On T-cell dynamics and the hyperactivation theory of AIDS pathogenesis

In an earlier article the author proposed a model of T cell dynamics in which rising activation rates produced falling T-cell counts. Here I show that, in a model of this type, apoptosis and proliferation must nearly balance. Hence, small perturbations of these processes could have large consequences, a fact that may be relevant to AIDS pathogenesis. In addition, I show the model can reproduce all decay rates found by McLean and Michie for cells with radiation-induced chromosome damage, provided that stable damage can proliferate. Memory-cell 'amnesia' is not required. T-cell lifetimes come out much shorter than previously estimated.     

The vascular-associated lymphoid tissue: a new site of local immunity

Recent data suggest that atherosclerosis might be a systemic (auto)immune reaction against heat shock protein 60, first occurring at notorious local predilection sites, i.e. the intima at arterial branching points. The local infiltration of mononuclear cells, mainly macrophage-derived foam cells, T cells and smooth-muscle cells in atheromatous plaques, have long been described. During the past few years, research has been concentrated on the early stages in the development of atherosclerosis, and on healthy arteries from young individuals unaffected by arterial disease. In this review, we summarize data characterizing pre-existing mononuclear cell infiltrations in healthy arteries from children and teenagers. These arterial accumulations at regions known to be predilection sites for the later development of atherosclerosis consist mostly of activated T cells, macrophages and dendritic cells, with only a few mast cells and virtually no B or natural killer cells. In analogy to the mucosa-associated lymphoid tissue, we termed these accumulations 'vascular-associated lymphoid tissue', and assumed a similar function as a local immunosurveillance system, monitoring the bloodstream for potentially harmful endogenous or exogenous antigens. In addition to the remarkable accumulation of mononuclear cells, the vascular-associated lymphoid tissue regions are characterized by a typical distribution of extracellular matrix proteins: collagen type I, collagen type III, fibronectin and tenascin are expressed preferentially in the vascular-associated lymphoid tissue region, whereas collagen type IV, collagen type V, collagen type VI and laminin show a homogenous distribution throughout all regions of the intima. Vascular adhesion molecules type 1, intercellular adhesion molecules type 1 and P-selectin are already present on the healthy endothelial cells of young children. Interactions between adhesion molecules, extracellular matrix components and cellular elements of the vascular-associated lymphoid tissue may provide the basis for the cellular accumulations in the vascular-associated lymphoid tissue regions and the possible development of atherosclerotic lesions later in life.     

Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.     

The innate immune response differs in primary and secondary Salmonella infection

This study examines innate immunity to oral Salmonella during primary infection and after secondary challenge of immune mice. Splenic NK and NKT cells plummeted early after primary infection, while neutrophils and macrophages (Mphi) increased 10- and 3-fold, respectively. In contrast, immune animals had only a modest reduction in NK cells, no loss of NKT cells, and a slight increase in phagocytes following secondary challenge. During primary infection, the dominant sources of IFN-gamma were, unexpectedly, neutrophils and Mphi, the former having intracellular stores of IFN-gamma that were released during infection. IFN-gamma-producing phagocytes greatly outnumbered IFN-gamma-producing NK cells, NKT cells, and T cells during the primary response. TNF-alpha production was also dominated by neutrophils and Mphi, which vastly outnumbered NKT cells producing this cytokine. Neither T cells nor NK cells produced TNF-alpha early during primary infection. The TNF-alpha response was reduced in a secondary response, but remained dominated by neutrophils and Mphi. Moreover, no significant IFN-gamma production by Mphi was associated with the secondary response. Indeed, only NK1.1(+) cells and T cells produced IFN-gamma in these mice. These studies provide a coherent view of innate immunity to oral Salmonella infection, reveal novel sources of IFN-gamma, and demonstrate that immune status influences the nature of the innate response.     

Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta

3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.     

Organic matter transformations and soil fertility in a treed pasture in semiarid NE Brazil

Planted silvo-pastoral systems are formed by sparing selected native trees when land is cleared for pasture establishment, or by planting selected species – often known agroforestry species – into the establishing pasture. Isolated trees within pastures and savannas are often associated with `resource islands', characterized by higher fertility and organic matter levels under the tree canopies. We here examine the processes underlying the differences in fertility and organic matter in a buffel grass (Cenchrus ciliaris L.) pasture that contained two tree species (Ziziphus joazeiro Mart., Spondias tuberosa Arruda Cam.) preserved from the native thorn forest and a planted agroforestry species (Prospois juliflora Swartz D.C). The objective is to distinguish effects of soil variability from those induced by the presence of trees or the planting of pasture. The ¹³C signatures of the original (largely C3) vegetation, the preserved and planted trees, and the planted C4 grass were used to distinguish the provenance of organic matter in the top soil (0–15 cm). This allowed the conclusion that all trees maintained C3 derived C at the original thorn forest level, while lower levels under pasture were due to mineralisation of organic matter. The net rates of forest-derived C loss under pasture varied with soil type amounting to between 25 and 50% in 13 years after pasture establishment. Only on Alfisol, C inputs from the pasture compensated for the C3-C losses. Analysis of organic and inorganic P fractions indicated Z. joazeiro and P. juliflora enriched the soil under their canopy with P, whereas S. tuberosa had no positive effect on fertility. A combination of ANOVA and spatial analysis and mapping was used to show vegetation effects.     

Mutual influences of Pseudomonas aeruginosa and Desulfovibrio desulfuricans on their adhesion to stainless steel

The mutual influences of Pseudomonas aeruginosa PAO1 and Desulfovibrio desulfuricans subsp. desulfuricans (ATCC 29577) on their adhesion to stainless steel were investigated in batch and column experiments. It was found that P. aeruginosa promoted the adhesion of D. desulfuricans under conditions of turbulence, but not under quiescent conditions. The enhancement involved the alignment of most D. desulfuricans along P. aeruginosa cells and was attributed to the additional interaction surface area provided by adhered P. aeruginosa to aligning D. desulfuricans cells. A slightly positive effect of preadhered D. desulfuricans on the adhesion of P. aeruginosa was found. Under condition of laminar flow, substantially better adhesion of D. desulfuricans to confluent P. aeruginosa biofilms than to steel was observed. The mutual influences are discussed in terms of more favorable adhesion energies and the influence of changed hydraulic conditions due to the roughness of P. aeruginosa biofilms.     

Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis

Epithelial cells of the mammary gland possess the inherent capacity to form epithelial monolayers in vitro. This requires coordination of cell migration, cell-cell contact formation, and cell proliferation. Using time-lapse phase contrast videomicroscopy we have observed mammary gland epithelial cells over different time scales. We show the generation of a complete polarized epithelial monolayer in real-time, starting from a few cells. We subsequently concentrated on the early stages of this process by tracking epithelial cells during phases of polarized migration. We performed migration analysis using fractal measures. With this technology the structure of seemingly random processes not accessible to the usual methods of linear analysis can be measured. As a control and proof of principle approach we applied infection of cells with an adenoviral vector, which is used as a gene targeting vector for many applications. Infection markedly influenced the patterns of migratory behavior. We, therefore, believe that time-lapse videomicroscopy in combination with fractal analysis can contribute to differential characterization of distinct cellular migration patterns. This will be useful in situations of long-term alterations in cell culture systems.     

Transforming growth factors beta(1) (TGF-beta(1)) and TGF-beta(2) promote glioma cell migration via Up-regulation of alpha(V)beta(3) integrin expression

The migratory behaviour of malignant gliomas relies on the interaction of integrins with extracellular matrix (ECM) components. Transforming growth factor-beta(1) (TGF-beta(1)) potently stimulates glioma cell motility whereas TGF-beta(2) is known for its immunosuppressive properties. Here, we show that both TGF-beta(1) and TGF-beta(2) promote migration of glioma cells. In parallel, TGF-beta(1) and TGF-beta(2) induce alpha(V) and beta(3) intergrin mRNA expression and enhance cell surface expression of alpha(V)beta(3) integrin. TGF-beta-mediated promotion of migration is abrogated by echistatin, a Arg-Gly-Asp (RGD) peptide antagonist of alpha(V)beta(3) integrin, and by a neutralizing anti-alpha(V)beta(3) integrin antibody. Taken together, we report a novel mechanism by which TGF-beta modulates cell ECM interactions and promotes glioma cell motility.