Neopterin release in human mixed lymphocyte culture: requirement of HLA-DR disparity

Recent evidence suggests that immune responses in vivo as well as in vitro are accompanied by release of neopterin [C. H. Huber, et al. (1983) J. Immunol. 130, 1047]. This investigation aimed to elucidate the genetic control of in vitro neopterin release in family mixed lymphocyte culture (MLC) studies. Neopterin levels and responder cell proliferation were assessed in a total of 92 MLCs established from different intrafamilial combinations. Results indicated a strong association between the magnitude of responder cell proliferation and the neopterin levels induced by stimulation with allogeneic cells. This conclusion was based on three findings: first, almost no neopterin release and proliferation was seen in MLCs established between HLA genotypically identical siblings; secondly, very low proliferation and neopterin levels were observed in MLCs between HLA-DR phenotypically identical family members; and thirdly, high neopterin release and strong cellular proliferation were a feature of MLCs established between individuals differing for at least one HLA-DR antigen. We thus conclude that T cell activation subsequent to recognition of HLA-DR disparity represents an essential prerequisite for induction of neopterin release in human MLC.     

Proliferation arrest in B-Raf mutant melanoma cell lines upon MAPK pathway activation

Due to elaborate control mechanisms, in benign tumors the activation of oncogenes primarily induces senescence, associated with cessation of cellular proliferation; for example, melanocytic nevi expressing mutant B-Raf. These mechanisms include the RB and/or the p53 pathway. The current model of melanomagenesis postulates that progression to immortal melanoma cells requires inactivating aberrations in signaling cascades controlling senescence. Thus, melanoma cells carrying mutant B-Raf should be resistant to mitogen-activated protein kinase (MAPK) pathway-induced senescence. Here, we demonstrate that hyperactivation of the MAPK pathway following activation of an inducible form of oncogenic C-Raf induces a senescence-like proliferation arrest in B-Raf mutant melanoma cells. This Raf-induced senescence is initially strictly dependent on MEK signaling, but seems to be independent of MAPK signaling after prolonged continuance. It is associated with reduced levels of RB phosphorylation and an increase in p21 expression, but is independent of p16(Ink4a) and p53. These data argue against the existence of fundamental changes in melanoma cells completely precluding senescence.     

Increase of MCP-1 (CCL2) in myelin mutant Schwann cells is mediated by MEK-ERK signaling pathway

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation of the Schwann cell-derived chemokine MCP-1 (CCL2) is a pathologically relevant mechanism for macrophage activation in mice heterozygously deficient for the myelin component P0 (P0+/-), we posed the question of the intracellular signaling cascade involved. By using western blot analysis of peripheral nerve lysates the MAP-kinases extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP kinase/ERK kinase 1/2 (MEK1/2) showed an early and constantly increasing activation in P0 mutants. Furthermore, in nerve fibers from the P0+/- mutants, Schwann cell nuclei were much more often positive for phosphorylated ERK1/2 than in nerve fibers from wild type mice. In vitro experiments using the MEK1/2-inhibitor CI-1040 decreased ERK1/2-phosphorylation and MCP-1 expression in a Schwann cell-derived cell line. Finally, systemic application of CI-1040 lead to a decreased ERK1/2-phosphorylation and substantially reduced MCP-1-production in peripheral nerves of P0+/- mutant mice. Our study identifies MEK1/2-ERK1/2 signaling as an important intracellular pathway that connects the Schwann cell mutation with the activation of pathogenetically relevant macrophages in the peripheral nerves. These findings may have important implications for the treatment of inherited peripheral neuropathies in humans.     

Signaling through RAS-RAF-MEK-ERK: from basics to bedside

Aberrant signaling caused by mutations in the RAS-RAF-MEK-ERK pathway and its upstream activators critically contributes to human tumor development. Strategies, which aim at inhibiting hyperactive signaling molecules, appear conceptually straight forward, but their translation into clinical practice has been hampered by many setbacks. Understanding structure, function and regulation of this intracellular pathway as well as its crosstalk with other signaling activities in the cell will be essential to ensure reasonable usage of new therapeutic possibilities. This review provides an understanding of this signaling cascade as revealed by genetic and biochemical approaches and discusses the existing or arising possibilities to interfere with unphysiological activation in cancer. Signaling aberrations and signal transduction therapies will be discussed exemplary for two types of hematological neoplasia, acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). In the future understanding the role of tumor stem cells, both as a source of tumor recurrence and tumor heterogeneity, the signals controlling their fate as well as epigenetic changes in cancer will be the next critical steps to further advance the applicability of these novel therapeutic strategies.     

Cold ischemia contributes to the development of chronic rejection and mitochondrial injury after cardiac transplantation

Chronic rejection (CR) remains an unsolved hurdle for long‐term heart transplant survival. The effect of cold ischemia (CI) on progression of CR and the mechanisms resulting in functional deficit were investigated by studying gene expression, mitochondrial function, and enzymatic activity. Allogeneic (Lew→F344) and syngeneic (Lew→Lew) heart transplantations were performed with or without 10 h of CI. After evaluation of myocardial contraction, hearts were excised at 2, 10, 40, and 60 days for investigation of vasculopathy, gene expression, enzymatic activities, and mitochondrial respiration. Gene expression studies identified a gene cluster coding for subunits of the mitochondrial electron transport chain regulated in response to CI and CR. Myocardial performance, mitochondrial function, and mitochondrial marker enzyme activities declined in all allografts with time after transplantation. These declines were more rapid and severe in CI allografts (CR‐CI) and correlated well with progression of vasculopathy and fibrosis. Mitochondria related gene expression and mitochondrial function are substantially compromised with the progression of CR and show that CI impacts on progression, gene profile, and mitochondrial function of CR. Monitoring mitochondrial function and enzyme activity might allow for earlier detection of CR and cardiac allograft dysfunction.     

Probing structure and function of the raf protein kinase domain with monoclonal antibodies.

Five monoclonal antibodies were generated against the raf kinase domain. All antibodies react with different isozymes of the raf family, as well as Raf proteins from different species, albeit with differential affinities. Epitope mapping showed all five epitopes clustered in the vicinity of the conserved APE sequence. Although thought to be an essential part of the catalytic site, antibody binding to that domain does not affect kinase activity in vitro or the capability to specifically associate with other cellular proteins. Based on a detailed dissection of the epitopes, a comparative analysis of secondary structure predictions indicates a common structural motif in that region, which is highly conserved amongst protein kinases of the serine/threonine as well as the tyrosine class.     

Results of a phase II study on the treatment of hairy cell leukemias with various doses of alpha-2-recombinant interferon

Hairy-cell leukemia has been shown to be extraordinary sensitive to treatment with alpha-interferon. In order to define clinically effective interferon doses associated with minimal toxicity two different dose regimens were applied in this clinical trial: firstly, a conventional dose schedule, and secondly, a biologically defined dose regimen. For dose finding in the latter group, neopterin, a GTP degradation product produced by macrophages under control of interferon, was chosen. Six patients (Group A) received conventional doses of recombinant interferon--alpha-2 (rIFN-alpha-2) 3 X 10(6) U/sqm/daily by the subcutaneous route. Five patients (Group B) were treated with the minimal dose of rIFN-alpha-2 which had previously been shown to induce maximum neopterin levels in urine. Already interferon doses in the range of 3 to 5 X 10(5) U/sqm2/daily administered subcutaneously proved to be sufficient for triggering maximum neopterin excretion in the urine. After six months of interferon treatment all patients were evaluable. At this time both doses regimens proved to be effective in terms of their anti-leukemic activity, but differed significantly in toxicity, which was only seen in Group A patients.     

Comparative study of alloimmune reactions induced by leukocyte and platelet transfusions in humans: characteristic changes of activation markers, gamma interferon, and FcR blocking antibody production

This study was undertaken to define immune responses induced by leukocyte and platelet transfusions. We offer evidence that activation by alloimmunization with intravenously administered "buffy coat" cells results in immune alterations similar to the early rejection episodes following kidney transplantation. Thus, IL-2 and HLA-DR expression increased on PBL 2 and 3 days after antigen exposure and paralleled elevations of IFN-gamma, neopterin, and beta 2 microglobulin levels. No significant changes were detected in the number of T-cell subpopulations. Alloimmunization by purified platelets alone that express only class I MHC antigens failed to induce the above alteration, even after repeated exposure. Repeated alloimmunization with "buffy coat" cells or isolated platelets stimulated a progressive increase up to 100% blocking activity on EA rosette formation. We conclude that the expression of activation markers on PBL and the production of IFN gamma and neopterin reflect the early phase of alloimmune response induced by leukocytes. This type of alloactivation is not a prerequisite for the development of FcR-blocking antibody, which is produced after pure platelet transfusion as well.     

Selective induction of mononuclear phagocytes to produce neopterin by interferons

Interferon-gamma (IFN-gamma) has been shown to be a potent inducer of neopterin secretion by human peripheral blood monocytes/macrophages (1). In this paper, it is shown that other known stimuli of monocytes (e.g., to secrete proteases or to migrate) such as zymosan-activated human serum, lipopolysaccharide, human C3/iC3 and zymosan coated with complement were unable to trigger monocytes/macrophages to release neopterin. Monocytes/macrophages could be stimulated solely by IFN-gamma (25 U/ml) and IFN-alpha at very high concentrations (10,000 U/ml). In the case of human peripheral blood mononuclear cells (PBMNC), basically the same pattern was observed. If however, in the buffer controls PBMNC showed some neopterin release, all stimuli triggered an increase of neopterin secretion: 10,000 U/ml IFN-alpha induced the same amount of secreted neopterin as did 25 U/ml of IFN-gamma. Both caused higher levels of neopterin secretion than ZAS, LPS and C3/iC3. Amongst the supernatants from PBMNC, only those which were obtained from cells activated with IFN-gamma or -alpha stimulated monocytes/macrophages to produce neopterin. Supernatants from lymphocytes activated with zymosan, lipopolysaccharide and interferon did not contain neopterin, nor did the latter induce monocytes/macrophages to generate and secrete neopterin. Antibodies against IFN-gamma inhibited the triggering effect of the supernatants except when generated by IFN-alpha at 10,000 U/ml. These results demonstrate that both interferons, IFN-gamma and IFN-alpha, the latter only at a 400-fold higher concentration, can trigger monocytes/macrophages directly to secrete neopterin. ZAS, LPS and C3/iC3 are weakly effective only on a mixture of lymphocytes and monocytes/macrophages, provided this cell mixture shows already a basic spontaneous neopterin release.(ABSTRACT TRUNCATED AT 250 WORDS)