DNA metabolism are consistent with in vivo observations in that epithelial cells are not regarded as the intrinsic proliferating cell in the hypercellularity observed in glomerular injury. The stimulation of RNA and protein synthesis may be related to the in vivo thickening of the glomerular basement membrane. In addition, it may be related to the production of molecules which may directly or indirectly affect endothelial or mesangial cells and/or affect the local charges in the glomeruli which are known to be important in permeselectivity of the capillary wall. In the case of mesangial cells, exposure to macrophage supernatants led to a significant increase in DNA snythesis as measured by the increase in uptake of 3HTdR. No stimulation was seen in RNA and protein synthesis as measured by the radioactive label technique. The increase in DNA synthesis correlates with in vivo observations of mesangial cell proliferation in glomerular injury. The factor(s) in the macrophage supernatant which affect the metabolism of glomerular cells in vitro is non-dialyzable and denatured by freezing and thawing. In addition, preliminary results indicate that the activity stimulating RNA and protein synthesis (epithelial cells) is insensitive to heat treatment while the one affecting DNA synthesis (mesangial cells) seems to be sensitive to heat treatment. Neither was shown to be species specific since both human and mouse macrophage supernatant induced the same changes in glomerular cell metabolism. The results of this investigation suggest that both cell types are selectively affected by a factor(s) present in the macrophage supernatant. It is likely that more than one factor is responsible for the metabolic changes observed. The possibility that this factor(s) may be identical with macrophage factors previously described in other cell systems cannot be ruled out at this time. These in vitro observations further support an active role for the monocyte/macrophage system in glomerular injury in experimental and clinical glomerulonephritis.

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

No description available.

The intracellular pathogen Legionella pneumophila thrives in both natural and man-made water habitats where it replicates inside freshwater amoebae. L. pneumophila follows a developmental cycle as it grows in amoebae. The actively-multiplying intracellular replicative forms (RFs) differentiate into highly virulent mature infectious forms (MIFs) late in the amoeba infection, and are then released extracellularly. L. pneumophila accidentally infects susceptible humans causing the non-communicable Legionnaires’ disease (LD). MIFs play a central role in the life cycle of L. pneumophila and are thought to be responsible for the transmission of LD. Early reports demonstrated that MIFs were poorly produced inside human macrophages, suggesting that the L. pneumophila progeny from human macrophages has fitness and infectivity disadvantages. Direct comparisons of the L. pneumophila progenies from amoebae and human macrophages have demonstrated that the progeny from amoebae is more morphologically differentiated, resistant to antibiotic challenges, and able to adhere to and initiate infections in host cells than the progeny from macrophages. Analysis of the transcriptomic and proteomic profiles of L. pneumophila inside different hosts has revealed a specific set of genes that are upregulated during differentiation of L. pneumophila into MIFs inside freshwater protozoa but not inside human macrophages, suggesting that these genes may be required for the full differentiation of L. pneumophila and, therefore, for the transmission of LD to susceptible humans. Since the expression of the gene lpg1669, which encodes a putative α-amylase, was upregulated in amoebae (highest level of upregulation among the tested genes) and inside Tetrahymena ciliates, but not inside human macrophages, the role of lpg1669 in the differentiation of L. pneumophila into MIFs was investigated. An isogenic lpg1669 deletion mutant did not display defects in morphological differentiation, in vitro (BYE broth) or in vivo (A. castellanii or U937 human macrophages) growth when compared to its parent strain, suggesting that the gene lpg1669 is not essential for the intracellular differentiation of L. pneumophila. Collectively, these findings demonstrate that L. pneumophila can reach different developmental end points in different hosts and could also provide a clue for the lack of transmission of LD among humans.

No description available.

No description available.

No description available.