A strong neuroprotective effect of the autonomous C-terminal peptide of IGF-1 Ec (MGF) in brain ischemia.

The ischemic stroke is the third leading cause of death in developed countries. The C-terminal peptide of mechano-growth factor (MGF), an alternatively spliced variant of insulin-like growth factor 1 (IGF-1), was found to function independently from the rest of the molecule and showed a neuroprotective effect in vivo and in vitro. In vivo, in a gerbil model of transient brain ischemia, treatment with the synthetic MGF C-terminal peptide provided very significant protection to the vulnerable neurons. In the same model, ischemia evoked increased expression of endogenous MGF in the ischemia-resistant hippocampal neurons, suggesting that the endogenous MGF might have an important neuroprotective function. In an in vitro organotypic hippocampal culture model of neurodegeneration, the synthetic peptide was as potent as the full-length IGF-1 while its effect lasted significantly longer than that of recombinant IGF-1. While two peptides showed an additive effect, the neuroprotective action of the C-terminal MGF was independent from the IGF-1 receptor, indicating a new mode of action for this molecule. Although MGF is known for its regenerative capability in skeletal muscle, our findings demonstrate for the first time a neuroprotective role against ischemia for this specific IGF-1 isoform. Therefore, the C-terminal MGF peptide has a potential to be developed into a therapeutic modality for the prevention of neuronal damage.

Transient cerebral ischemia induces delayed proapoptotic Bad translocation to mitochondria in CA1 sector of hippocampus.

Delayed ischemic brain damage is associated with mitochondrial dysfunction, but the underlying mechanisms are not known in detail. Recent data suggest that the process is associated with multidirectional changes in the activities of various proteins located in mitochondria. Of these, the stress-activated kinase JNK is delay-activated postischemia. We induced 5 min cerebral ischemia in gerbils followed by 3, 24, 48, 72 and 96 h of reperfusion. Here we show the postischemic translocation of proapoptotic protein Bad to mitochondria. Immunoelectron microscopic examination revealed the co-appearance of Bad and Bcl-2 proteins in postischemic mitochondria in ischemia-vulnerable CA1 sector of hippocampus as opposed to the ischemia-resistant DG region. Mitochondrial increase of Bad protein is coincident with a transient decrease of the active, phosphorylated form of prosurvival kinase, Raf-1, under conditions of long reperfusion. The above demonstrated sequence of events is likely to play a role in delayed postischemic nerve cell death.

Opposite reaction of ERK and JNK in ischemia vulnerable and resistant regions of hippocampus: involvement of mitochondria.

Delayed ischemic death of neurones is observed selectively in CA1 region of hippocampus at 3-4 days of reperfusion. Signals generated immediately during and after ischemia are further propagated by a variety of kinases, proteases and phosphatases. Tissue samples from dorsal (vulnerable) and abdominal (resistant) parts of gerbil hippocampi were collected to determine the activation state of key signaling molecules: Akt, Raf-1, JNK, ERK1/2 in the course of reperfusion after 5 min of global cerebral ischemia. Western blot analysis of phosphorylated forms of the kinases revealed persistent activation of JNK, being limited mostly to vulnerable CA1 region. On the contrary, activation of ERK, although observed transiently in both parts, was enhanced for a longer time in the abdominal hippocampus. The levels of the active/phosphorylated Akt and Raf-1 kinases did not change significantly during the recovery period. No significant correlation between postischemic JNK activation and c-Jun phosphorylation or its contribution to AP1-like complex formation was found. In contrast, the amount of active JNK linked with mitochondrial membranes was significantly increased and preceded neuronal death in CA1. In the same period of time the AP1 complex, augmented in CA1 region, did not appear to contain a classical c-Fos protein. These results are consistent with the theory that either long-lasting activation of JNK and/or contrasting ERK and JNK activities in critical time of reperfusion, contribute to selective apoptosis of CA1 neurons. This, in connection with the translocation of activated JNK to mitochondria and time/regional differences in AP1 binding protein complexes can affect final postischemic outcome.

Neuroprotection by cyclosporin A following transient brain ischemia correlates with the inhibition of the early efflux of cytochrome C to cytoplasm.

The efflux of mitochondrial protein cytochrome C to cytoplasm is one of the key events of mitochondrial dysfunction observed in post-ischemic pathology. We investigated the effect of intra-carotid infusion of 5-10 mg/kg of cyclosporin A (CsA) on the neuronal survival in CA1 sector of hippocampus and on the subcellular localization of cytochrome C in the model of 5 min gerbil brain ischemia. To discriminate between the immunosuppressive and the mitochondria protecting component of CsA action, we compared the effect of CsA with one other immunosuppressant FK506. Almost 75% of neurons in ischemia-affected brain area were saved after CsA but not after FK506 treatment. This protective effect was only observed when the drug was infused immediately upon reperfusion. Early CsA treatment was able to block an initial phase of cytochrome C release, occurring transiently at 30 min post-ischemia, an effect never observed after FK506 administration. We assessed the neuroprotective potency of CsA vs. FK506 in rat cortical primary culture treated with compounds that mimic destructive signals induced by brain ischemia. In all cases, neuronal death and cytochrome C release were evidently suppressed by CsA applied not later than 30 min after the initial insult. Thus, early treatment with CsA in vitro and after bolus intra-carotid injection in vivo can save neurons by inhibition of cytochrome C efflux to cytoplasm.

Immature hematopoietic cells display selective requirements for adhesion- and degranulation-promoting adaptor protein in development and homeostatsis.

Adhesion- and degranulation-promoting adaptor protein (ADAP) modulates T cell development and function and promotes TCR signaling. Regulation of ADAP protein expression during thymopoiesis and in development of other hematopoietic lineages has not been explored. Using intracellular staining, we detected ADAP protein in bone marrow lymphocyte precursors. Like its binding partner SH2-containing leukocyte phosphoprotein of 76 kDa, ADAP is dynamically regulated during thymocyte positive selection. ADAP is also found in unconventional thymocytes, including NKT, CD8alphaalpha, and TCRgammadelta T cells. In peripheral T cells, ADAP is up-regulated after TCR stimulation and with acquisition of memory status. Although absent in splenic B cells, ADAP is present in pro-B cells, as well as in BM erythrocyte and myeloid progenitors. Studies with radiation chimeras show that ADAP is dispensable for NKT, CD8alphaalpha and TCRgammadelta T cell development, while confirming that ADAP is required for optimal development of conventional TCRalphabeta T cells in the thymus. Interestingly, ADAP is necessary for CD8alphaalpha homeostasis in the small intestinal epithelium, yet is dispensable for optimal reconstitution of splenic B cell populations. Our observations highlight the dynamic regulation of ADAP during T cell maturation and document expression patterns that suggest a possible role for ADAP in development of non-T hematopoietic lineages.

ADAP is dispensable for NK cell development and function.

NK cells are key mediators of the innate immune response and anti-tumor surveillance. Adhesion and degranulation-promoting adapter protein (ADAP, formerly known as SLAP-130 or Fyb) is a hematopoietic-specific adapter that is required for efficient TCR signaling and T cell activation. Herein, we examine a potential role for ADAP in NK development and function. ADAP is expressed in primary NK cells and in IL-2 stimulated lymphokine-activated killers. However, ADAP-deficient mice show no defects in NK development. Further, ADAP is dispensable for key NK functions, including cytotoxicity in response to engagement of activating receptors, cytokine production, conjugate formation and tumor suppression in vivo. These results indicate that, unlike events stimulated by TCR engagement, signaling events engaged by immunoreceptor tyrosine-based activation motif-associated and cytokine receptors on NK cells can occur independently of ADAP.

Hepatocyte growth factor levels in liver and blood, and post-operative liver cell proliferation in patients with benign and malignant liver tumors after partial hepatectomy.

BACKGROUND: The purpose of our study was to investigate hepatocyte proliferation and the expression of hepatocyte growth factor (HGF) in liver tissue and blood from patients with benign and malignant liver tumors after partial hepatectomy. MATERIAL/METHODS: We studied 25 consecutive patients undergoing partial hepatectomy for metastatic colorectal adenocarcinoma (15 cases) and benign liver tumors (10 cases). Immunohistochemical examination for the presence of PCNA and HGF, c-MET/HGF-receptor expression was performed on formalin-fixed samples from: a) sections of resected fragments of liver tissue remote from the tumor; b) tumor tissue; c) remnant liver, 30 min after hepatectomy; d) fine needle aspiration liver biopsy, 7 days after liver resection. Circulating HGF and the level of AFP and GGT as biomarkers for liver cell regeneration were measured in the patients' blood at the same time. RESULTS: The proliferation rate of liver cells was higher in patients with malignant than benign liver tumors. This correlated with increased HGF in blood, but not with the expression of HGF and c-MET/HGF-R in liver tissue. The expression of HGF was detected in specimens from colorectal liver metastases. CONCLUSIONS: The mutual interactions between tumor and other cells may influence the proliferation of hepatocytes throughout the regenerative process in patients with colorectal carcinoma metastases after partial hepatectomy.