Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex

Background

The human hemoglobin switch (HbF→HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor.

Design and Methods

We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34⁺ cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production.

Results

In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80–90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects.

Conclusions

Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221–222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.     

Effect of antioxidants on mitochondrial function in HIV-1-related lipoatrophy: a pilot study

We investigated the effect of antioxidant supplementation on mitochondrial function, fat distribution, and lipid and glucose metabolism in HIV-1-infected patients with antiretroviral therapy (ART)-related lipoatrophy. 61 ART-treated HIV-1-infected patients with lipoatrophy were randomized to receive either n-acetyl-L-carnitine (n?=?21), lipoic acid?+?n-acetylcisteine (LA/NAC) (n?=?20), or no supplementation (n?=?20) for 48 weeks. At baseline and at the end of treatment, mitochondrial function was studied by (13)C-methionine breath test and by mitochondrial (mt)-DNA quantification on circulating T-cells and subcutaneous adipose tissue. Body composition was assessed by dual-energy X-ray absorpiometry (DEXA). (13)CO(2)-exhalation increased between baseline and week 48 in both supplementation arms as evidenced by a higher delta over baseline excretion at 45?min (from mean?±?SEM of 7.8?±?1.08 to 9.9?±?0.6, p?=?0.04 in the n-acetyl-carnitine arm, and from 7.4?±?0.8 to 11.5?±?1.6, p?=?0.01 in LA/NAC arm). Cumulative (13)CO2 excretion increased from median (interquartile range; IQR) of 3.25 (2.55-4.2) to 4.51 (4.12-5.2) in the carnitine arm; from 3.79 (2.67-4.37) to 4.83 (4.25-5.56) in the LA/NAC arm; p?=?0.004, 0.02, respectively. mtDNA content increased in CD4+ T-cells from patients who received n-acetyl-carnitine (+30 copies/cell; p?=?0.03), without significant difference by the overall comparison of the study groups. Fat body mass and lipid profile did not change significantly in any of the arms. Our study showed that antioxidant supplementation may have a protective role on mitochondrial function, with limited effects on the reversal of clinical lipodystrophic abnormalities in HIV-1-infected patients.     

Phenomenon of new drugs on the Internet: the case of ketamine derivative methoxetamine

On the basis of the material available both in the scientific literature and on the web, this paper aims to provide a pharmacological, chemical and behavioural overview of the novel compound methoxetamine. This is a dissociative drug related to ketamine, with a much longer duration of action and intensity of effects. A critical discussion of the availability of information on the web of methoxetamine as a new recreational trend is here provided. Those methodological limitations, which are intrinsically associated with the analysis of online, non-peer reviewed, material, are here discussed as well. It is concluded that the online availability of information on novel psychoactive drugs, such as methoxethanine, may constitute a pressing public health challenge. Better international collaboration levels and novel forms of intervention are necessary to tackle this fast-growing phenomenon.     

Neuromuscular paralysis and recovery in mice injected with botulinum neurotoxins A and C

Botulinum neurotoxin type A (BoNT/A) is commonly used in human therapy. This treatment may induce immunoresistance and preliminary evaluation of other botulinum neurotoxin serotypes suggested botulinum neurotoxin type C (BoNT/C) to be a good alternative to BoNT/A. Here, we have further characterized the biological activities of BoNT/C using a variety of experimental approaches. Muscle paralysis and time of recovery of mouse hind limb injected with BoNT/A or BoNT/C were assayed with the Digit Abduction Scoring assay. The extent and duration of paralysis were similar with the two toxin serotypes. Extensor digitorum longus or tibialis anterior muscles were dissected at times of complete paralysis and of complete recovery. Muscle weight and force were significantly reduced in mice injected with BoNT/A and BoNT/C, and some atrophy persisted for a long time. In BoNT/C-treated junctions, nerve terminal sprouting was prominent, indicating that the capacity to extend the field of innervation is not hampered by BoNT/C. BoNT/C induced a marked decrease in the frequency of miniature endplate potentials and in the amplitude of endplate potentials. 3,4-diaminopyridine reversed the effect of BoNT/C by increasing the amplitude of synchronized endplate potentials. The present study shows an extensive similarity in the biological activities of BoNT/A and BoNT/C, further supporting the suggestion that BoNT/C is a valid alternative to BoNT/A.     

Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins

Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca²⁺ which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of β-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca²⁺ influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca²⁺ overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins.     

The young mouse heart is composed of myocytes heterogeneous in age and function

The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated protein p16(INK4a) and telomere length. The heart was characterized by the coexistence of young, aged, and senescent myocytes. Old nonreplicating, p16(INK4a)-positive, hypertrophied myocytes with severe telomeric shortening were present together with young, dividing, p16(INK4a)-negative, small myocytes with long telomeres. A class of myocytes with intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action potential (AP) duration and I(CaL) play in potentiating Ca(2+) cycling and the mechanical behavior of young myocytes or in decreasing Ca(2+) transients and the performance of senescent hypertrophied cells. The characteristics of the AP appeared to be modulated by the transient outward K(+) current I(to) which was influenced by the different expression of the K(+) channels subunits. Collectively, these observations at the physiological and structural cellular level document that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.     

Basal and hyperaemic myocardial blood flow in regionally denervated canine hearts: an in vivo study with positron emission tomography

Purpose Positron emission tomography (PET) studies in patients with diabetic autonomic neuropathy (DAN) have demonstrated the impact of this disease on cardiac sympathetic innervation and myocardial blood flow (MBF). To investigate the effects of selective partial sympathetic denervation of the left ventricle (LV) on baseline and hyperaemic MBF, we measured myocardial presynaptic catecholamine re-uptake (uptake-1), β-adrenoceptor (β-AR) density and MBF non-invasively by means of PET in a canine model of regional sympathetic denervation. Methods In 11 anaesthetised dogs, the sympathetic nerves of the free wall and septum of the LV were removed by means of dissection and phenol painting. Three weeks later, the animals were studied with PET. MBF was measured at baseline and following i.v. adenosine (140 μg kg⁻¹ min⁻¹) and dobutamine (20 μg kg⁻¹ min⁻¹) using¹⁵O-labelled water. Sympathetic denervation was confirmed by an 80±12% decrease in the volume of distribution (V_d) of [¹¹C]hydroxyephedrine (HED) compared with innervated regions. Myocardial β-AR density was measured using [¹¹C]CGP12177. Results Innervated and denervated regions showed no differences in MBF at baseline and during adenosine or dobutamine. [¹¹C]HED V_dwas inversely correlated with MBF in both regions at baseline, and the correlation was lost during hyperaemia in denervated regions. However, for any given value of MBF, [¹¹C]HED V_dwas significantly lower in the denervated regions. β-AR density was comparable in denervated and innervated regions (17.9±4.2 vs 18.4±3.3 pmol g⁻¹;p=NS). Conclusion In this experimental model, selective, regional sympathetic denervation of the LV, which results in a profound reduction in [¹¹C]HED V_d, did not affect baseline or hyperaemic MBF. In addition, we demonstrated that, under baseline conditions, there was a significant inverse correlation between [¹¹C]HED V_dand MBF in both denervated and innervated regions.     

Pre- and post-synaptic sympathetic function in human hibernating myocardium

Purpose Impaired pre-synaptic noradrenaline uptake-1 mechanism has been reported in a swine model of hibernating myocardium (HM). To ascertain whether adrenergic neuroeffector abnormalities are present in human HM, we combined functional measurements in vivo using cardiovascular magnetic resonance (CMR) and positron emission tomography (PET) to assess pre- and post-synaptic sympathetic function. Methods Twelve patients with coronary artery disease and chronic left ventricular (LV) dysfunction underwent CMR at baseline and 6 months after bypass for assessment of regional and global LV function and identification of segments with reversible dysfunction. Before surgery, myocardial noradrenaline uptake-1 ([¹¹C]meta-hydroxy-ephedrine; HED) and β-adrenoceptor (β-AR) density ([¹¹C]CGP-12177) were measured with PET. Patient PET data were compared with those in 18 healthy controls. Results The volume of distribution (V_d) of HED in HM (47.95±28.05 ml/g) and infarcted myocardium (42.69±25.76 ml/g) was significantly reduced compared with controls (66.09±14.48 ml/g). The V_d of HED in normal myocardium (49.93±20.48 ml/g) of patients was also lower than that in controls and the difference was close to statistical significance (p=0.06). Myocardial β-AR density was significantly lower in HM (5.49±2.35 pmol/g), infarcted (4.82±2.61 pmol/g) and normal (5.86±1.81 pmol/g) segments of patients compared with healthy controls (8.61±1.32 pmol/g). Conclusion Noradrenaline uptake-1 mechanism and β-AR density are reduced in the myocardium of patients with chronic LV dysfunction and evidence of HM. The increased sympathetic activity to the heart in these patients is a generalised rather than regional phenomenon which is likely to contribute to the remodelling process of the whole LV rather than playing a causative role in HM.     

Down-regulation of the HGF/MET autocrine loop induced by celecoxib and mediated by P-gp in MDR-positive human hepatocellular carcinoma cell line

Many tumors are resistant to drug-induced cell-cycle arrest and apoptosis. We have reported that apoptosis can be restored in human multidrug-resistant (MDR) hepatocellular carcinoma cell lines by celecoxib. Here we show that P-glycoprotein (P-gp) mediates cell-cycle arrest and autophagy induced by celecoxib in human MDR overexpressing hepatocellular carcinoma cell line by down-regulation of the HGF/MET autocrine loop and Bcl-2 expression. Exposure of cells to a low concentration of celecoxib down-regulated the expression of mTOR and caused G1 arrest and autophagy, while higher concentration triggered apoptosis. Cell growth inhibition and autophagy were associated with up-regulation of the expression of TGFβ1, p16^INK4b, p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, cyclin E, pRb and E2F. The role of P-glycoprotein expression in resistance of MDR cell clone to cell-cycle arrest, autophagy and apoptosis was shown in cells transfected with MDR1 small interfering RNA. These findings demonstrate that the constitutive expression of P-gp is involved in the HGF/MET autocrine loop that leads to increased expression of Bcl-2 and mTor, inhibition of eIF2α expression, resistance to autophagy/apoptosis and progression in the cell-cycle. Since mTor inhibitors have been proposed in treatment of “drug resistant” cancer, these data may help explain the reversing effect of mTor inhibitors.