Bleeding and thrombosis outcomes in hospitalised COVID-19 patients on low-molecular-weight heparin and antiplatelet therapy.

BACKGROUND: An increased incidence of thromboembolic events in hospitalised COVID­19 patients has been demonstrated despite the use of low-molecular-weight heparin (LMWH). Antiplatelet therapy prior to admission and early in the disease course has been hypothesised to be protective against thrombosis. OBJECTIVES: To describe the bleeding and thrombosis outcomes in hospitalised patients with confirmed COVID­19 receiving LMWH, with and without concomitant antiplatelet therapy. Secondary objectives were to explore predictors of bleeding and thrombosis outcomes, and dosing practices of antiplatelet therapy and LMWH. METHODS: We conducted a descriptive, cross-sectional study of bleeding and thrombosis outcomes at Tygerberg Academic Hospital, Cape Town, South Africa, during the first COVID­19 wave, in 808 hospitalised patients with confirmed COVID­19 receiving LMWH with and without concomitant antiplatelet therapy. Multivariate logistic regression analysis was performed if predictors were deemed statistically and clinically significant. RESULTS: Patients receiving both LMWH and antiplatelet therapy had similar bleeding outcomes compared with patients only receiving LMWH (odds ratio (OR) 1.5; 95% confidence interval (CI) 0.6 - 4.0). Patients receiving both LMWH and antiplatelet therapy had increased odds of developing thrombosis compared with patients only receiving LMWH (OR 4.8; 95% CI 2.1 - 10.7). CONCLUSION: The bleeding risk in COVID­19 patients receiving both LMWH and antiplatelet therapy was not significantly increased. A potentially higher risk of thrombosis in patients receiving LMWH and antiplatelet therapy was observed. However, this could reflect confounding by indication. Randomised studies are required to further evaluate the use of antiplatelet therapy to treat hospitalised patients with COVID­19.

Ivermectin exposures reported to the Poisons Information Helpline in South Africa during the COVID-19 pandemic.

BACKGROUND: Ivermectin is an antiparasitic drug that has shown in vitro activity against COVID­19. Clinical studies supporting ivermectin for COVID­19 prevention and treatment are conflicting, with important limitations. Public support for ivermectin is significant, with extensive off-label use despite the conflicting views on its efficacy. Ivermectin tablets and injectable formulations are not registered in South Africa for human use by the South African Health Products Regulatory Authority. The National Department of Health does not currently recommend the use of ivermectin for COVID­19. OBJECTIVES: To describe cases of ivermectin exposure reported to the Poisons Information Helpline of the Western Cape (PIHWC) before and after publication of the drug's in vitro activity against SARS-CoV-2. METHODS: In a retrospective review, ivermectin-related calls reported to the PIHWC from 1 June 2015 to 30 June 2020 (period 1) were compared with calls received from 1 July 2020 to 31 July 2021 (period 2), dichotomised according to the first publication indicating ivermectin activity against SARS-CoV-2. RESULTS: Seventy-one cases were screened, and 65 were included for analysis; 19 cases were reported during period 1 and 46 during period 2. During period 2, 25 ivermectin cases (54.3%) were related to COVID­19 use. Of these, 24 cases (52.2%) involved veterinary preparations, 3 (6.5%) human preparations and 19 (41.3%) unknown preparations. Fourteen cases (73.7%) during period 1 and 30 (65.2%) during period 2 were reported to be symptomatic. The most common organ systems involved were the central nervous (n=26 cases; 40.0%), gastrointestinal (n=18; 27.7%), ocular (n=9; 13.8%) and dermatological (n=5; 7.7%) systems. CONCLUSION: Ivermectin-related exposure calls increased during study period 2, probably as a result of ivermectin being used as preventive and definitive therapy for COVID­19 in the absence of robust evidence on efficacy, dosing recommendations or appropriate formulations.

Boomslang (Dispholidus typus) envenomation in a patient on warfarin therapy.

The venom of the boomslang (Dispholidus typus) has potent effects on the coagulation system. It is known to produce a venom-induced consumptive coagulopathy (VICC) through the proposed activation of clotting factor II (prothrombin), factor X, and possibly factor IX. Warfarin, an anticoagulant medication, decreases the circulating vitamin K-dependent clotting factors II, VII, IX and X. We report a unique case of a boomslang bite in a patient on warfarin therapy. During the patient's hospital stay he developed abnormal clotting profiles indicating an underlying VICC, but without major bleeding. He received monovalent antivenom and recovered with no complications. We discuss two possible outcomes of a boomslang bite in a patient on warfarin therapy, exploring the underlying pathophysiology that could lead to the presentation of a reduced risk of overall bleeding or, alternatively, that the bleeding could be compounded and exacerbated. It is possible that in our case the anticoagulant effect of warfarin was wholly obscured by the VICC of the boomslang venom. The composition of the snake venom may have been a contributory factor in the reduced clinical bleeding observed.

Vasopressor therapy in atypical antipsychotic overdose.

Hypotension is a common presentation following an overdose of quetiapine. Adrenaline is often used as the vasopressor of choice for hypotension not responding to intravenous fluids. We present a case of quetiapine overdose with hypotension unresponsive to high-dose adrenaline. The patient was commenced on noradrenaline and made a full recovery. We highlight learning points about vasopressor therapy for atypical antipsychotic overdose. Quetiapine-induced hypotension is thought to be mediated by α1-receptor antagonism. Adrenaline is unlikely to improve blood pressure, as it is an agonist at both α- and ß-receptors. Alpha-2- and ß2-agonism can reduce sympathetic outflow and cause vasodilation, respectively, further exacerbating the hypotension. Noradrenaline is the preferred vasopressor of choice for hypotension caused by quetiapine overdose, as it has less affinity for α2- and ß2-receptors, but maintains α1-receptor agonism. Drugs with a similar mechanism of inducing hypotension should also be treated with noradrenaline as the vasopressor of choice.

Medical cannabis: What practitioners need to know.

The South African (SA) Constitutional Court recently decriminalised the private cultivation, possession and use of cannabis by adults. Cannabis contains varying amounts of the cannabinoids delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), depending on various cultivation factors. No commercial plant-derived cannabis products are currently registered by the SA Health Products Regulatory Authority (SAHPRA) for medical use. Such products are therefore unregulated, but are freely available in SA, and may be of inadequate quality and unverified composition, and not guaranteed to be safe or effective. SAHPRA has to date approved only one synthetic medical cannabis product, dronabinol. Evidence supporting benefit from medical cannabis exists for two drug-resistant childhood forms of epilepsy, Dravet syndrome and Lennox-Gastaut syndrome. Adjuvant therapy with medical cannabis can reduce seizure frequency for Lennox-Gastaut syndrome and Dravet syndrome by 18.8% and 22.8%, respectively, and may be beneficial for other rare forms of epilepsy. There is moderate evidence for chemotherapy-induced nausea and vomiting with the synthetic cannabinoids. Multiple sclerosis-associated spasticity showed a small clinical improvement in self-reported spasticity when a purified form of THC/CBD was added to existing therapy. Currently, low-level or no convincing evidence exists for the use of medical cannabis for chronic pain, sleep and weight disorders, and neuropsychiatric disorders. Cannabis is associated with a greater risk of adverse effects than active and placebo controls, and may be involved in clinically significant drug-drug interactions. The evolving regulatory and legal landscape on the use of medical cannabis will guide prescription and recreational use in the coming years.

Current evidence for directed and supportive investigational therapies against COVID-19.

Coronavirus disease 2019 (COVID-19) is a global health crisis. There is currently a great need for effective and safe therapies directed at the disease, but no drugs are presently registered for use in COVID-19. Several directed therapies have been proposed, and most are still in clinical trials. Currently available published, peer-reviewed results mostly involve small sample sizes with study limitations restricting the interpretation of the findings. Many trials currently published also do not have a control group, limiting the interpretation of the effect of the intervention. Investigational directed therapies as well as investigational supportive therapies against COVID-19 are reviewed here. Chloroquine and hydroxychloroquine show promise as directed therapies, but current trial results are conflicting. Lopinavir/ritonavir also shows potential, but was started late in the disease course in most trials. No randomised controlled evidence is currently available for remdesivir and favipiravir. Corticosteroid use is not recommended for directed therapy against COVID-19, and the role of tocilizumab is currently unclear, based on limited evidence. Early initiation of investigational directed therapies may provide benefit in selected patients. The results from larger randomised controlled trials will clarify the place of these therapies in COVID-19 treatment.

Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells.

Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site-specific endonucleases. Two genomic sites within the MBS85 and chemokine (C-C motif) receptor 5 (CCR5) genes (AAVS1 and CCR5 zinc-finger nuclease (CCR5-ZFN) sites, respectively) have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells (HSCs). Matrix chromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptional inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and HSCs. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a ZFN was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.

MK-801 increases the baseline level of anxiety in mice introduced to a spatial memory task without prior habituation.

C57BL/6J mice were introduced to a nine arm radial maze without prior habituation and trained in the acquisition of a working memory task in 16 sessions, one session per day. In this maze mice need to climb onto an upward inclined bridge in order to reach and cross onto an arm. They received in each session an i.p. injection of MK-801 (0.1 mg/kg) 30 min before training or immediately after training. MK-801 pre-treated mice made significantly more entries onto the bridges, fewer entries onto the arms and took significantly longer time to make a first arm visit compared to saline and MK-801 post-treated mice during the first 3 session blocks (4 sessions per block). These results indicate that MK-801 induced anxiety which was extended throughout the first 3 session blocks. MK-801 pre-treated mice made also significantly more errors and required more sessions to reach the criterion compared to saline and MK-801 post-treated mice. Administration of MK-801 after training did not affect the acquisition of the task. The present results indicate that MK-801 pre-treatment impaired the acquisition of a spatial task and this can be accounted for by its effect on the baseline level of anxiety which was elevated. The introduction of mice to the acquisition of the task without prior habituation demonstrates that a drug treatment can affect learning and memory by increasing and/or prolonging anxiety. Such effect may be confounded with learning and memory performance and not detected with pre-habituation training procedures, particularly when the number of sessions is determined a-priori.

Tolerance, sensitization and dependence to diazepam in Balb/c mice exposed to a novel open space anxiety test.

Balb/c mice were exposed to an elevated platform that is extended on two opposite sides with lowered steep slopes. They were tested for 12min per session in 6 successive days. They received i.p. administration of either saline or one dose of diazepam (DZP 0.5, 1, 3mg/kg) in sessions 1-3, and saline in sessions 4 and 5. All groups of mice received a single dose of DZP (1mg/kg) in session 6. DZP produced inverted U-shaped dose-responses on the number of entries into different areas of the apparatus, with a peak in mean response at 1mg/kg whereas its effect on the duration of entries was mostly comparable between the 3 doses. It increased the number of crossings on the surface of the platform and facilitated entries onto the slopes. DZP-treated mice crossed frequently onto and spent longer time on the slopes in sessions 1-3 whereas saline-treated mice remained on the platform in sessions 1-6. Withdrawal of DZP in sessions 4-5 increased the latency of first entry and decreased the number and duration of entries onto the slopes which was reversed with the administration of 1mg/kg of DZP in the next session. This ON-OFF the drug may be due to the half-life of DZP which is very short in mice and rats ( approximately 0.88h). It also indicates that DZP-treated mice did not benefit from previous experience of entries onto the slopes which suggests a possible "state-dependent" effect. Administration of DZP after repeated exposures to the test did not facilitate entries onto the slopes but instead increased significantly the number of crossings on the surface of the platform; this increase was much higher than that observed in mice initially treated with DZP and exposed to the test. There is no evidence of habituation in saline-treated mice: the number of crossings on the platform was comparable between the first 5 sessions of the test. These results demonstrate that repeated exposures to the same anxiogenic environment resulted in avoidance responses developing tolerance and approach responses developing sensitization. They suggest that tolerance and sensitization are two opposite sides of the habituation process to the same stimulus and may account for the maintained state of anxiety.

Anxiety responses in Balb/c, c57 and CD-1 mice exposed to a novel open space test.

In the present report we describe the behavior of two albinos (BALB/c and CD-1) and one pigmented (c57/BL6) strains of mice exposed to a novel open space anxiety test in a single 12 min session. The test is based on exposure of mice to an unfamiliar elevated platform which is extended on two opposite sides with steep slopes presented downward or upward. In the first experiment, the behavior of mice was examined on the elevated platform at two different heights (75 and 100 cm) with downward slopes. In the second experiment, we examined the behavior of mice on the platform at the lowest height (75 cm) but with upward slopes which lead to a stand. In the third experiment, we examined the behavior of Balb/c mice on the platform at the lowest height (75 cm) with downward slopes, and a hub enclosure providing a protected space located in the centre of the platform. The least anxious strain of mice was expected to take risks and cross onto the slopes (experiments 1 and 3) and onto the stands (experiment 2). The results of experiment 1 show that Balb/c mice did not cross onto the slope, and CD-1 mice made more crossings into and spent more time on the slopes than c57 mice. The increase in the heights of the platform reduced the number of crossings on the platform in all three strains of mice, and decreased the time spent on the platform before first entry onto a slope in c57 and in CD-1 mice. It also decreased the number of entries and duration of entries onto the slopes in CD-1 mice. In experiment 2, Balb/c mice did cross onto the upward slopes but significantly less than c57 and CD-1 mice but they did not cross onto the stands attached to the end of the slopes. CD-1 mice made more entries onto and spent more time on the stands than c57 mice. In the third experiment, Balb/c and c57 mice spent most of their time inside a protective space (cylinder) placed in the centre of the platform demonstrating strong avoidance responses of the outer area of the platform, and only three c57 mice crossed onto the slopes for a very brief duration in one or two entries. In all three experiments, mice entered more frequently and spent more time in the outer areas than in the inner areas of the platform, particularly in the areas adjacent to slopes than in the areas adjacent to a void space. CD-1 mice appears the least anxious taking more risks by venturing onto the slopes and onto the stands while Balb/c appears the most anxious spending a large amount of time in the areas adjacent to the slopes. The different configurations of the test apparatus (experiments 1 and 2) seem to provide different incentives for the drive to explore and escape which may account for differences in anxiety responses whereas the presence of a protective space (experiment 3) appears to encourage avoidance responses.

Distinguishing anxiolysis and hyperactivity in an open space behavioral test.

This report describes the emotional responses of mice exposed to an unfamiliar elevated platform that is extended on two opposite sides by downward lowered steep slopes. Balb/c mice were exposed to the test for 12 min per session in 3 successive days. They received i.p. administration of diazepam (0, 0.5, 1 and 3 mg/kg) or amphetamine (0, 1, 2.5, 5 and 10 mg/kg) 30 min prior to test sessions. Separate groups of Balb/c mice were used for each dose of the drugs. Both drugs increased the number of crossings on the platform, indicating increased motor activity, and the effects were dose-dependent. Diazepam also significantly increased the number and duration of entries onto the slopes indicating an anxiolytic effect, whereas none of the saline or amphetamine-treated mice adventured onto the slopes. Amphetamine and diazepam produced an inverted U-shaped dose-response effect on different parameters of the test and demonstrate that the drug concentration which elicited a peak in mean number of entries is different from the drug concentration which elicited a peak in mean duration of entries. This study demonstrates the sensitivity and discriminatory power of an open space anxiety test for future pharmacological studies.

A new model for the study of high-K(+)-induced preconditioning in cultured neurones: role of N-methyl-d-aspartate and alpha7-nicotinic acetylcholine receptors.

Spreading depression (SD), whether elicited by local application of high K(+) medium to the cortical surface or by other stimuli, can increase the brain's tolerance to a subsequent, severe ischaemic insult in vivo, a phenomenon termed preconditioning. Herein, we have developed and validated a robust in vitro protocol for high-K(+)-preconditioning of cultured neurones. This new model is especially appropriate to unravel the molecular mechanisms underlying neuronal preconditioning and subsequent ischaemic tolerance. With this new, optimised preparation, preconditioning was found to be dependent upon culture day in vitro, cell density, K(+) concentration and duration of treatment. Finally, preconditioning was shown to be dependent upon N-methyl-d-aspartate (NMDA), CAM-kinase II signalling and alpha7-nicotinic (alpha7 nACh) receptor function, which is analogous to in vivo preconditioning induced by various stimuli.

Are benzodiazepines really anxiolytic? Evidence from a 3D maze spatial navigation task.

The effects of diazepam and chlordiazepoxide were assessed in a 3D maze which is a modification of an 8-arm radial maze. Each arm of the maze is attached to a bridge radiating from a central platform. Animals exposed for the first time to the maze do not venture beyond the line that separate a bridge from an arm. The prime criteria set for an anxiolytic effect is whether mice would increase the frequency of entries onto arms and increase arm/bridge entries ratio. C57 mice readily cross the line on first exposure and make more than 8 arm visits onto arms on second exposure, while other strains (CD-1 and Balb/c) hold back and rarely cross the line on first exposure and require more sessions to make more than 8 arm entries. An anxiolytic drug is expected to encourage intermediate (CD-1) and high (Balb/c) anxiety mice to adventure onto the arms of the maze and make more visits to the arms to comparable levels seen with low anxiety c57 mice. In the present report, administration of different doses of diazepam (0.625, 1.25, 2.5 and 5 mg kg(-1) i.p.) and chlordiazepoxide (5, 10 and 15 mg kg(-1) i.p.) did not reduce anxiety in animals, with the lowest dose of diazepam increasing motor activity in Balb/c and increasing anxiety in c57 mice while the highest doses of both diazepam (2.5 and 5 mg kg(-1) i.p.) and chlordiazepoxide (15 mg kg(-1) i.p.) induced mild sedation. Our results raise some concerns about the methodological foundations in the current assessment of anxiety and anxiolytic compounds both in animal and human studies.

Emotional responses and memory performance of middle-aged CD1 mice in a 3D maze: effects of low infrared light.

Non-thermal near infra-red (IR) has been shown to have many beneficial photobiological effects on a range of cell types, including neurons. In the present study, a pretreatment with a daily 6 min exposure to IR1072 for 10 days yielded a number of significant behavioral effects on middle-aged female CD-1 mice (12-months) tested in a 3D-maze. Middle-aged mice show significant deficits in a working memory test and IR treatment reversed this deficit. Interestingly, the IR treated middle-aged group despite making less memory errors than sham middle-aged group spent longer time in different parts of the maze than both the young group (3-months) and sham-middle-aged group (12-months). Young mice appeared more anxious than middle-aged mice in the first sessions of the test. Exposure to IR appeared to have no significant effects upon exploratory activity or anxiety responses. However, it elicited significant effects on working memory, with the IR middle-aged mice being more considerate in their decision making, which results in an overall improved cognitive performance which is comparable to that of young CD-1 mice. The present study describes a novel method for assessing emotional responses and memory performance in a 3D spatial navigation task and demonstrates the validity of our new all-in-one test and its sensitivity to ageing and non-invasive beneficial IR treatment.

Detailed analysis of the behavior and memory performance of middle-aged male and female CD-1 mice in a 3D maze.

Fifty percent of CD-1 mice from both sex die by the end of 2 years. The survival rate is higher in females than in males. This high mortality rate is associated to the high susceptibility of this strain of mice to some immuno-pathologies and the high incidence of systemic amyloidosis. It is therefore possible that premature cognitive deficits can be observed in CD-1 mice. In the present study, we describe a novel method for assessing emotional responses and memory performance of young (4 months) and middle-aged (12 months) CD-1 mice of both sexes in a 3D spatial navigation task. Animals are introduced to the maze without preliminary habituation and trained in a working memory test. As expected CD-1 mice have a low number of entries to arms on their first exposure to the maze which confirm our previous report on the anxious trait of this strain compared to C57/BL6 mice. The measure of arm/bridge ratio suggests that anxiety induced by exposure to the maze persists much longer in middle-aged male mice compared to middle-aged female mice and compared to both young male and female mice. The measure of memory revealed that young female mice made significantly less arm repeats and more unique arm visits before first arm repeat than middle-aged female and male mice. There are also significant differences between young female and young male mice with the former committing fewer errors than the latter.

Models of anxiety: responses of mice to novelty and open spaces in a 3D maze.

The present report describes the emotional responses of different strains of mice to exposure to a novel open space model of anxiety using a 3D spatial navigation task. The 3D maze is modification of the radial maze with flexible arms that can be raised above or lowered below the horizontal level of a central platform. To access the arms animals need to cross a bridge linking the arms to the central platform. In this model, mice are exposed to novelty in an unfamiliar open space setting with no safe alternative. Fear from novelty is compounded with the need to explore. The drive to escape and the drive to approach are intermingled making this open space model radically different from the current models of anxiety which provide animals with the choice between safe and anxiogenic spaces. In a series of experiments, we examined the behaviour of different groups of mice from C57, C3H, CD1 and Balb/c strains. In the first experiment, different groups of C57 mice were tested in one of the three arms configurations. In the second experiment, C57 mice were compared to C3H mice. In the third experiment, C57 mice were compared to CD1 and Balb/c mice in the raised arm configuration over three successive sessions. In the fourth experiment, we examined the behaviour of C57 mice in the lowered arm configuration with an open and an enclosed central. In the final experiment, we examined the difference between C57 and C3H mice of both genders. Using several spatio-temporal parameters of the transition responses between central platform, bridges and arms, we have been able to show consistent results demonstrating significant differences between C57 and C3H mice, and between Balb/c and both C57 and CD1 mice. C3H appear more anxious than C57 mice, and Balb/c mice seem more anxious than C57 and CD1 mice. We also observed significant differences between sexes in C3H mice but not in C57 mice. C3H male mice appear more anxious than C3H female mice and than both C57 male and female mice. In the lowered arm configuration with an enclosed central platform, C57 mice took longer time to make a first entry to an arm, made more visits to bridges before first entry to an arm and required longer time between re-entries to arms, spent longer time on the central platform and shorter time on arms compared to mice in the other arm configurations. They also made frequent entries to the centre and bridges compared to mice in the lowered arm with an open central platform. These results demonstrate not only the sensitivity of the parameters of the test but also the consistencies and concordances of the results which make this 3D maze a valuable new tool in the study of the underlying neural mechanisms of anxiety responses in addition to learning and memory, and in assessing the effects of potential anxiolytic drugs. In this report we examine methodological issues related to the design of animal behavioural paradigms and question the value and the construct validity of the current models of human anxiety.