Jens Gerwien

Reduced functional deficits, neuroinflammation, and secondary tissue damage after treatment of stroke by nonerythropoietic erythropoietin derivatives

Carbamylerythropoietin (CEPO) does not bind to the classical erythropoietin (EPO) receptor. Nevertheless, similarly to EPO, CEPO promotes neuroprotection on the histologic level in short-term stroke models. In the present study, we investigated whether CEPO and other nonerythropoietic EPO analogs could enhance functional recovery and promote long-term histologic protection after experimental focal cerebral ischemia. Rats were treated with the compounds after focal cerebral ischemia. Animals survived 1, 7, or 60 days and underwent behavioral testing (sensorimotor and foot-fault tests). Brain sections were stained and analyzed for Iba-1, myeloperoxidase, Tau-1, CD68 (ED1), glial fibrillary acidic protein (GFAP), Fluoro-Jade B staining, and overall infarct volumes. Treatment with CEPO reduced perifocal microglial activation (P<0.05), polymorphomonuclear cell infiltration (P<0.05), and white matter damage (P<0.01) at 1 day after occlusion. Carbamylerythropoietin-treated rats showed better functional recovery relative to vehicle-treated animals as assessed 1, 7, 14, 28, and 50 days after stroke. Both GFAP and CD68 were decreased within the ipsilateral thalamus of CEPO-treated animals 60 days postoperatively (P<0.01 and P<0.05, respectively). Furthermore, behavioral analysis showed efficacy of CEPO treatment even if administered 24 h after the stroke. Other nonerythropoietic derivatives such as carbamylated darbepoetin alfa and the mutant EPO-S100E were also found to protect against ischemic damage and to improve postischemic neurologic function. In conclusion, these results show that postischemic intravenous treatment with nonerythropoietic EPO derivatives leads to improved functional recovery, which may be linked to their long-term effects against neuroinflammation and secondary tissue damage.

The Sonic Hedgehog Pathway Mediates Carbamylated Erythropoietin-enhanced Proliferation and Differentiation of Adult Neural Progenitor Cells

Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation.

The nonerythropoietic asialoerythropoietin protects against neonatal hypoxia-ischemia as potently as erythropoietin

Recently, erythropoietin (EPO) and the nonerythropoietic derivative asialoEPO have been linked to tissue protection in the nervous system. In this study, we tested their effects in a model of neonatal hypoxia‐ischemia (HI) in 7‐day‐old rats (unilateral carotid ligation and exposure to 7.7% O2 for 50 min). EPO (10 U/g body weight = 80 ng/g; n = 24), asialoEPO (80 ng/g; n = 23) or vehicle (phosphate‐buffered saline with 0.1% human serum albumin; n = 24) was injected intraperitoneally 4 h before HI. Both drugs were protective, as judged by measuring the infarct volumes, neuropathological score and gross morphological score. The infarct volumes were significantly reduced by both EPO (52%) and asialoEPO (55%) treatment, even though the plasma levels of asialoEPO had dropped below the detection limit (1 pm) at the onset of HI, while those of EPO were in the nanomolar range. Thus, a brief trigger by asialoEPO before the insult appears to be sufficient for protection. Proteomics analysis after asialoEPO treatment alone (no HI) revealed at least one differentially up‐regulated protein, synaptosome‐associated protein of 25 kDa (SNAP‐25). Activation (phosphorylation) of ERK was significantly reduced in asialoEPO‐treated animals after HI. EPO and the nonerythropoietic asialoEPO both provided significant and equal neuroprotection when administered 4 h prior to HI in 7‐day‐old rats. The protection might be related to reduced ERK activation and up‐regulation of SNAP‐25.

MHC class II ligation induces CD58 (LFA-3)-mediated adhesion in human T cells

MHC class II positive T cells found in areas of inflammation are believed to play an important pathogenetic role in autoimmunity. In experimental models , class II molecules have been shown to regulate adhesion between human T cells. It is, however, not known in detail how class II molecules are functionally linked to adhesion molecules. Some data suggest that β2 integrin (CD11a/CD18) molecules play a role in class-II-induced homotypic adhesion in B cells, monocytes, and virus-transformed or neoplastic cell lines. We have previously obtained evidence that adhesion molecules other than β2 integrins might play a role in class-II-mediated adhesion in T cells. To study further class-II-mediated adhesion in T cells, we have taken advantage of (allo)antigen-specific β2-integrin-negative, CD4-positive T cell lines obtained from a leukocyte adhesion deficiency patient. We show that class II ligation induces homotypic adhesion in both β2-integrin-positive and negative, CD4-positive T cell lines. Anti-CD18 monoclonal antibody (mAb) weakly inhibited the adhesion response in β2-integrin-positive T cells and had no effect on β2-integrin-negative T cells. In contrast, an anti-CD58 (LFA-3) mAb almost completely inhibited the adhesion response in β2-integrin-negative T cells. Antibodies against the CD58 ligand, CD2, partly inhibited the adhesion response in β2-integrin-negative T cells whereas antibodies against other adhesion molecules did not. The adhesion response in β2-integrin-positive T cells was partly inhibited by antibodies against CD58 and CD2. Taken together, these data provide the first evidence that CD58 molecules are involved in class-II-induced homotypic adhesion between T cells.