Peroxicretion: a novel secretion pathway in the eukaryotic cell.

BACKGROUND: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. RESULTS: Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. CONCLUSION: Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

Qualitative T-helper responses to multiple viral antigens correlate with vaccine-induced immunity to simian/human immunodeficiency virus infection.

Evidence is accumulating that CD4(+) T-helper (Th) responses play a critical role in facilitating effector responses which are capable of controlling and even preventing human immunodeficiency virus (HIV) infection. The present work was undertaken to determine whether immunization with multiple antigens influenced individual Th responses and increased protection relative to a single antigen. Rhesus macaques were primed with DNA and boosted (immune-stimulating complex-formulated protein) with a combination of regulatory and structural antigens (Tat-Env-Gag) or with Tat alone. Immunization with combined antigens reduced the magnitude of the responses to Tat compared to the single-antigen immunization. Interestingly, the Th immune responses to the individual antigens were noticeably different. To determine whether the qualitative differences in vaccine-induced Th responses correlated with vaccine efficacy, animals were challenged intravenously with simian/human immunodeficiency virus (strain SHIV(89.6p)) 2 months following the final immunization. Animals that developed combined Th1- and Th2-like responses to Gag and Th2 dominant Env-specific responses were protected from disease progression. Interestingly, one animal that was completely protected from infection had the strongest IFN-gamma and interleukin-2 (IL-2) responses prior to challenge, in addition to very strong IL-4 responses to Gag and Env. In contrast, animals with only a marked vaccine-induced Tat-specific Th2 response (no IFN-gamma) were not protected from infection or disease. These data support the rationale that effective HIV vaccine-induced immunity requires a combination of potent Th1- and Th2-like responses best directed to multiple antigens.

Specific passage of simian immunodeficiency virus from end-stage disease results in accelerated progression to AIDS in rhesus macaques.

To determine whether passage of late-stage variants of simian immunodeficiency virus (SIV) would lead to a more virulent infection and rapid disease progression, a study was designed to examine the effects of selective transmission of SIV from late-stage cases of AIDS in Macaca mulatta. In a uniform group of 10 age-matched animals from the same genetic breeding stock infected with SIV(B670), it took 7 months before one of the ten animals developed AIDS. Passage of virus taken from this animal immediately prior to death resulted in death of the recipient due to AIDS within 4 months. Again, subsequent passage of virus taken late in disease resulted in an accelerated disease course, with AIDS developing within 2.5 and 1.8 months in two recipients. The fourth passage of virus taken late in disease from the most rapid progressor (1.8 months) resulted in AIDS developing in this recipient within 1 month of infection. During each consecutive passage in vivo, the loss of memory T cells became more acute. Evidence that the virus became more virulent with selective passage of late-stage variants was provided by the markedly increased levels of both plasma antigen and viral RNA. Subsequent in vivo passage from end-stage AIDS selected for a strain of SIV capable of causing the acute development of AIDS as rapidly as 1 month post-infection. The pathology of acute AIDS in these cases closely resembled that seen after a chronic disease course.