The primary objective of the study was to explore safety and tolerability of hyperimmune caprine serum (AIMSPRO) in established diffuse cutaneous systemic sclerosis (SSc). Secondary objectives ...included assessment of potential efficacy and biological activity and exploration of candidate biomarkers.
This was a double-blind parallel group randomised placebo-controlled clinical trial. After informed consent 20 patients with established diffuse cutaneous SSc of greater than 3 years duration not receiving immunosuppressive therapy were randomised to receive either active (n=10) or placebo formulation (n=10) by subcutaneous twice weekly injection over 26 weeks. Clinical assessments were evaluated over 26 weeks.
There were no safety concerns during this study. Frequency of adverse events was not different between active and placebo groups. Mean modified Rodnan Skin Score (mRSS) fell by 1.4±4.7 units with active treatment but increased by 2.1±6.4 units on placebo when baseline values were compared with 26 weeks and responder analysis showed clinically meaningful improvement in mRSS at 26 weeks in 5 (50%) of actively treated patients compared with 1 (10%) in the control group (p=0.062). PIIINP (µg/L) showed a comparatively larger increase in the treatment group compared with the placebo group, (p=0.0118).
These results confirm tolerability and safety of this novel biological agent in established diffuse SSc. The value of a placebo treated control group in small clinical trials evaluating skin disease in SSc is confirmed. Potential improvement in mRSS and changes in PIIINP in cases receiving active therapy suggest that this intervention may be of clinical benefit and warrants further evaluation.
We have studied endothelin-1 (ET-1) levels and ET-1 ligand and receptor tissue expression in scleroderma renal crisis (SRC) and undertaken a pilot open label safety study of bosentan, a non-selective ...ET-1 receptor antagonist, in SRC Bosentan in Renal Disease-1 (BIRD-1).
Serum levels of ET-1 were measured in healthy controls (n = 20) or systemic sclerosis (SSc) (n = 80) with or without SRC, including cases of pulmonary arterial hypertension (PAH). Renal biopsies (n = 27) from patients with SRC were stained for endothelin ligand and receptors. Six cases of SRC received 6 months bosentan. Outcome measures were compared with SRC cases managed at our centre from 2000 to 2004 (n = 49).
Serum ET-1 was elevated in SRC but less than in PAH. ET-1 and both endothelin A and endothelin B receptor expression was increased in SRC biopsies in glomeruli, interstitium and hallmark vascular lesions of SRC. In the BIRD-1 cohort, serum ET-1 was elevated in all cases at SRC (median healthy controls 0.50 pg/ml; SRC 1.48 pg/ml; P < 0.0005), and increased further with bosentan therapy (1.46 vs. 3.05 pg/ml; t-test P < 0.05). Bosentan was well tolerated with no significant drug-related serious adverse events and long-term outcomes were favourable compared with historic cases. Three patients developed rebound hypertension on withdrawal of bosentan and one appeared to further benefit from maintenance therapy.
Upregulation of ET-1 ligand axis suggests that ET-1 receptor blockade is logical and treatment with bosentan appears to be safe in SRC. Future studies to assess therapeutic benefit and compare selective or non-selective receptor antagonists are justified.
► TRPM2 inhibition decrease OGD-induced hippocampal cell death in male preferentially. ► No sex difference in TRPM2 contribution to H2O2-induced hippocampal cell death. ► Male and female neurons ...express similar levels of functional TRPM2 channels. ► TRPM2 channels activated during reperfusion in male neurons.
Hippocampal CA1 neurons are particularly sensitive to ischemic damage, such as experienced following cardiac arrest and cardiopulmonary resuscitation. In recent years transient receptor potential M2 (TRPM2) channels have been identified as mediators of ischemic damage. We previously demonstrated that neuroprotective strategies targeting TRPM2 channels preferentially protect male cortical neurons from ischemic injury both in vitro and in vivo. It is important to determine the role of TRPM2 in ischemic injury of hippocampal neurons as this population of neurons are particularly sensitive to ischemic injury and are therapeutic targets. Here we report significantly decreased neuronal cell death following in vitro ischemia preferentially in male hippocampal neurons using TRPM2 inhibitors or knockdown of TRPM2 expression. Electrophysiological characterization of sex-stratified cultures shows similar levels of functional TRPM2 channel expression in male and female hippocampal neurons under basal conditions. In contrast, recordings made during reperfusion following in vitro ischemia revealed that TRPM2 channels are activated only in male neurons, resulting in rapid and complete depolarization. These findings provide strong evidence for TRPM2 as a target for protection against cerebral ischemia in male brain and helps define a molecular cell death pathway that is differentially engaged in male and female neurons.
Highlights • Immunohistochemistry and cell density analysis of ischemic injury in cerebellum. • Purkinje cell death rapid and not via apoptosis following CA/CPR. • GluN2B antagonist selectively ...protects CA1 neurons.
Systemic sclerosis (SSc) is a multisystem autoimmune rheumatic disorder with high morbidity and the highest case specific mortality of the rheumatic diseases. There is no currently approved ...unequivocally effective treatment for SSc and therefore there is a huge unmet medical need for novel and effective therapies. Hyperimmune caprine serum (HCS) is a goat serum extract derivative produced from goats vaccinated with a detergent-inactivated HIV viral lysate. It contains caprine immunoglobulins and small molecular weight proteins as well as a CRH, α-2 macroglobulin (α-2M) and lipoprotein-related peptide-1 complex. In this thesis we explore the hypothesis that hyperimmune caprine serum improves skin and other measures of disease severity in established dcSSc by modulating immunological function that determines persistence of clinical disease. This hypothesis is explored through 1) a prospective clinical trial, 2) long-term clinical use and 3) detailed assessment of serum growth factors and cytokines, as well as established and exploratory markers of disease. The primary objective of the clinical trial was to explore safety and tolerability of HCS in established diffuse cutaneous systemic sclerosis (dcSSc). Secondary objectives included assessment of potential efficacy and biological activity and exploration of candidate biomarkers. There were no safety concerns and frequency of adverse events was not different between HCS and placebo group. MRSS improved in the HCS group and worsened in the placebo group, with more responders in the HCS group at 26 weeks. Neuropathic pain improved in the HCS group compared to placebo. There was a trend to benefit for lung function indices. Cluster analysis revealed changes in a number of cytokines in the HCS group compared to placebo, in parallel with the skin changes. In particular, α-MSH and ACTH were significantly increased in the HCS group leading use to hypothesise that improvement in MRSS may have been mediated through the melanocortin system.
Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca2+‐activated ...potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long‐term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1‐Ethyl‐2‐benzimidazolinone (1‐EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post‐synaptic mechanisms. Real‐time quantitative reverse transcriptase‐polymerase chain reaction indicated that CA/CPR did not cause a loss of N‐methyl‐D‐aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch‐clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1‐EBIO did not prevent the CA/CPR‐induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post‐synaptic signaling that were prevented by treatment with the SK2 agonist 1‐EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.
Sustained protection of hippocampal plasticity by 1‐EBIO.
Each year there are approximately 7000 out of hospital cardiac arrests in the pediatric population, with 30% resuscitation rate and a 6–10% rate of survival to hospital discharge. Survivors of ...cardiac arrest exhibit learning and memory deficits that are devastating during the school years. Delayed neuronal cell death occurs in the hippocampus following cardiac arrest and likely contributes to memory impairments. Circulating endogenous estrogen in young adult females has been shown to provide protection against ischemic cell death, as does chronic exogenous administration of 17β-estradiol (E2). Chronic estrogen benefit can have undesirable feminizing effects, particularly in pre-adolescents. Here, we tested if a single-dose of E2 is neuroprotective in our pediatric cardiac arrest mouse model performed in juvenile mice. We subjected P21P25 C57Blk6 male and female mice to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). This developmental stage preceded the hormonal onset and serum estradiol and testosterone levels were not different in males and females. A single dose of E2 (100μg/kg) or vehicle was administered 30 min after resuscitation. Neuronal cell death measured 3 days after CA/CPR showed reduced hippocampal cell death in E2-treated females, but not males. Benefit of E2 in females was blocked by the P38 MAPK inhibitor, SB203580. Hippocampal-dependent memory function was equally impaired in E2-and vehicle-treated females measured in the contextual fear conditioning task at 7 days. Our findings demonstrate female-specific transient neuroprotection with E2 that does not provide sustained functional benefit.
•Single dose estradiol provided neuroprotection only in females.•Estradiol neuroprotection was blocked by a P38 MAPK inhibitor.•Acute neuroprotection did not provide sustained histological or functional benefit.
Neonatal stroke is common and causes life‐long motor and cognitive sequelae. Because neonates with stroke are not diagnosed until days‐months after the injury, chronic targets for repair are needed. ...We evaluated oligodendrocyte maturity and myelination and assessed oligodendrocyte gene expression changes using single cell RNA sequencing (scRNA seq) at chronic timepoints in a mouse model of neonatal arterial ischemic stroke. Mice underwent 60 min of transient right middle cerebral artery occlusion (MCAO) on postnatal day 10 (p10) and received 5‐ethynyl‐2′‐deoxyuridine (EdU) on post‐MCAO days 3–7 to label dividing cells. Animals were sacrificed 14 and 28–30 days post‐MCAO for immunohistochemistry and electron microscopy. Oligodendrocytes were isolated from striatum 14 days post‐MCAO for scRNA seq and differential gene expression analysis. The density of Olig2+EdU+ cells was significantly increased in ipsilateral striatum 14 days post‐MCAO and the majority of oligodendrocytes were immature. Density of Olig2+EdU+ cells declined significantly between 14 and 28 days post‐MCAO without a concurrent increase in mature Olig2+EdU+ cells. By 28 days post‐MCAO there were significantly fewer myelinated axons in ipsilateral striatum. scRNA seq identified a cluster of “disease associated oligodendrocytes (DOLs)” specific to the ischemic striatum, with increased expression of MHC class I genes. Gene ontology analysis suggested decreased enrichment of pathways involved in myelin production in the reactive cluster. Oligodendrocytes proliferate 3–7 days post‐MCAO and persist at 14 days, but fail to mature by 28 days. MCAO induces a subset of oligodendrocytes with reactive phenotype, which may be a therapeutic target to promote white matter repair.
Main Points
Focal arterial ischemic stroke in full‐term equivalent neonatal mice alters proliferation and maturation of oligodendrocytes and myelination.
Single cell transcriptomics reveal a unique subset of oligodendrocytes with an immune phenotype that persist 2 weeks after ischemia.
•New mouse model of pediatric cardiac arrest & CPR to study global cerebral ischemia.•Pediatric brain less sensitive to global ischemia than adult brain.•Pediatric striatum relatively resistant to ...ischemia.•Mild hypothermia protects pediatric mouse brain from ischemic damage.
Pediatric sudden cardiac arrest (CA) is an unfortunate and devastating condition, often leading to poor neurologic outcomes. However, little experimental data on the pathophysiology of pediatric CA is currently available due to the scarcity of animal models.
We developed a novel experimental model of pediatric cardiac arrest and cardiopulmonary resuscitation (CA/CPR) using postnatal day 20–25 mice. Adult (8–12 weeks) and pediatric (P20-25) mice were subjected to 6min CA/CPR. Hippocampal CA1 and striatal neuronal injury were quantified 3 days after resuscitation by hematoxylin and eosin (H&E) and Fluoro-Jade B staining, respectively.
Pediatric mice exhibited less neuronal injury in both CA1 hippocampal and striatal neurons compared to adult mice. Increasing ischemia time to 8min CA/CPR resulted in an increase in hippocampal injury in pediatric mice, resulting in similar damage in adult and pediatric brains. In contrast, striatal injury in the pediatric brain following 6 or 8min CA/CPR remained extremely low. As observed in adult mice, cardiac arrest causes delayed neuronal death in pediatric mice, with hippocampal CA1 neuronal damage maturing at 72h after insult. Finally, mild therapeutic hypothermia reduced hippocampal CA1 neuronal injury after pediatric CA/CPR.
This is the first report of a cardiac arrest and CPR model of global cerebral ischemia in mice.
Therefore, the mouse pediatric CA/CPR model we developed is unique and will provide an important new tool to the research community for the study of pediatric brain injury.
Female sex steroids, particularly estrogens, contribute to the sexually dimorphic response observed in cerebral ischemic outcome, with females being relatively protected compared to males. Using a ...mouse model of cardiac arrest and cardiopulmonary resuscitation, we previously demonstrated that estrogen neuroprotection is mediated in part by the estrogen receptor β, with no involvement of estrogen receptor α. In this study, we examined the neuroprotective effect of the novel estrogen receptor, G protein-coupled estrogen receptor 1 (GPER1/GPR30). Male mice administered with the GPR30 agonist G1 exhibited significantly reduced neuronal injury in the hippocampal CA1 region and striatum. The magnitude of neuroprotection observed in G1-treated mice was indistinguishable from estrogen-treated mice, implicating GPR30 in estrogen neuroprotection. Real-time quantitative RT-PCR indicates that G1 treatment increases expression of the neuroprotective ion channel, small-conductance calcium-activated potassium channel 2. We conclude that GPR30 agonists show promise in reducing brain injury following global cerebral ischemia.
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