Cardiovascular risk in Cuban adolescents and young adults with congenital adrenal hyperplasia.

BACKGROUND: Hyperandrogenism and supraphysiologic glucocorticoid replacement may lead to subclinical atherosclerosis in people with congenital adrenal hyperplasia (CAH) and predispose the development of cardiovascular diseases from an early age. OBJECTIVES: To determine if cardiometabolic risk factors and subclinical atherosclerosis are more frequent in patients with CAH due to 21-hydroxylase deficiency (21OHD) and if there is an association with clinical, hormonal and treatment of 21OHD. MATERIAL AND METHODS: A descriptive prospective cross-sectional study exploring clinical variables, biochemical, hormonal variables, endothelial dysfunction (flow-mediated dilation < 5%) and carotid intima media thickness (≥ 95 percentile in adolescents and ≥ 75 percentile in adults) and epicardial fat. Adolescents and young patients with 21OHD were compared with controls matched by age, sex, body mass index and Tanner stage. RESULTS: Forty four subjects (22 with CAH), 36 (82%) females, with a mean age of 17.1 ± 5.5 years (range 10-30 years) were included. Family history revealed diabetes, hypertension, and hypercholesterolemia with high frequencies in both groups. The blood pressure was similar in both groups. Blood glucose levels were lower and triglycerides higher in patient (both p < 0.01). Epicardial fat was similar between groups and in patients with CAH it was related to cholesterol levels ​​(r = 0.679, p < 0.01), time since CAH diagnosis (r = 0.462, p = 0.03) and glucocorticoid dose (r = 0.499, p = 0.04). Carotid intima media thickness (CIMT) had a tendency to be increased in patients (p = 0.07) and was directly related to 17-hydroxyprogesterone (r = 0.510, p = 0.018), diastolic blood pressure (r = 0.444, p = 0.04) and the homeostatic model assessment (HOMA) index (r = 0.507, p = 0.01). Endothelial dysfunction was not different between groups. CONCLUSIONS: Some cardiometabolic risk factors were increased in patients with CAH and were associated with clinical, hormonal and treatment parameters of CAH. Cardiometabolic risk should be evaluated regularly in patients with CAH.

Dihydropyrimidinase-Related Protein 2 Is a New Partner in the Binding between 4E-BP2 and eIF4E Related to Neuronal Death after Cerebral Ischemia.

Transient cerebral ischemia induces neuronal degeneration, followed in time by secondary delayed neuronal death that is strongly correlated with a permanent inhibition of protein synthesis in vulnerable brain regions, while protein translational rates are recovered in resistant areas. In the translation-regulation initiation step, the eukaryotic initiation factor (eIF) 4E is a key player regulated by its association with eIF4E-binding proteins (4E-BPs), mostly 4E-BP2 in brain tissue. In a previous work, we identified dihydropyrimidinase-related protein 2 (DRP2) as a 4E-BP2-interacting protein. Here, using a proteomic approach in a model of transient cerebral ischemia, a detailed study of DRP2 was performed in order to address the challenge of translation restoration in vulnerable regions. In this report, several DRP2 isoforms that have a specific interaction with both 4E-BP2 and eIF4E were identified, showing significant and opposite differences in this association, and being differentially detected in resistant and vulnerable regions in response to ischemia reperfusion. Our results provide the first evidence of DRP2 isoforms as potential regulators of the 4E-BP2-eIF4E association that would have consequences in the delayed neuronal death under ischemic-reperfusion stress. The new knowledge reported here identifies DRP2 as a new target to promote neuronal survival after cerebral ischemia.

Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia.

Ischemic strokes are caused by a reduction in cerebral blood flow and both the ischemic period and subsequent reperfusion induce brain injury, with different tissue damage depending on the severity of the ischemic insult, its duration, and the particular areas of the brain affected. In those areas vulnerable to cerebral ischemia, the inhibition of protein translation is an essential process of the cellular response leading to delayed neuronal death. In particular, translation initiation is rate-limiting for protein synthesis and the eukaryotic initiation factor (eIF) 4F complex is indispensable for cap-dependent protein translation. In the eIF4F complex, eIF4G is a scaffolding protein that provides docking sites for the assembly of eIF4A and eIF4E, binding to the cap structure of the mRNA and stabilizing all proteins of the complex. The eIF4F complex constituents, eIF4A, eIF4E, and eIF4G, participate in translation regulation by their phosphorylation at specific sites under cellular stress conditions, modulating the activity of the cap-binding complex and protein translation. This work investigates the phosphorylation of eIF4G1 involved in the eIF4E/eIF4G1 association complex, and their regulation in ischemia-reperfusion (IR) as a stress-inducing condition. IR was induced in an animal model of transient cerebral ischemia and the results were studied in the resistant cortical region and in the vulnerable hippocampal CA1 region. The presented data demonstrate the phosphorylation of eIF4G1 at Ser1147, Ser1185, and Ser1231 in both brain regions and in control and ischemic conditions, being the phosphorylation of eIF4G1 at Ser1147 the only one found in the eIF4E/eIF4G association complex from the cap-containing matrix (m7GTP-Sepharose). In addition, our work reveals the specific modulation of the phosphorylation of eIF4G1 at Ser1147 in the vulnerable region, with increased levels and colocalization with eIF4E in response to IR. These findings contribute to elucidate the molecular mechanism of protein translation regulation that underlies in the balance of cell survival/death during pathophysiological stress, such as cerebral ischemia.

Postischemic Neuroprotection of Aminoethoxydiphenyl Borate Associates Shortening of Peri-Infarct Depolarizations.

Brain stroke is a highly prevalent pathology and a main cause of disability among older adults. If not promptly treated with recanalization therapies, primary and secondary mechanisms of injury contribute to an increase in the lesion, enhancing neurological deficits. Targeting excitotoxicity and oxidative stress are very promising approaches, but only a few compounds have reached the clinic with relatively good positive outcomes. The exploration of novel targets might overcome the lack of clinical translation of previous efficient preclinical neuroprotective treatments. In this study, we examined the neuroprotective properties of 2-aminoethoxydiphenyl borate (2-APB), a molecule that interferes with intracellular calcium dynamics by the antagonization of several channels and receptors. In a permanent model of cerebral ischemia, we showed that 2-APB reduces the extent of the damage and preserves the functionality of the cortical territory, as evaluated by somatosensory evoked potentials (SSEPs). While in this permanent ischemia model, the neuroprotective effect exerted by the antioxidant scavenger cholesteronitrone F2 was associated with a reduction in reactive oxygen species (ROS) and better neuronal survival in the penumbra, 2-APB did not modify the inflammatory response or decrease the content of ROS and was mostly associated with a shortening of peri-infarct depolarizations, which translated into better cerebral blood perfusion in the penumbra. Our study highlights the potential of 2-APB to target spreading depolarization events and their associated inverse hemodynamic changes, which mainly contribute to extension of the area of lesion in cerebrovascular pathologies.

Focus on the Small GTPase Rab1: A Key Player in the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of large aggregates in the survival neurons called Lewy bodies, which mainly contain α-synuclein (α-syn). The cause of cell death is not known but could be due to mitochondrial dysfunction, protein homeostasis failure, and alterations in the secretory/endolysosomal/autophagic pathways. Survival nigral neurons overexpress the small GTPase Rab1. This protein is considered a housekeeping Rab that is necessary to support the secretory pathway, the maintenance of the Golgi complex structure, and the regulation of macroautophagy from yeast to humans. It is also involved in signaling, carcinogenesis, and infection for some pathogens. It has been shown that it is directly linked to the pathogenesis of PD and other neurodegenerative diseases. It has a protective effect against α-σψν toxicity and has recently been shown to be a substrate of LRRK2, which is the most common cause of familial PD and the risk of sporadic disease. In this review, we analyze the key aspects of Rab1 function in dopamine neurons and its implications in PD neurodegeneration/restauration. The results of the current and former research support the notion that this GTPase is a good candidate for therapeutic strategies.

Differential Association of 4E-BP2-Interacting Proteins Is Related to Selective Delayed Neuronal Death after Ischemia.

Cerebral ischemia induces an inhibition of protein synthesis and causes cell death and neuronal deficits. These deleterious effects do not occur in resilient areas of the brain, where protein synthesis is restored. In cellular stress conditions, as brain ischemia, translational repressors named eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) specifically bind to eIF4E and are critical in the translational control. We previously described that 4E-BP2 protein, highly expressed in brain, can be a molecular target for the control of cell death or survival in the reperfusion after ischemia in an animal model of transient cerebral ischemia. Since these previous studies showed that phosphorylation would not be the regulation that controls the binding of 4E-BP2 to eIF4E under ischemic stress, we decided to investigate the differential detection of 4E-BP2-interacting proteins in two brain regions with different vulnerability to ischemia-reperfusion (IR) in this animal model, to discover new potential 4E-BP2 modulators and biomarkers of cerebral ischemia. For this purpose, 4E-BP2 immunoprecipitates from the resistant cortical region and the vulnerable hippocampal cornu ammonis 1 (CA1) region were analyzed by two-dimensional (2-D) fluorescence difference in gel electrophoresis (DIGE), and after a biological variation analysis, 4E-BP2-interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Interestingly, among the 4E-BP2-interacting proteins identified, heat shock 70 kDa protein-8 (HSC70), dihydropyrimidinase-related protein-2 (DRP2), enolase-1, ubiquitin carboxyl-terminal hydrolase isozyme-L1 (UCHL1), adenylate kinase isoenzyme-1 (ADK1), nucleoside diphosphate kinase-A (NDKA), and Rho GDP-dissociation inhibitor-1 (Rho-GDI), were of notable interest, showing significant differences in their association with 4E-BP2 between resistant and vulnerable regions to ischemic stress. Our data contributes to the first characterization of the 4E-BP2 interactome, increasing the knowledge in the molecular basis of the protection and vulnerability of the ischemic regions and opens the way to detect new biomarkers and therapeutic targets for diagnosis and treatment of cerebral ischemia.

Molecular diagnosis of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive group of diseases. 21-Hydroxylase deficiency (21OHD) accounts for between 95 and 99% of all CAH cases. OBJECTIVES: To characterize the genotype of patients clinically diagnosed with 21OHD and to identify the most frequent mutations in the Cuban population. METHODS: Cross-sectional descriptive study that included all patients diagnosed with 21OHD from January 2000 to December 2018. For the molecular analysis of the CYP21A2 gene, a protocol was used that used the polymerase chain reaction in 2 stages; in the first stage genomic DNA was amplified and 5 point mutations were detected in the second stage (Intron 2, Deletion of 8 bp, G318X, I172N and P30L). RESULTS: The 5 point mutations were identified in 31 of the 55 (56%) studied patients, 16/21 (76%) in the salt-wasting, 12/18 (67%) in the simple virilizing and 3/16 (19%) in the nonclassical form. The Intron 2 mutation was the most frequent, followed by G318X and 8 bp deletion. Compound heterozygotes were found in 10 patients, all corresponded to classic forms of the disease. CONCLUSIONS: The causal CYP21A2 gene mutation was detected in 56% (72% in classic CAH), which makes the method encouraging. The most frequent mutations observed were Intron 2 and G318X. The detection of mutations offers confirmation of diagnosis, prediction of phenotype and genetic counseling.

Stress Granule Induction after Brain Ischemia Is Independent of Eukaryotic Translation Initiation Factor (eIF) 2α Phosphorylation and Is Correlated with a Decrease in eIF4B and eIF4E Proteins.

Stress granules (SGs) are cytoplasmic ribonucleoprotein aggregates that are directly connected with the translation initiation arrest response to cellular stresses. Translation inhibition (TI) is observed in transient brain ischemia, a condition that induces persistent TI even after reperfusion, i.e. when blood flow is restored, and causes delayed neuronal death (DND) in selective vulnerable regions. We previously described a connection between TI and DND in the hippocampal cornu ammonis 1 (CA1) in an animal model of transient brain ischemia. To link the formation of SGs to TI and DND after brain ischemia, we investigated SG induction in brain regions with differential vulnerabilities to ischemia-reperfusion (IR) in this animal model. SG formation is triggered by both eukaryotic translation initiation factor (eIF) 2α phosphorylation and eIF4F complex dysfunction. We analyzed SGs by immunofluorescence colocalization of granule-associated protein T-cell internal antigen-1 with eIF3b, eIF4E, and ribosomal protein S6 and studied eIF2 and eIF4F complex. The results showed that IR stress induced SG formation in the CA1 region after 3-day reperfusion, consistent with TI and DND in CA1. SGs were formed independently of eIF2α phosphorylation, and their appearance was correlated with a decrease in the levels of eIF4F compounds, the cap-binding protein eIF4E, and eIF4B, suggesting that remodeling of the eIF4F complex was required for SG formation. Finally, pharmacological protection of CA1 ischemic neurons with cycloheximide decreased the formation of SGs and restored eIF4E and eIF4B levels in CA1. These findings link changes in eIF4B and eIF4E to SG induction in regions vulnerable to death after IR.

A novel ATTR L32V mutation causes familial amyloid polyneuropathy in a Bolivian family.

We report a new transthyretin (ATTR) gene c.272C>G mutation and variant protein, p.Leu32Val, in a kindred of Bolivian origin with a rapid progressive peripheral neuropathy and cardiomyopathy. Three individuals from a kindred with peripheral nerve and cardiac amyloidosis were examined. Analysis of the TTR gene was performed by Sanger direct sequencing. Neuropathologic examination was obtained on the index patient with mass spectrometry study of the ATTR deposition. Direct DNA sequence analysis of exons 2, 3, and 4 of the TTR gene demonstrated a c.272 C>G mutation in exon 2 (p.L32V). Sural nerve biopsy revealed massive amyloid deposition in the perineurium, endoneurium and vasa nervorum. Mass spectrometric analyses of ATTR immunoprecipitated from nerve biopsy showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data. The ATTR L32V is associated with a severe course. This has implications for treatment of affected individuals and counseling of family members.

Cutting Edge: Regulation of Exosome Secretion by the Integral MAL Protein in T Cells.

Exosomes secreted by T cells play an important role in coordinating the immune response. HIV-1 Nef hijacks the route of exosome secretion of T cells to modulate the functioning of uninfected cells. Despite the importance of the process, the protein machinery involved in exosome biogenesis is yet to be identified. In this study, we show that MAL, a tetraspanning membrane protein expressed in human T cells, is present in endosomes that travel toward the plasma membrane for exosome secretion. In the absence of MAL, the release of exosome particles and markers was greatly impaired. This effect was accompanied by protein sorting defects at multivesicular endosomes that divert the exosomal marker CD63 to autophagic vacuoles. Exosome release induced by HIV-1 Nef was also dependent on MAL expression. Therefore, MAL is a critical element of the machinery for exosome secretion and may constitute a target for modulating exosome secretion by human T cells.

Synthesis, antioxidant and neuroprotective analysis of diversely functionalized α-aryl-N-alkyl nitrones as potential agents for ischemic stroke therapy.

Herein, we report the synthesis, antioxidant and biological evaluation of 32 monosubstituted α-arylnitrones derived from α-phenyl-tert-butyl nitrone (PBN) in the search for neuroprotective compounds for ischemic stroke therapy, trying to elucidate the structural patterns responsible for their neuroprotective activity. Not surprisingly, the N-tert-butyl moiety plays beneficious role in comparison to other differently N-substituted nitrone groups. It seems that electron donor substituents at the ortho position and electron withdrawing substituents at the meta position of the aryl ring induce good neuroprotective activity. As a result, (Z)-N-tert-butyl-1-(2-hydroxyphenyl)methanimine oxide (21a) and (Z)-N-tert-butyl-1-(2-(prop-2-yn-1-yloxy)phenyl)methanimine oxide (24a) showed a significant increase in neuronal viability in an experimental ischemia model in primary neuronal cultures, and induced neuroprotection and improved neurodeficit score in an in vivo model of transient cerebral ischemia. These results showed that nitrones 21a and 24a are new effective small and readily available antioxidants, and suitable candidates for further structure optimization in the search for new phenyl-derived nitrones for the treatment of ischemic stroke and related diseases.

The Golgi complex of dopaminergic enteric neurons is fragmented in a hemiparkinsonian rat model.

Since gastrointestinal disorders are early consequences of Parkinson's disease (PD), this disease is clearly not restricted to the central nervous system (CNS), but also significantly affects the enteric nervous system (ENS). Large aggregates of the protein α-synuclein forming Lewy bodies, the prototypical cytopathological marker of this disease, have been observed in enteric nervous plexuses. However, their value in early prognosis is controversial. The Golgi complex (GC) of nigral neurons appears fragmented in Parkinson's disease, a characteristic common in most neurodegenerative diseases. In addition, the distribution and levels of regulatory proteins such as Rabs and SNAREs are altered, suggesting that PD is a membrane traffic-related pathology. Whether the GC of enteric dopaminergic neurons is affected by the disease has not yet been analyzed. In the present study, dopaminergic neurons in colon nervous plexuses behave as nigral neurons in a hemiparkinsonian rat model based on the injection of the toxin 6-OHDA. Their GCs are fragmented, and some regulatory proteins' distribution and expression levels are altered. The putative mechanisms of the transmission of the neurotoxin to the ENS are discussed. Our results support the possibility that GC structure and the level of some proteins, especially syntaxin 5, could be helpful as early indicators of the disease. RESEARCH HIGHLIGHTS: The Golgi complexes of enteric dopaminergic neurons appear fragmented in a Parkinson's disease rat model. Our results support the hypothesis that the Golgi complex structure and levels of Rab1 and syntaxin 5 could be helpful as early indicators of the disease.

Golgi tubules: their structure, formation and role in intra-Golgi transport.

Tubules are common Golgi elements that can form extensive networks associated with the cis-, lateral and trans-Golgi sides, but despite this, they have almost been forgotten for decades. The molecular mechanisms involved in their formation, elongation and fission are only just beginning to be understood. However, the role of these membranes is not well understood. In the present review, we analyze the mechanisms that induce Golgi tubulation or, conversely, disrupt tubules in order to throw some lights on the nature of these elements. The putative role of these elements in the framework of current models for intra-Golgi transport is also discussed.

Golgi fragmentation is Rab and SNARE dependent in cellular models of Parkinson's disease.

Fragmentation of the Golgi ribbon is a common feature of many neurodegenerative diseases but little is known about the causes of this alteration. In Parkinson's disease, it is believed to be the consequence of an ER-Golgi transport imbalance and/or of cytoskeleton alterations. In the present study, we analyze the mechanisms involved in Golgi fragmentation in differentiated PC12 cells treated with 6-hydroxydopamine or methamphetamine as cellular models of Parkinson's disease. Our data demonstrate that Golgi fragmentation precedes and might trigger the aggregation of α-synuclein and the formation of inclusions, alterations in anterograde and retrograde transport between the endoplasmic reticulum and Golgi complex, and cytoskeleton damage. In contrast, fragmentation is directly related with alterations in the levels of Rab1, 2 and 8 and the SNARE protein syntaxin 5. Thus, overexpression of Rab1 and 8 and depletion of Rab2 and syntaxin 5 rescue the Golgi morphology. In conclusion, the homeostasis of a limited number of Rab and SNARE proteins is important for understanding the cytopathology of Parkinson's disease.

Development of Pharmacological Strategies with Therapeutic Potential in Ischemic Stroke.

Acute ischemic stroke constitutes a health challenge with great social impact due to its high incidence, with the social dependency that it generates being an important source of inequality. The lack of treatments serving as effective neuroprotective therapies beyond thrombolysis and thrombectomy is presented as a need. With this goal in mind, our research group's collaborative studies into cerebral ischemia and subsequent reperfusion concluded that there is a need to develop compounds with antioxidant and radical scavenger features. In this review, we summarize the path taken toward the identification of lead compounds as potential candidates for the treatment of acute ischemic stroke. Evaluations of the antioxidant capacity, neuroprotection of primary neuronal cultures and in vivo experimental models of cerebral ischemia, including neurological deficit score assessments, are conducted to characterize the biological efficacy of the various neuroprotective compounds developed. Moreover, the initial results in preclinical development, including dose-response studies, the therapeutic window, the long-term neuroprotective effect and in vivo antioxidant evaluation, are reported. The results prompt these compounds for clinical trials and are encouraging regarding new drug developments aimed at a successful therapy for ischemic stroke.

The hidden microbial ecosystem in the perennial ice from a Pyrenean ice cave.

Over the last years, perennial ice deposits located within caves have awakened interest as places to study microbial communities since they represent unique cryospheric archives of climate change. Since the beginning of the twentieth century, the temperature has gradually increased, and it is estimated that by the end of this century the increase in average temperature could be around 4.0°C. In this context of global warming the ice deposits of the Pyrenean caves are undergoing a significant regression. Among this type of caves, that on the Cotiella Massif in the Southern Pyrenees is one of the southernmost studied in Europe. These types of caves house microbial communities which have so far been barely explored, and therefore their study is necessary. In this work, the microbial communities of the Pyrenean ice cave A294 were identified using metabarcoding techniques. In addition, research work was carried out to analyze how the age and composition of the ice affect the composition of the bacterial and microeukaryotic populations. Finally, the in vivo effect of climate change on the cellular machinery that allow microorganisms to live with increasing temperatures has been studied using proteomic techniques.

Regulation of ER-Golgi intermediate compartment tubulation and mobility by COPI coats, motor proteins and microtubules.

Little is known about the formation and regulation of endoplasmic reticulum (ER)-Golgi transport intermediates, although previous studies suggest that cargo is the main regulator of their morphology. In this study, we analyze the role of coat protein I (COPI) and cytoskeleton in the formation of tubular ER-Golgi intermediate compartment (ERGIC) and also show that partial COPI detachment by means of low temperature (15 degrees C) or brefeldin A induces the formation of transient tubular ERGIC elements. Most of them moved from the cell periphery to the perinuclear area and were 2.5x slower than vesicles. Time-lapse analysis of living cells demonstrates that the ERGIC elements are able to shift very fast from tubular to vesicular forms and vice versa, suggesting that the amount of cargo is not the determining factor for ERGIC morphology. Both the partial microtubule depolymerization and the inhibition of uncoating of the membranes result in the formation of long tubules that grow from round ERGICs and form at complex network. Interestingly, both COPI detachment and microtubule depolymerization induce a redistribution of kinesin from peripheral ERGIC elements to the Golgi area, while dynein distribution is not affected. However, both kinesin and dynein downregulation by RNA interference induced ERGIC tubulation. The tubules induced by kinesin depletion were static, whereas those resulting from dynein depletion were highly mobile. Our results strongly suggest that the interaction of motor proteins with COPI-coated membranes and microtubules is a key regulator of ERGIC morphology and mobility.

Alcohol induces Golgi fragmentation in differentiated PC12 cells by deregulating Rab1-dependent ER-to-Golgi transport.

In the present study, we analyze the effects of ethanol on the Golgi structure and membrane transport in differentiated PC12 cells, which are used as a model of neurons. Chronic exposure to moderate doses of ethanol induces Golgi fragmentation, a common characteristic of many neurodegenerative diseases. Alcohol impaired the lateral linking of stacks without causing microtubule damage. Extensive immunocytochemical and western blot analyses of representative Golgi proteins showed that few, but important, proteins are significantly affected. Thus, alcohol exposure induced a significant ER-to-Golgi transport delay, the retention of the GTPase Rab1 in the Golgi membranes and the accumulation of tethering factor p115 in the cytosol. These modifications would explain the observed fragmentation. The amount of p115 and the stacking protein GRASP65 increased in alcohol-treated cells, which might be a mechanism to reverse Golgi damage. Importantly, the overexpression of GTP-tagged Rab1 but not of a dominant-negative Rab1 mutant, restored the Golgi morphology, suggesting that this protein is the main target of alcohol. Taken together, our results support the view that alcohol and neurodegenerative diseases such as Parkinson have similar effects on intracellular trafficking and provide new clues on the neuropathology of alcoholism.

Preclinical Characterization of Antioxidant Quinolyl Nitrone QN23 as a New Candidate for the Treatment of Ischemic Stroke.

Nitrones are encouraging drug candidates for the treatment of oxidative stress-driven diseases such as acute ischemic stroke (AIS). In a previous study, we found a promising quinolylnitrone, QN23, which exerted a neuroprotective effect in neuronal cell cultures subjected to oxygen-glucose deprivation and in experimental models of cerebral ischemia. In this paper, we update the biological and pharmacological characterization of QN23. We describe the suitability of intravenous administration of QN23 to induce neuroprotection in transitory four-vessel occlusion (4VO) and middle cerebral artery occlusion (tMCAO) experimental models of brain ischemia by assessing neuronal death, apoptosis induction, and infarct area, as well as neurofunctional outcomes. QN23 significantly decreased the neuronal death and apoptosis induced by the ischemic episode in a dose-dependent manner and showed a therapeutic effect when administered up to 3 h after post-ischemic reperfusion onset, effects that remained 11 weeks after the ischemic episode. In addition, QN23 significantly reduced infarct volume, thus recovering the motor function in a tMCAO model. Remarkably, we assessed the antioxidant activity of QN23 in vivo using dihydroethidium as a molecular probe for radical species. Finally, we describe QN23 pharmacokinetic parameters. All these results pointing to QN23 as an interesting and promising preclinical candidate for the treatment of AIS.

An Exploratory Study of Itolizumab on the Preservation of Beta Cell Function in Adults with Recent-Onset Type 1 Diabetes.

We conducted a phase I-IIa, randomized, monocentric, double-blind, placebo-controlled clinical trial to evaluate the safety and impact of the combination treatment of Itolizumab and insulin on preserving beta cell function in adults with recent-onset type 1 diabetes. Twelve patients were randomly assigned to three treatment groups, each receiving a different Itolizumab dose (0.4/0.8/1.6 mg/kg body weight, respectively) and a placebo group. All patients received concomitant intensive multiple-dose insulin therapy. Endogenous insulin secretion was assessed by the measurement of C-peptide during the mixed-meal tolerance test. No serious adverse events were reported. No changes in the total daily insulin doses, glycated hemoglobin levels, and stimulated C-peptide were observed between the Itolizumab and placebo groups at 52 weeks. A significant decrease in stimulated C-peptide was observed during the follow-up period (p = 0.012). One subject treated with 1.6 mg of Itolizumab showed a marked increase in the levels of stimulated C-peptide three years after completion of the trial. Taken together, this is the first study to demonstrate that combination treatment with Itolizumab and insulin is safe in humans and does not affect the residual function of beta cells up to 52 weeks. The findings from our study show preliminary evidence that high doses of Itolizumab could potentially arrest the loss of beta cell function in the long term. Further studies with a longer follow-up and larger numbers of patients are envisaged to assess the effect with high dose Itolizumab.