A Role for Perforin in Downregulating T-Cell Responses during Chronic Viral Infection

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf −/−) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf −/− mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral “exhaustion” of activated CD8 T cells occurred less efficiently in perf −/− mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf −/− mice; ~50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.     

Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease.

Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.     

Gastrin inhibits motility,decreases cell death levels and increases proliferation in human glioblastoma cell lines

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 373–382, 1998     

Granal stacking of thylakoid membranes in higher plant chloroplasts: the physicochemical forces at work and the functional consequences that ensue.

The formation of grana in chloroplasts of higher plants is examined in terms of the subtle interplay of physicochemical forces of attraction and repulsion. The attractive forces between two adjacent membranes comprise (1) van der Waals attraction that depends on the abundance and type of atoms in each membrane, on the distance between the membranes and on the dielectric constant, (2) depletion attraction that generates local order by granal stacking at the expense of greater disorder (i.e. entropy) in the stroma, and (3) an electrostatic attraction of opposite charges located on adjacent membranes. The repulsive forces comprise (1) electrostatic repulsion due to the net negative charge on the outer surface of thylakoid membranes, (2) hydration repulsion that operates at small separations between thylakoid membranes due to layers of bound water molecules, and (3) steric hindrance due to bulky protrusions of Photosystem I (PSI) and ATP synthase into the stroma. In addition, specific interactions may occur, but they await experimental demonstration. Although grana are not essential for photosynthesis, they are ubiquitous in higher plants. Grana may have been selected during evolution for the functional advantages that they confer on higher plants. The functional consequences of grana stacking include (1) enhancement of light capture through a vastly increased area-to-volume ratio and connectivity of several PSIIs with large functional antenna size, (2) the ability to control the lateral separation of PSI from PSII and, therefore, the balanced distribution of excitation energy between two photosystems working in series, (3) the reversible fine-tuning of energy distribution between the photosystems by State 1-State 2 transitions, (4) the ability to regulate light-harvesting via controlled thermal dissipation of excess excitation energy, detected as non-photochemical quenching, (5) dynamic flexibility in the light reactions mediated by a granal structure in response to regulation by a trans-thylakoid pH gradient, (6) delaying the premature degradation of D1 and D2 reaction-centre protein(s) in PSII by harbouring photoinactived PSIIs in appressed granal domains, (7) enhancement of the rate of non-cyclic synthesis of adenosine triphosphate (ATP) as well as the regulation of non-cyclic vs. cyclic ATP synthesis, and (8) the potential increase of photosynthetic capacity for a given composition of chloroplast constituents in full sunlight, concomitantly with enhancement of photochemical efficiency in canopy shade. Hence chloroplast ultrastructure and function are intimately intertwined.     

The ArfGAP Glo3 is required for the generation of COPI vesicles

The small GTPase Arf and coatomer (COPI) are required for the generation of retrograde transport vesicles. Arf activity is regulated by guanine exchange factors (ArfGEF) and GTPase-activating proteins (ArfGAPs). The ArfGAPs Gcs1 and Glo3 provide essential overlapping function for retrograde vesicular transport from the Golgi to the endoplasmic reticulum. We have identified Glo3 as a component of COPI vesicles. Furthermore, we find that a mutant version of the Glo3 protein exerts a negative effect on retrograde transport, even in the presence of the ArfGAP Gcs1. Finally, we present evidence supporting a role for ArfGAP protein in the generation of COPI retrograde transport vesicles.