Pegfilgrastim in children with severe congenital neutropenia.

Two pediatric patients affected by severe congenital neutropenia (SCN) were treated with 100 mcg/L/dose every 9-12 days within a pilot study (Observatory of the Italian Ministry of Health, Eudract Code 2005-003096-20) on the use of pegfilgrastim in patients with chronic neutropenia. Both children increased their absolute neutrophil count, reduced their infectious load, and improved their quality of life. Serum concentrations of G-CSF observed in pegfilgrastim mirrored those seen in filgrastim. These data suggest that pegfilgrastim may be beneficial in SCN patients with an exposure of hematopoietic cells to G-CSF similar to that on filgrastim.

Etanercept as a salvage treatment for refractory aplastic anemia.

About 10-15% of patients with acquired aplastic anemia (AAA) have resistant/recurrent disease not eligible for standard treatment like hematopoietic stem cell transplantation and/or combined immunosuppression. We report a 17-year-old male with an 11 years history of AAA who, after two courses of immunosuppression, was red cell transfusion-dependent, severely thrombocytopenic, refractory to platelet transfusion, had iron overload and post-transfusion HCV infection. This patient achieved transfusion independence from platelets and normalized Hb after treatment with the anti-TNF agent Etanercept. Over a 12 months follow-up he experienced only transient increase of liver transaminases.

Recombinant human TNF-binding protein-1 (rhTBP-1) treatment delays both symptoms progression and motor neuron loss in the wobbler mouse.

TNF-alpha overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-alpha in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-alpha and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-alpha was further confirmed by the protective effect of treatment with rhTNF-alpha binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-alpha and TNFR1 levels. rhTBP-1 might possibly bind TNF-alpha and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.

Regulation of redox-sensitive exofacial protein thiols in CHO cells.

Thiols affect a variety of cell functions, an effect known as redox regulation, largely attributed to modification of transcription factors and intracellular signaling mechanisms. Since exofacial protein thiols are more exposed to redox-acting molecules used in cell culture and may represent sensors of the redox state of the environment, we investigated their susceptibility to redox regulation. Exofacial protein thiols were measured using cell-impermeable Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB]. For quantification, we also set up an ELISA assay based on the cell-impermeable biotinylated SH reagent, N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM). Exposure of CHO cells to H(2)O(2) induces oxidation of surface thiols at concentrations not affecting intracellular GSH. Depletion (50%) of GSH decreases surface thiols by 88%. Surface thiols are also highly sensitive to thiol antioxidants, since exposure to 5 mM N-acetyl-L-cysteine (NAC) for 2 h augmented their expression without increasing GSH levels. Using BIAM labeling and two-dimensional gel electrophoresis, we show that this increase in surface thiols is due to the reduction of specific membrane proteins. Peptide mass fingerprinting by MALDI mass spectrometry allowed us to identify two of these proteins as Erp57 and vimentin.

Redox regulation of cyclophilin A by glutathionylation.

Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo. Using MALDI-MS we identified Cys52 and Cys62 as targets of glutathionylation in T lymphocytes, and, using bioinformatic tools, we defined the reasons for the susceptibility of these residues to the modification. In addition, we found by circular dichroism spectroscopy that glutathionylation has an important impact on the secondary structure of CypA. Finally, we suggest that glutathionylation of CypA may have biological implications and that CypA may play a key role in redox regulation of immunity.

Gene expression profiling reveals a signaling role of glutathione in redox regulation.

Proteins can form reversible mixed disulfides with glutathione (GSH). It has been hypothesized that protein glutathionylation may represent a mechanism of redox regulation, in a fashion similar to that mediated by protein phosphorylation. We investigated whether GSH has a signaling role in the response of HL60 cells to hydrogen peroxide (H2O2), in addition to its obvious antioxidant role. We identified early changes in gene expression induced at different times by H2O2 treatment, under conditions that increase protein glutathionylation and minimal toxicity. We then investigated the effect of prior GSH depletion by buthionine sulfoximine and diethylmaleate on this response. The analysis revealed 2,016 genes regulated by H2O2. Of these, 215 genes showed GSH-dependent expression changes, classifiable into four clusters displaying down- or up-regulation by H2O2, either potentiated or inhibited by GSH depletion. The modulation of 20 selected genes was validated by real-time RT-PCR. The biological process categories overrepresented in the largest cluster (genes whose up-regulation was inhibited by GSH depletion) were NF-kappaB activation, transcription, and DNA methylation. This cluster also included several cytokine and chemokine ligands and receptors, the redox regulator thioredoxin interacting protein, and the histone deacetylase sirtuin. The cluster of genes whose up-regulation was potentiated by GSH depletion included two HSPs (HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB. This work demonstrates that GSH, in addition to its antioxidant and protective function against oxidative stress, has a specific signaling role in redox regulation.

A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury.

The cytokine erythropoietin (EPO) protects the heart from ischemic injury, in part by preventing apoptosis. However, EPO administration can also raise the hemoglobin concentration, which, by increasing oxygen delivery, confounds assignment of cause and effect. The availability of EPO analogs that do not bind to the dimeric EPO receptor and lack erythropoietic activity, e.g., carbamylated EPO (CEPO), provides an opportunity to determine whether EPO possesses direct cardioprotective activity. In vivo, cardiomyocyte loss after experimental myocardial infarction (MI) of rats (40 min of occlusion with reperfusion) was reduced from approximately 57% in MI-control to approximately 45% in animals that were administered CEPO daily for 1 week (50 microg/kg of body weight s.c.) with the first dose administered intravenously 5 min before reperfusion. CEPO did not increase the hematocrit, yet it prevented increases in left ventricular (LV) end-diastolic pressure, reduced LV wall stress in systole and diastole, and improved LV response to dobutamine infusion compared with vehicle-treated animals. In agreement with the cardioprotective effect observed in vivo, staurosporine-induced apoptosis of adult rat or mouse cardiomyocytes in vitro was also significantly attenuated ( approximately 35%) by CEPO, which is comparable with the effect of EPO. These data indicate that prevention of cardiomyocyte apoptosis, in the absence of an increase in hemoglobin concentration, explains EPO's cardioprotection. Nonerythropoietic derivatives such as CEPO, devoid of the undesirable effects of EPO, e.g., thrombogenesis, could represent safer and more effective alternatives for treatment of cardiovascular diseases, such as MI and heart failure. Furthermore, these findings expand the activity spectrum of CEPO to tissues outside the nervous system.