High incidence of severe cyclosporine neurotoxicity in children affected by haemoglobinopaties undergoing myeloablative haematopoietic stem cell transplantation: early diagnosis and prompt intervention ameliorates neurological outcome

<p>Abstract</p> <p>Background</p> <p>Neurotoxicity is a recognized complication of cyclosporine A (CSA) treatment. The incidence of severe CSA-related neurological complications following hematopoietic stem cell transplantation (HSCT) is 4-11%.</p> <p>Methods</p> <p>We describe 6 cases of CSA related neurotoxicity out of 67 matched related HSCT performed in paediatric Middle East patients affected by haemoglobinopaties (5 beta thalassemia major, 1 sickle cell disease-SCD). Conditioning regimen consisted of iv busulphan, cyclophosphamide and graft-versus-host-disease (GvHD) prophylaxis with CSA, methylprednisolone, methotrexate and ATG.</p> <p>Results</p> <p>All 6 patients presented prodromes such as arterial hypertension, headache, visual disturbances and vomiting, one to two days before overt CSA neurotoxicity. CSA neurotoxicity consisted of generalized seizures, signs of endocranial hypertension and visual disturbances at a median day of onset of 11 days after HSCT (range +1 to +40). Brain magnetic resonance imaging (MRI) performed in all subjects showed reversible leukoencephalopathy predominantly in the posterior regions of the brain (PRES) in 5/6 patients. EEG performed in 5/6 patients was always abnormal. Neurotoxicity was not explainable by high CSA blood levels, as all patients had CSA in the therapeutic range with a median of 178 ng/ml (range 69-250). CSA was promptly stopped and switched to tacrolimus with disappearance of clinical and radiological findings. All patients are symptoms-free at a median follow up of 882 days (range 60-1065).</p> <p>Conclusions</p> <p>Our experience suggests that paediatric patients with haemoglobinopaties have a high incidence of CSA related neurological events with no correlation between serum CSA levels and neurotoxicity. Prognosis is good following CSA removal. Specific prodromes such as arterial hypertension, headache or visual disturbances occurring in the early post-transplant period should be carefully evaluated with electrophysiological and MRI-based imaging in order to intervene promptly and avoid irreversible sequels.</p

Effect of TRPM7 inhibition on HUVEC migration and NO production.

<p>HUVEC were treated with 2-APB and Co(III)hexaammine or transfected with a shRNA against <i>TRPM7</i> for 24 h. (A) The cells were wounded and migration was allowed for 10 h. Microphotographs show the result of a representative assay (20×magnification). (B) The wound area was calculated by ImageJ software and expressed using an arbitrary value scale to quantify the results. The values are expressed as the mean of 3 different experiments ± standard deviation. *p<0.05, **p<0.01. (C) NOS activity was measured by using the Griess method.</p

TRPM7 amounts in quiescent, proliferating and senescent HUVEC.

<p>(A) HUVEC were either cultured in complete growth medium (proliferating cells, P) or in starvation medium (quiescent cells, Q) for 2 days. (B) HUVEC were lyzed 2 days before (−2, preconfluent), 2 days after reaching confluence (+2, post-confluent) and on the day (T0) they reached confluence. (C) Subconfluent HUVEC were starved as described for 48 h. Then the cells were exposed for different times to complete growth medium (CGM) to induce the re-entry in the cell cycle. (D) High and low PD HUVEC were analyzed for TRPM7 levels. Cell extracts were utilized for Western blot using anti-TRPM7 antibodies. Actin was used to show that equal amounts of proteins were loaded per lane.</p

TRPM7 expression in endothelial cells cultured in different concentrations of Mg.

<p>(A) RT-PCR was performed on RNA extracted from HUVEC exposed to different concentrations of Mg for 6 and 24 h using primers designed on <i>TRPM7</i> sequence. (B) HUVEC were cultured in media containing different concentrations of Mg for 6 and 24 h. After lysis cell extracts were utilized for Western blot using anti-TRPM7 antibodies. In A and B actin was used as a control of loading.</p

Blocking the rise of intracellular calcium inhibits the growth of cells cultured in different concentrations of magnesium

Divalent cations, especially calcium and magnesium, have been shown to play an important regulatory role in endothelial and immune cells. To learn more about the interaction of these two metals in the regulation of cell growth, we altered the calcium/magnesium ratio by culturing human endothelial cells, macrophages, and T lymphocytes in media containing different concentrations of magnesium. We observed that the growth of the three cell types was retarded in low extracellular magnesium, and this retardation is particularly evident in highly proliferating cells. High concentrations of magnesium does not exert any effect on cell growth. When (i) calcium influx was blocked by adding the calcium antagonist verapamil, and (ii) calcium release from intracellular stores was inhibited by exposure to TMB-8, the growth of endothelial cells, macrophages, and T lymphocytes was inhibited. In particular, the release of calcium from intracellular stores seems to be more important than its influx in sustaining cell proliferation. Our results indicate that calcium plays a crucial role in mediating cell proliferation independently from the extracellular concentrations of magnesium

Magnesium deficiency promotes a pro-atherogenic phenotype in cultured human endothelial cells via activation of NFkB.

Contains fulltext : 87400.pdf (publisher's version ) (Closed access)Phenotypic modulation of endothelium to a dysfunctional state contributes to the pathogenesis of atherosclerosis, partly through the activation of the transcription factor NFkB. Several data indicate that magnesium deficiency caused by prolonged insufficient intake and/or defects in its homeostasis may be a missing link between diverse cardiovascular risk factors and atherosclerosis. Here we report that endothelial cells cultured in low magnesium rapidly activate NFkB, an event which is prevented by exposure to the anti-oxidant trolox. It is well known that NFkB activation correlates with marked alterations of the cytokine network. In the present study, we show that exposure of endothelial cells to low magnesium increases the secretion of RANTES, interleukin 8 and platelet derived growth factor BB, all important players in atherogenesis. Moreover, we describe the increased secretion of matrix metalloprotease-2 and -9 and of their inhibitor TIMP-2. Interestingly, by zymography we show that metalloprotease activity predominated over the inhibitory effect of TIMP-2. These results indicate that low magnesium promotes endothelial dysfunction by inducing pro-inflammatory and pro-atherogenic events.1 november 201