M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.     

Artificial antigen-presenting cells as a tool to exploit the immune 'synapse'

Recent progress in molecular medicine has provided important tools to identify antigen-specific T cells. In most cases, the approach is based on oligomeric combinations of recombinant major histocompatibility complex-peptide complexes fixed to various rigid supports available for binding by the T-cell receptor. These tools have greatly increased our insight into mechanisms of immune responses mediated by CD8+ T cells. Examples of the diverse fields of application for this technology include immunization, viral infections and oral tolerance induction.     

Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses

Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.     

Lactate adversely affects the in vitro formation of endothelial cell tubular structures through the action of TGF-beta1

When lactate accumulation in a tumor microenvironment reaches an average concentration of 10-20 mM, it tends to reflect a high degree of malignancy. However, the hypothesis that tumor-derived lactate has a number of partially adverse biological effects on malignant and tumor-associated host cells requires further evidence. The present study attempted to evaluate the impact of lactate on the process of angiogenesis, in particular on the formation of tubular structures. The endothelial cell (EC) network in desmoplastic breast tumors is primarily located in areas of reactive fibroblastic stroma. We employed a fibroblast-endothelial cell co-culture model as in vitro angiogenesis system normally producing florid in vitro tubule formation to analyze this situation. In contrast to previous studies, we found that lactate significantly reduces EC network formation in a dose-dependent manner as quantified by semi-automated morphometric analyses following immunohistochemical staining. The decrease in CD31-positive tubular structures and the number of intersections was independent of VEGF supplementation and became more pronounced in the presence of protons. The number of cells, primarily of the fibroblast population, was reduced but cell loss could not be attributed to a decrease in proliferative activity or pronounced apoptotic cell death. Treatment with 10 mM lactate was accompanied by enhanced mRNA expression and release of TGF-beta1, which also shows anti-angiogenic activity in the model. Both TGF-beta1 and lactate induced myofibroblastic differentiation adjacent to the EC tubular structures. The lactate response on the EC network was diminished by TGF-beta1 neutralization, indicating a causal relationship between lactate and TGF-beta1 in the finely tuned processes of vessel formation and maturation which may also occur in vivo within tumor tissue.     

Hormone responsiveness of plasma membrane-bound enzymes in normal and regenerating rat liver.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.     

Heat shock protein 70-reactivity is associated with increased cell surface density of CD94/CD56 on primary natural killer cells

Previously we described an involvement of the C-type lectin receptor CD94 and the neuronal adhesion molecule CD56 in the interaction of natural killer (NK) cells with Hsp70-protein and Hsp70-peptide TKD. Therefore, differences in the cell surface density of these NK cell-specific markers were investigated comparatively in CD94-sorted, primary NK cells and in established NK cell lines NK-92, NKL, and YT after TKD stimulation. Initially, all NK cell types were positive for CD94; the CD56 expression varied. After stimulation with TKD, the mean fluorescence intensity (mfi) of CD94 and CD56 was upregulated selectively in primary NK cells but not in NK cell lines. Other cell surface markers including natural cytotoxicity receptors remained unaffected in all cell types. CD3-enriched T cells neither expressing CD94 nor CD56 served as a negative control. High receptor densities of CD94/CD56 were associated with an increased cytolytic response against Hsp70 membrane-positive tumor target cells. The major histocompatibility complex (MHC) class I-negative, Hsp70-positive target cell line K562 was efficiently lysed by primary NK cells and to a lower extent by NK lines NK-92 and NKL. YT and CD3-positive T cells were unable to kill K562 cells. MHC class-I and Hsp70-positive, Cx + tumor target cells were efficiently lysed only by CD94-sorted, TKD-stimulated NK cells with high CD94/CD56 mfi values. Hsp70-specificity was demonstrated by antibody blocking assays, comparative phenotyping of the tumor target cells, and by correlating the amount of membrane-bound Hsp70 with the sensitivity to lysis. Remarkably, a 14-mer peptide (LKD), exhibiting only 1 amino acid exchange at position 1 (T to L), neither stimulated Hsp70-reactivity nor resulted in an upregulated CD94 expression on primary NK cells. Taken together our findings indicate that an MHC class I-independent, Hsp70 reactivity could be associated with elevated cell surface densities of CD94 and CD56 after TKD stimulation.