Mycobacterium requires an all-around closely apposing phagosome membrane to maintain the maturation block and this apposition is re-established when it rescues itself from phagolysosomes

Pathogenic mycobacteria survive in macrophages of the host organism by residing in phagosomes which they prevent from undergoing maturation and fusion with lysosomes. Several molecular mechanisms have been associated with the phagosome maturation block. Here we show for Mycobacterium avium in mouse bone marrow-derived macrophages that the maturation block required an all-around close apposition between the mycobacterial surface and the phagosome membrane. When small (0.1 μm) latex beads were covalently attached to the mycobacterial surface to act as a spacer that interfered with a close apposition, phagosomes rapidly acquired lysosomal characteristics as indicators for maturation and fusion with lysosomes. As a result, several mycobacteria were delivered into single phagolysosomes. Detailed electron-microscope observations of phagosome morphology over a 7-day post-infection period showed a linear correlation between bead attachment and phagosome–lysosome fusion. After about 3 days post infection, conditions inside phagolysosomes caused a gradual release of beads. This allowed mycobacteria to re-establish a close apposition with the surrounding membrane and sequester themselves into individual, non-maturing phagosomes which had lost lysosomal characteristics. By rescuing themselves from phagolysosomes, mycobacteria remained fully viable and able to multiply at the normal rate. In order to unify the present observations and previously reported mechanisms for the maturation block, we discuss evidence that they may act synergistically to interfere with 'Phagosome Membrane Economics' by causing relative changes in incoming and outgoing endocytic membrane fluxes.     

Tumor Reliance on Cytosolic versus Mitochondrial One-Carbon Flux Depends on Folate Availability

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Human tumor cells, modified by a novel pressure/crosslinking methodology, promote autologous lymphocyte proliferation and modulate cytokine secretion

 Hydrostatic pressure (P) combined with membrane protein crosslinking (CL) by adenosine dialdehyde (AdA) can render tumor cells immunogenic. We have recently shown that PCL treatment of murine tumor cells augmented the presentation of MHC-restricted tumor-associated antigens and enhanced cell-mediated immunity. In cancer patients inoculated with autologous PCL-modified tumor cells, a significant delayed-type hypersensitivity response was elicited. Since the balance between cell-mediated immunity and humoral immunity is reciprocally controlled by immunoregulatory cytokines, we have examined the proliferative response and cytokine secretion pattern in cultures of human peripheral blood mononuclear cells (PBMC) stimulated by autologous PCL-modified and unmodified tumor cells. These tumor cells were obtained from freshly resected tumor tissue of 16 patients with colon (8), lung (4) and renal (4) carcinomas. The results demonstrated that PCL-modified tumor cells promoted an increase in PBMC proliferation in 5 out of 8 (63%), 1 out of 4 (25%) and 4 out of 4 (100%) colon, lung and renal cell carcinomas. Fourteen of the above cultures were also analyzed for the secretion of interleukin-10 and interferon-γ. Overall, a substantial decrease in IL-10 secretion was detected in 9 out of 14 (64%) cultures while a reciprocal increase in interferon-γ secretion was noted in 8 out of 14 (57%) cultures. Our results confirmed that PCL-modified human tumor cells of different etiologies can modulate the pattern of cytokines released from stimulated autologous lymphocytes. Such a procedure could prove valuable in the production of autologous tumor vaccines. Received: 8 January 1998 / Accepted: 9 April 1998