Activation or suppression of bactericidal activity of macrophages during a graft-versus-host reaction againstI-A andI-J-region differences,respectively

Systemic graft-versus-host reactions (GVHR) were induced in F₁ heterozygous mice by injecting 10⁸ parental lymphocytes. The Anti-Thy 1.2-sensitive, T-cell mediated activation of macrophages was assessed by their increased capacity to destroy a facultative intracellular bacteriumListeria monocytogenes. The difference inMHC regions causing a GVHR that induced high levels of macrophage activation mapped toI-A. In contrast, differences atK orD, in any of the otherH-2 subregions or in the non-H-2 background, includingMls alone or in combination, did not induce a GVHR leading to macrophage activation, unless these differences were combined with a difference atI-A. The numbers of parental cells needed to activate macrophages via a GVHR caused byI-A vs. non-I-A differences, varied at least 30- to 100-fold. When parental cells were injected into F₁ offspring of parents differing atI-J, growth ofListeria was enhanced significantly; this negative effect on macrophages was not seen when parental combinations differing atI-A alone were compared with those differing atI-A plusI-J orI-J plus otherH-2 regions.     

Activation or suppression of bactericidal activity of macrophages during a graft-versus-host reaction against I-A and I-J-region differences, respectively

Systemic graft-versus-host reactions (GVHR) were induced in F1 heterozygous mice by injecting 10(8) parental lymphocytes. The Anti-Thy 1.2-sensitive, T-cell mediated activation of macrophages was assessed by their increased capacity to destroy a facultative intracellular bacterium Listeria monocytogenes. The difference in MHC regions causing a GVHR that induced high levels of macrophage activation mapped to I-A. In contrast, differences at K or D, in any of the other H-2 subregions or in the non-H-2 background, including Mls alone or in combination, did not induce a GVHR leading to macrophage activation, unless these differences were combined with a difference at I-A. The numbers of parental cells needed to activate macrophages via a GVHR caused by I-A vs. non-I-A differences, varied at least 30- to 100-fold. When parental cells were injected into F1 offspring of parents differing at I-J, growth of Listeria was enhanced significantly; this negative effect on macrophages was not seen when parental combinations differing at I-A alone were compared with those differing at I-A plus I-J or I-J plus other H-2 regions.     

Antiviral immune responses in CTLA4 transgenic mice.

The role of B7 binding CD28 in the regulation of T- and B-cell responses against viral antigens was assessed in transgenic mice expressing soluble CTLA4-Hgamma1 (CTLA4-Ig tg mice) that blocks B7-CD28 interactions. The results indicate that transgenic soluble CTLA4 does not significantly alter cytotoxic T-cell responses against replicating lymphocytic choriomeningitis virus (LCMV) or vaccinia virus but drastically impairs the induction of cytotoxic T-cell responses against abortively replicating vesicular stomatitis virus (VSV). While the T-independent neutralizing immunoglobulin M (IgM) responses were within normal ranges, the switch to IgG was reduced 4- to 16-fold after immunization with abortively replicating VSV and more than 30-fold after immunization with an inert VSV glycoprotein antigen in transgenic mice. IgG antibody responses to LCMV, as detected by enzyme-linked immunosorbent assay and by neutralizing action, were reduced about 3- to 20-fold and more than 50-fold, respectively. These results suggest that responses in CTLA4-Ig tg mice are mounted according to their independence of T help. While immune responses to nonreplicating or poorly replicating antigens are in general most dependent on T help and B7-CD28 interactions, they are most impaired in CTLA4-Ig tg mice. The results of the present experiments also indicate that highly replicating viruses, because of greater quantities of available antigens and by inducing as-yet-undefined factors and/or cell surface changes, are capable of compensating for the decrease in T help caused by the blocking effects of soluble CTLA4.     

In vivo selection of neutralization-resistant virus variants but no evidence of B cell tolerance in lymphocytic choriomeningitis virus carrier mice expressing a transgenic virus-neutralizing antibody

B cell tolerance is maintained by active deletion and functional anergy of self-reactive B cells depending on the time, amount, and site of the self-antigen expression. To study B cell tolerance toward a transplacentally transmitted viral Ag, we crossed transgenic mice expressing the mu heavy and the kappa light chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing mAb KL25 (HL25-transgenic mice) with persistently infected LCMV carrier mice. Although HL25-transgenic LCMV carrier mice exhibited the same high virus titers as nontransgenic LCMV carrier mice, no evidence for B cell tolerance was found. In contrast, enhanced LCMV-neutralizing Ab titers were measured that, however, did not clear the virus. Instead, LCMV isolates from different tissues turned out to be neutralization resistant Ab escape variants expressing different substitutions of amino acid Asn119 of the LCMV-glycoprotein 1 that displays the neutralizing B cell epitope. Virus variants with the same mutations were also selected in vitro in the presence of the transgenic mAb KL25 confirming that substitutions of Asn119 have been selected by LCMV-neutralizing Abs. Thus, despite abundant expression of viral neo-self-antigen in HL25-transgenic LCMV carrier mice, transgenic B cells expressing LCMV-neutralizing Abs were rather stimulated than tolerized and neutralization resistant Ab escape variants were selected in vivo.     

Alleviation of water and osmotic stress-induced changes in nitrogen metabolizing enzymes in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. cultivars by potassium

Present communication reports laboratory and pot experiments conducted to study the influence of water and osmotic stress on nitrogen uptake and metabolism in two wheat (Triticum aestivum L) cultivars with and without potassium supplementation. Polyethylene glycol 6000-induced osmotic stress/restricted irrigation caused a considerable decline in the activity of nitrate reductase, glutamate synthase, alanine and aspartate aminotransferases, and glutamate dehydrogenase. Potassium considerably improved nitrogen metabolism under normal water supply conditions and also resulted in amelioration of the negative impact of water and osmotic stresses indicating that potassium supplementation can be used as a potential tool for enhancing the nitrogen use efficiency in wheat for exploiting its genetic potential.     

Protective antiviral cytotoxic T cell memory is most efficiently maintained by restimulation via dendritic cells.

Dendritic cells (DC) play a key role in the initiation of T cell-mediated immune responses and may therefore be successfully used in antiviral and antitumor vaccination strategies. Because both strength and duration of an immune response determines the outcome of a vaccination protocol, we evaluated the life span of DC-induced antiviral CTL memory against systemic and peripheral challenge infections with lymphocytic choriomeningitis virus (LCMV). We found that expansion and activation of CTL by DC was transient. Protection against systemic LCMV infection after DC immunization was relatively long-lived (>60 days), whereas complete protection against peripheral infection via intracerebral infection or infection into the footpad with LCMV, where rapid recruitment of effector T cells to the site of infection and elimination of viral pathogen plays a major role, was short-lived (<30 days). Protective immunity was most efficiently restored by administration of antigenic peptides via DC, rather than in combination with IFA or in liposomes. These results suggest that Ag presentation by DC may be crucial for both initiation and maintenance of protective CTL-mediated immunity against viruses infecting solid organs or against peripheral mesenchymal or epithelial tumors.     

The CXC Chemokine-degrading Protease SpyCep of Streptococcus pyogenes Promotes Its Uptake into Endothelial Cells

Streptococcus pyogenes expresses the LPXTG motif-containing cell envelope serine protease SpyCep (also called ScpC, PrtS) that degrades and inactivates the major chemoattractant interleukin 8 (IL-8), thereby impairing host neutrophil recruitment. In this study, we identified a novel function of SpyCep: the ability to mediate uptake into primary human endothelial cells. SpyCep triggered its uptake into endothelial cells but not into human epithelial cells originating from pharynx or lung, indicating an endothelial cell-specific uptake mechanism. SpyCep mediated cellular invasion by an endosomal/lysosomal pathway distinct from the caveolae-mediated invasion pathway of S. pyogenes. Recombinant expression and purification of proteolytically active SpyCep and a series of subfragments allowed functional dissection of the domains responsible for endothelial cell invasion and IL-8 degradation. The N-terminal PR domain was sufficient to mediate endothelial cell invasion, whereas for IL-8-degrading activity, the protease domain and the flanking A domain were required. A polyclonal rabbit serum raised against the recombinant protease efficiently blocked the invasion-mediating activity of SpyCep but not its proteolytic function, further indicating that SpyCep-mediated internalization is independent from its enzymatic activity. SpyCep may thus specifically mediate its own uptake as secreted protein into human endothelial cells.