Properties of donated red blood cell components from patients with hereditary hemochromatosis.

BACKGROUND: Red blood cells (RBCs) contain large amounts of iron, and periodic therapeutic phlebotomy is thus the main treatment for hereditary hemochromatosis (HH). However, the donation of therapeutic phlebotomy products from asymptomatic patients for transfusion purposes remains controversial. In this study, we compared the quality of RBCs obtained from HH patients with those of non-HH RBCs, within the allowed 42-day storage period. STUDY DESIGN AND METHODS: RBCs were obtained from HH patient donors and random regular blood donors by whole blood collection. RBCs were stored for up to 42 days, according to national regulations and standard blood bank conditions in France. The following variables were assessed: hematologic and biochemical results, RBC membrane and soluble inflammatory markers, and the proinflammatory potential of HH RBC supernatant toward endothelial cells in an in vitro model. RESULTS: There were no major differences between the two groups in terms of biophysical, biochemical, or soluble immunomodulatory factors. However, we observed small but significant differences in changes in RBC membrane proteins during storage, including increased phosphatidylserine expression and decreased hemolysis in HH compared with normal RBCs. However, there were no differences in terms of bioactivity of soluble immunomodulatory factors in the RBC supernatant during storage between HH and control donors, as determined by their effects on endothelial cells in vitro. CONCLUSIONS: These in vitro studies suggest that RBCs from HH patients appear, while exhibiting subtle differences, to be suitable for transfusion purposes according to currently accepted criteria.

Antifibroblast growth factor receptor 3 antibodies identify a subgroup of patients with sensory neuropathy.

BACKGROUND: Immunological mechanisms are suspected in sensory neuropathy (SN) occurring with systemic autoimmune diseases and in some idiopathic cases, but so far there are no antibodies (Abs) identifying these neuropathies. METHODS: In the search for such specific antibodies, serum samples were collected from 106 patients with SN of these 72 fulfilled the diagnosis criteria of sensory neuronopathy (SNN) and 211 control subjects including patients with sensorimotor neuropathies, other neurological diseases (ONDs), systemic autoimmune diseases and healthy blood donors. RESULTS: In the first step, a protein array with 8000 human proteins allowed identification of the intracellular domain of the fibroblast growth factor receptor 3 (FGFR3) as a target of Abs in 7/16 SNN and 0/30 controls. In the second step, an ELISA method was used to test the 317 patients and controls for anti-FGFR3 Abs. Abs were detected in 16/106 patients with SN and 1/211 controls (p<0.001). Among the 106 patients with SN, anti-FGFR3 Abs were found in 11/38 patients with autoimmune context, 5/46 with idiopathic neuropathy and 0/22 with neuropathy of other aetiology (p=0.006). The only control patient with anti-FGFR3 Abs had lupus and no recorded neuropathy. Sensitivity, specificity, and positive and negative predictive values of anti-FGFR3 Abs for a diagnosis of idiopathic or dysimmune SN were 19%, 99.6%, 94.1% and 77.3%, respectively. A cell-based assay confirmed serum reactivity against the intracellular domain of FGFR3. The neuropathy in patients with anti-FGF3 Abs was non-length dependent in 87% of patients and fulfilled the criteria of probable SNN in 82%. Trigeminal nerve involvement and pain were frequent features. CONCLUSIONS: A anti-FGFR3 Abs identify a subgroup of patients with SN in whom an underlying autoimmune disorder affecting sensory neurons in the dorsal root and trigeminal nerve ganglia is suspected.

The collapsin response mediator protein 5 onconeural protein is expressed in Schwann cells under axonal signals and regulates axon-Schwann cell interactions.

Collapsin response mediator protein 5 (CRMP5) is one of the rare peripheral nerve antigens that is a target of autoantibodies in a paraneoplastic peripheral neuropathy. The pattern of axonal and myelin alterations suggests that CRMP5 is involved in axon-Schwann cell interaction. We examined CRMP5 expression and function in primary cultures of Schwann cells and neurons and at various developmental and regenerating stages of rat sciatic nerve and in CRMP5-deficient mice in vivo. Collapsin response mediator protein 5 was strongly expressed during postnatal development and regeneration and decreased with myelination. It was mainly expressed by immature Schwann cells and persisted in Remak cells in the adult; however, a subpopulation of Schwann cells that were induced to myelinate also expressed CRMP5. We identified 2 axonal molecular cues regulating CRMP5 expression: human neuregulin type 1, which induces CRMP5 expression in immature and premyelinating Schwann cells, and cyclic adenosine monophosphate, which inhibits CRMP5 expression when Schwann cells begin myelination. Collapsin response mediator protein 5-deficient mice showed abnormal Schwann process extension resulting in abnormal cell-axon segregation, indicating that CRMP5 is involved in the morphologic adaptation of Schwann cells to surround axons. These results demonstrate the importance of CRMP5 in axon-Schwann cell cooperation during development and regeneration.

Physical fitness is independently related to blood leptin concentration and insulin sensitivity index in male subjects with central adiposity.

AIM: To compare the maximal power output (MPO) of subjects presenting a central adiposity to those of controls and to study the links between plasma leptin or indices of insulin sensitivity (QUICKI) and physical fitness (PF). METHODS: MPO was determined for 169 middle-aged men divided into two groups according to waist circumference (WC- < 94 cm, WC+ ≥ 94 cm) each subdivided in two subgroups with low and high PF (WC-L, WC-H, WC+L, WC+H) determined from the median MPO relative to fat free mass (3.06 W/kg(FFM)). RESULTS: MPO (W/kg(FFM)) was lower in WC+ than in WC-. Expressed relative to fat mass, leptin was lower and QUICKI higher in WC- than in WC+. In WC+H, leptin and QUICKI were significantly less disturbed than in WC+L and were independently correlated to MPO (r = -0.36 and r = 0.32 respectively; p < 0.001). In WC+, when visceral perimeter was added to the analysis, the relationships MPO/leptin remained significant but not MPO/QUICKI. CONCLUSION: The low PF in subjects with abdominal obesity is independently linked to plasma leptin and insulin sensitivity even if leptin and insulin may share common pathways in their peripheral effects. Visceral adiposity participates to the link between MPO and QUICKI, but not between MPO and leptin.

Differential effect of oxidative or excitotoxic stress on the transcriptional profile of amyotrophic lateral sclerosis-linked mutant SOD1 cultured neurons.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, degenerative disorder of motor neurons. The causes of most cases of ALS are as yet undefined. In a previous study, it was shown that N-methyl-D-aspartate (NMDA) and H(2)O(2) stimuli reduce neuronal survival in cortical neurons in culture (Boutahar et al., 2008). To identify variations in gene expression in response to these neurotoxins in transgenic vs. control cortical neurons cultures, both microarray and RT-PCR analysis were performed. High-density oligonucleotide microarrays showed changes in the expression of about 600 genes involved in protein degradation, neurotrophic factors pathway, cell cycle, inflammation, cytoskeleton, cell adhesion, transcription, or signalling. The most up-regulated genes following H(2)O(2) treatment were involved in cytoskeletal organization and axonal transport, such as ARAP2, KIF17, and DKK2, or in trophic factors pathways, such as insulin-like growth factor-binding protein 4 (IGFBP4), FGF17, and serpin2. The most down-regulated genes were involved in ion transport, such as TRPV1. After NMDA treatment, the most up-regulated genes were involved in protein degradation, such as ubiquitin-conjugating enzyme E2I and cathepsin H, and the most down-regulated genes were involved in ion transport, such as SCN7A. We conclude that these neurotoxins act through different transcriptional inductions, and these changes may reflect an adaptative cellular response to the cellular stress induced by the neurotoxins involved in ALS in the presence of mutant human SOD1.

Brain-derived neurotrophic factor inhibits cell cycle reentry but not endoplasmic reticulum stress in cultured neurons following oxidative or excitotoxic stress.

Neurotrophins protect neurons against glutamate and oxidative stress, but the underlying mechanism remains unclear. We investigated the neuroprotective role of the neurotrophin brain-derived neurotrophic factor (BDNF) in neuronal cultures subjected to NMDA or H(2)O(2) toxicity and analyzed the molecular mechanisms involved, particularly those related to regulation of cell cycle or endoplasmic reticulum (ER) stress. Preincubation with BDNF of cortical neuron cultures prevented NMDA- or H(2)O(2)-induced neuronal death as well as MAPK-ERK1/2 activation. Inhibition of phosphatidylinositol 3-kinase (PI3-K) abolished the protective effect of BDNF. NMDA and H(2)O(2) induced activation of cell cycle reentry regulators such as retinoblastoma (Rb) protein and E2F1 transcription factor. However, BDNF abolished the activation of both factors. NMDA-induced expression of chaperone encoding gene BIP was slightly inhibited by BDNF, but it did not affect expression of ER stress protein CHOP. Our results suggest that BDNF neuroprotection may be mediated through inhibition of Ras-MAPK pathway and cell cycle reentry during oxidative or excitotoxic stress responses. However, BDNF did not modify expression of ER stress signal induced by NMDA.

Timing differences of signaling response in neuron cultures activated by glutamate analogue or free radicals.

Oxidative stress and excitotoxicity are both involved in the pathogenesis of neuronal degenerative diseases like ALS. In order to compare their action, some key proteins involved in their respective signaling pathways, particularly ERK and p53, were analyzed in primary cultures of cortical neurons subjected to NMDA or H(2)O(2) treatment. Early ERK activation was detected after NMDA treatment and was maintained during 24 h, but not after H(2)O(2) treatment. Early p53 expression was also found after NMDA treatment but diminished later. On the other hand, it progressively increased from 6 h to 24 h after H(2)O(2) treatment. Blocking ERK1/2 activation with the upstream inhibitor U0126 inhibited NMDA-mediated p53 expression, suggesting that ERK1/2 signals drive the cells to apoptosis under these conditions. In order to identify the initial membrane target of these neurotoxins, PAK1 was analyzed. Early increase of PAK1 expression was measured after NMDA treatment and was still present after 24 h. Conversely increased PAK1 expression was only detected 24 h after H(2)O(2) treatment. In order to define the components through which NMDA or H(2)O(2) induce the final elements of these pathways, p21 and c-jun, we have performed a detailed functional analysis of c-jun and p21 promoters following plasmid transfection. Both p21 and c-jun were activated after NMDA treatment, but this activation was abolished after H(2)O(2) treatment. We conclude that NMDA induces an early effect that involves activation of p53, ERK, PAK1, p21 and c-jun. On the other hand, H(2)O(2) induces long-term p53 expression, late expression of PAK1 without activation of p21 promoter. The timing differences of the action of these neurotoxins may explain why the presence of both compounds is needed to induce neuronal death.

Cyclic strain promotes shuttling of PYK2/Hic-5 complex from focal contacts in osteoblast-like cells.

We showed that cyclic strain (CS) of osteoblastic cells induced tyrosine phosphorylation of two homologous tyrosine kinases FAK and PYK2, and of two homologous adaptor proteins paxillin and Hic5, with similar kinetics. Immunostaining showed that all four proteins were localized to focal contacts in controls. In contrast, the dynamics of their subcellular localization observed after CS differed. While FAK and paxillin remained at the focal contact, Hic-5 and PYK2 translocated outside ventral focal contacts as early as 30 min after CS and were sequestered by the cytoskeleton. Co-immunoprecipitation showed that the association of PYK2/Hic-5 and PYK2/FAK increased with time after strain while that of paxillin and Hic-5 decreased. Altogether these results suggested that CS regulates focal contact activity in osteoblasts by modulating PYK2-containing complexes in particular by shuttling out of the focal contact the adaptor Hic-5 and favoring the anchorage of FAK within contacts.

Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation.

The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.