Effects of monoclonal anti-H-2Ld and anti-H-2Dd alloantibodies in the immunological enhancement of skin grafts and neonatal heart grafts in the mouse

Three anti-H-2Ld and two anti-H-2Dd monoclonal alloantibodies were analyzed for their capacity to enhance skin graft and neonatal heart graft survival. Of two anti-H-2Ld antibodies with the same specificity but with different isotypes, IgG2a antibody 30-5-7S prolonged graft survival in a skin graft combination with an Ld difference, whereas IgM antibodies did not. A second IgG2a antibody, but with a specificity different from 30-5-7S, was ineffective on its own. However, when mixed with 30-5-7S, skin graft survival was augmented as compared with the prolongation by 30-5-7S alone. Enhancement by anti-H-2Ld antibodies was dependent on the extent of the H-2 graft barrier. It was abrogated on extension of the graft barrier to a D-end H-2 difference by using the B10.A----B10.BR combination. Also, anti-Dd antibodies, either alone or in combination with anti-Ld, were ineffective in this skin graft combination. By using the same graft combination but the less immunogenic neonatal heart graft model, anti-Ld antibodies were still ineffective, but both anti-Dd antibodies were able to enhance graft survival from 15 to 22 days. When mixed with anti-Ld antibody 30-5-7S, graft survival was augmented further to 30 days. These results indicate that two kinds of enhancing alloantibodies may be distinguished. One category interacts with immunodominant epitopes on H-2 molecules, but their effectiveness may be limited to a particular H-2 difference, because immunodominance may vary from one graft barrier to another. In the second category, antibodies are ineffective on their own but they are able to potentiate the effects of antibodies of the first kind. These allocations are relative, however, because they are dependent on the type of graft examined.     

Induction of nonspecific cytotoxicity by monoclonal anti-T3 antibodies

The effects of monoclonal anti-T3 antibodies on the effector phase of cytotoxic T lymphocytes (CTL) were studied with respect to antigen-specific and antigen-nonspecific lysis of different target cells. Anti-T3 antibodies inhibited the antigen-specific lysis by CTL generated in mixed lymphocyte cultures (MLC), but they concomitantly augmented the nonspecific killing of third-party cells such as the cell lines Daudi, Raji, and K562. This nonspecific cytotoxicity was induced by various anti-T3 antibodies, whereas antibodies reactive with other antigens expressed on the cytotoxic effector cells lacked any such activity. Anti-T3 antibodies induced nonspecific cytotoxicity only when activated T cells, obtained by primary MLC, by repeated restimulation, or after cloning, were used. The antibodies had no effect on unstimulated peripheral T lymphocytes or thymocytes. The inhibition of the antigen-specific lysis and the induction of nonspecific lysis by anti-T3 was dose dependent, and both effects occurred at the same concentration range of anti-T3. F(ab')2 fragments of anti-T3 inhibited the specific lysis but were not able to induce cytotoxic activity, indicating that this induction is an Fc-dependent process. When different target cells were tested, only Fc receptor-positive cells were susceptible for this nonspecific cytotoxicity. Thus, anti-T3 antibodies have a dual effect on effector CTL: they inhibit antigen-specific lysis and concomitantly induce nonspecific lysis in an Fc-dependent way.     

Effect of protease inhibitors on human monocyte IgG Fc receptor II. Evidence that serine esterase activity is essential for Fc gamma RII-mediated binding

We have shown previously that certain proteases can modulate the affinity of human Fc gamma RII for IgG. To study whether proteolytic events not only increase FcR affinity, but are essential for Fc gamma R functioning, we evaluated the effect of different protease inhibitors on binding mediated by two classes of human monocyte IgG FcR. These R, Fc gamma RI and Fc gamma RII, can be analyzed selectively in rosetting assays by employing E sensitized by either human IgG or mouse IgG1. Rosetting by both classes of R was inhibited profoundly by incubation of monocytes with different types of serine protease inhibitors such as diisopropylfluorophosphate, PMSF, or N alpha-tosyl-L-lysyl-chloromethylketone. The type II Fc gamma R was much more sensitive to inhibition than Fc gamma RI. We, therefore, studied these effects in more detail by using cell line K562, which expresses only Fc gamma RII. PMSF, diisopropylfluorophosphate, and N alpha-tosyl-L-lysyl-chloromethylketone were, again, inhibiting Fc gamma RII-mediated binding dose-dependently, whereas several inhibitors of metal, aspartic, or thiol proteases proved ineffective. Furthermore, Fc gamma RII-mediated rosetting on both cell types was profoundly inhibited by the addition of different small synthetic substrates of serine esterases. In an attempt to discriminate whether the proteolytic event is an intra- or extracellular process, macromolecular antiproteases such as soybean or ovomucoid trypsin inhibitor or alpha 1-antiprotease were tested. Fc gamma RII-mediated binding by K562 cells was not susceptible to macromolecular antiproteases, in contrast to monocytes. In the presence of drugs which interfere both with receptor recycling and intracellular traffic between endosomal compartments (e.g., primaquine or monensin), the effects of inhibitors were largely abrogated. This showed that endocytosis of inhibitors might be essential, indicating the proteolytic event to be intracellular. Our findings suggest that human monocyte Fc gamma RII-mediated functioning is dependent upon the action of one or more serine proteases.     

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins,a family of putative kinase regulators

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.     

Male-specific cell migration into the developing gonad

Background: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing β-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for β-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad.Results: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5–16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells.Conclusions: Migration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.     

Mutations at the Arabidopsis CHM locus promote rearrangements of the mitochondrial genome.

Nuclear recessive mutations at the chloroplast mutator (CHM) locus of Arabidopsis produce a variegated phenotype that is inherited in a non-Mendelian fashion. Molecular analysis of the cytoplasmic genomes of variegated plants from two independent chm mutant lines, using specific chloroplast and mitochondrial probes, showed that the chm mutations reproducibly induce the appearance of specific new restriction fragments in the mitochondrial genome. The presence of these restriction fragments cosegregated with the variegated phenotype in the progeny of crosses between mutant and wild-type plants. Sequence analysis of one of the new restriction fragments found in the variegated plants suggested that it was the product of a rearrangement event involving regions of the mitochondrial genome. Thus, it appears that the CHM locus may encode a protein involved in the control of specific mitochondrial DNA reorganization events.     

Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.