Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

The Effects of Fingolimod (FTY720) on Leukocyte Subset Circulation cannot be Behaviourally Conditioned in Rats.

Suppression of immune functions can be elicited by behavioural conditioning using drugs such as cyclosporin A or rapamycin. Nevertheless, little is known about the underlying mechanisms and generalisability of this phenomenon. Against this background, the present study investigated whether the pharmacological properties of fingolimod (FTY720), an immunosuppressive drug widely applied to treat multiple sclerosis, can be conditioned in rats by means of taste-immune associative learning. For this purpose, a conditioned taste avoidance paradigm was used, pairing the presentation of a novel sweet drinking solution (saccharin or sucrose) as conditioned stimulus (CS) with therapeutically effective doses of FTY720 as unconditioned stimulus (US). Subsequent re-exposure to the CS at a later time point revealed that conditioning with FTY720 induced a mild conditioned taste avoidance only when saccharin was employed as CS. However, on an immunological level, neither re-exposure with saccharin nor sucrose altered blood immune cell subsets or splenic cytokine production. Despite the fact that intraperitonally administered FTY720 could be detected in brain regions known to mediate neuro-immune interactions, the present findings show that the physiological action of FTY720 is not inducible by mere taste-immune associative learning. Whether conditioning generalises across all small-molecule drugs with immunosuppressive properties still needs to be investigated with modified paradigms probably using distinct sensory CS. Moreover, these findings emphasize the need to further investigate the underlying mechanisms of conditioned immunomodulation to assess the generalisability and usability of associative learning protocols as supportive therapies in clinical contexts.

Preclinical evaluation of single domain antibody efficacy in mitigating local tissue damage induced by Bothrops snake envenomation.

Camelid single-domain antibodies (VHH) represent a promising class of immunobiologicals for therapeutic applications due to their remarkable stability, specificity, and therapeutic potential. To enhance the effectiveness of antivenoms for snakebites, various methods have been explored to address limitations associated with serum therapy, particularly focusing on mitigating local damage and ensuring sustainable production. Our study aimed to characterize the pharmacological profile and neutralization capacity of anti-Phospholipase A2 (PLA2) monomeric VHH (Genbank accessions: KC329718). Using a post-envenoming mouse model, we used intravital microscopy to assess leukocyte influx, measured CK and LDH levels, and conducted a histopathology analysis to evaluate VHH KC329718's ability to neutralize myotoxic activity. Our findings demonstrated that VHH KC329718 exhibited heterogeneous distribution in muscle tissue. Treatment with VHH KC329718 reduced leukocyte influx caused by BthTX-I (a Lys-49 PLA2) by 28 %, as observed through intravital microscopy. When administered at a 1:10 ratio [venom or toxin:VHH (w/w)], VHH KC329718 significantly decreased myotoxicity, resulting in a 35-40 % reduction in CK levels from BthTX-I and BthTX-II (an Asp-49 PLA2) and a 60 % decrease in CK levels from B. jararacussu venom. LDH levels also showed reductions of 60%, 80%, and 60% induced by BthTX-I, BthTX-II, and B. jararacussu venom, respectively. Histological analysis confirmed the neutralization potential, displaying a significant reduction in tissue damage and inflammatory cell count in mice treated with VHH KC329718 post B. jararacussu venom inoculation. This study underscores the potential of monomeric anti-PLA2 VHH in mitigating myotoxic effects, suggesting a promising avenue for the development of new generation antivenoms to address current therapeutic limitations.

Multi-layered effects of Panax notoginseng on immune system.

Recent research has demonstrated the immunomodulatory potential of Panax notoginseng in the treatment of chronic inflammatory diseases and cerebral hemorrhage, suggesting its significance in clinical practice. Nevertheless, the complex immune activity of various components has hindered a comprehensive understanding of the immune-regulating properties of Panax notoginseng, impeding its broader utilization. This review evaluates the effect of Panax notoginseng to various types of white blood cells, elucidates the underlying mechanisms, and compares the immunomodulatory effects of different Panax notoginseng active fractions, aiming to provide the theory basis for future immunomodulatory investigation.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

The role of leukocyte and platelet-rich fibrin in the prevention of medication-related osteonecrosis of the jaw, in patients requiring dental extractions: an observational study.

PURPOSE: Medication-related osteonecrosis of the jaw (MRONJ) is a significant complication which can present following a dental extraction in patients receiving anti-resorptive and anti-angiogenic medications. The purpose of this study was to investigate the possible beneficial effect of L-PRF in the prevention of MRONJ in patients receiving these medications and requiring dental extractions. METHODS: Thirty-nine patients were included and divided into two groups, depending on whether L-PRF was used after the required dental extraction or not. Subsequently, the patients were categorised into low and high-risk for developing MRONJ, as recommended by the SDCEP guidance. RESULTS: None of the patients in the L-PRF group returned with established MRONJ. Five high-risk patients in the control group presented with established MRONJ in the follow-up appointment. A significant statistical difference (p = 0.04) was observed following a comparison of the high-risk patients of the two groups. CONCLUSION: These encouraging results suggest that L-PRF may be useful in the prevention of MRONJ following a dental extraction especially in patients of the higher risk category. A protocol for the management of this type of patients is also introduced.

Vitamin B12 attenuates leukocyte inflammatory signature in COVID-19 via methyl-dependent changes in epigenetic markings.

COVID-19 induces chromatin remodeling in host immune cells, and it had previously been shown that vitamin B12 downregulates some inflammatory genes via methyl-dependent epigenetic mechanisms. In this work, whole blood cultures from moderate or severe COVID-19 patients were used to assess the potential of B12 as adjuvant drug. The vitamin normalized the expression of a panel of inflammatory genes still dysregulated in the leukocytes despite glucocorticoid therapy during hospitalization. B12 also increased the flux of the sulfur amino acid pathway, that regulates the bioavailability of methyl. Accordingly, B12-induced downregulation of CCL3 strongly and negatively correlated with the hypermethylation of CpGs in its regulatory regions. Transcriptome analysis revealed that B12 attenuates the effects of COVID-19 on most inflammation-related pathways affected by the disease. As far as we are aware, this is the first study to demonstrate that pharmacological modulation of epigenetic markings in leukocytes favorably regulates central components of COVID-19 physiopathology.

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

Interleukin-6 inhibition in ST-elevation myocardial infarction: Immune cell profile in the randomised ASSAIL-MI trial.

BACKGROUND: We recently showed that interleukin (IL)-6 inhibition by tocilizumab improves myocardial salvage in ST-elevation myocardial infarction (STEMI). However, the mechanisms for this effect are not clear. METHODS: In this exploratory sub-study of the ASSAIL-MI trial, we examined leukocyte differential counts and their relation to myocardial salvage and peak troponin T (TnT) in STEMI patients randomised to tocilizumab (n = 101) or placebo (n = 98). We performed RNA-sequencing on whole blood (n = 40) and T cells (n = 20). B and T cell subpopulations were examined by flow cytometry (n = 69). FINDINGS: (i) STEMI patients had higher neutrophil counts at hospitalisation compared with stable angina patients. (ii) After percutaneous coronary intervention there was a gradual decline in neutrophils, which was significantly more pronounced in the tocilizumab group. (iii) The decrease in neutrophils in the tocilizumab group was associated with improved myocardial salvage and lower peak TnT. (iv) RNA-sequencing suggested that neutrophil function was also attenuated by tocilizumab. (v) B and T cell sub-populations changed only minimally after STEMI with minor effects of tocilizumab, supported as well by RNA-sequencing analyses of T cells. (vi) However, a low CD8+ count was associated with improved myocardial salvage in patients admitted to the hospital > 3 h after symptom onset. INTERPRETATION: Tocilizumab induced a rapid reduction in neutrophils and seemed to attenuate neutrophil function in STEMI patients potentially related to the beneficial effects of tocilizumab on myocardial salvage. FUNDING: South-Eastern Norway Regional Health Authority (Nos. 2019067, 2017084), the Central Norway Regional Health Authority and Norwegian Research Council (No. 283867).

Methods for Assessing the Effects of Galectins on Leukocyte Trafficking and Clearance.

Numerous protocols exist for investigating leukocyte recruitment and clearance both in vitro and in vivo. Here we describe an in vitro flow chamber assay typically used for studying the mechanisms underpinning leukocyte movement through the endothelium and zymosan-induced peritonitis, an acute in vivo model of inflammation that enables both leukocyte trafficking and clearance to be monitored. Insight is given as to how these models can be used to study the actions of galectins on the inflammatory process.

Susceptibility of Sea Bream (Sparus aurata) to AIP56, an AB-Type Toxin Secreted by Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural environment. Among the affected hosts are economically important cultured fish, such as sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), yellowtail (Seriola quinqueradiata), and cobia (Rachycentron canadum). The best characterized virulence factor of Phdp is the Apoptosis-Inducing Protein of 56 kDa (AIP56), a secreted AB-type toxin that has been shown to induce apoptosis of sea bass phagocytes during infection. AIP56 has an A subunit that displays metalloprotease activity against NF-kB p65 and a B subunit that mediates binding and internalization of the A subunit in susceptible cells. Despite the fact that the aip56 gene is highly prevalent in Phdp isolates from different fish species, the toxicity of AIP56 has only been studied in sea bass. In the present study, the toxicity of AIP56 for sea bream was evaluated. Ex vivo assays showed that sea bream phagocytes are resistant to AIP56 cytotoxicity and that resistance was associated with an inefficient internalization of the toxin by those cells. Accordingly, in vivo intoxication assays revealed that sea bream is much more resistant to AIP56-induced lethality than sea bass. These findings, showing that the effect of AIP56 is different in sea bass and sea bream, set the basis for future studies to characterize the effects of AIP56 and to fully elucidate its virulence role in different Phdp susceptible hosts.

Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models.

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1ß. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.

Drug delivery of 6-bromoindirubin-3'-glycerol-oxime ether employing poly(D,L-lactide-co-glycolide)-based nanoencapsulation techniques with sustainable solvents.

BACKGROUND: Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. RESULTS: For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around - 15 to - 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen's egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. CONCLUSIONS: Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential.

Cordycepin prevents and ameliorates experimental autoimmune encephalomyelitis by inhibiting leukocyte infiltration and reducing neuroinflammation.

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease characterized by multifocal perivascular infiltration of immune cells in the central nervous system (CNS). Cordycepin (3'-deoxyadenosine), an adenosine analogue initially extracted from the fungus Cordyceps militarisa, is one of the candidates that has multiple actions. We investigated that cordycepin attenuated the activation of LPS-induced mouse bone marrow-derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells (MoDCs) through the inhibition of the AKT, ERK, NFκB, and ROS pathways and impaired the migration of BMDCs through the downregulation of adhesion molecules and chemokine receptors in vitro. In experimental autoimmune encephalomyelitis (EAE) model, preventive treatment with cordycepin decreased the expression of trafficking factors in the CNS, inhibited the secretion of inflammatory cytokines (IFN-γ, IL-6, TNF-α, and IL-17), and attenuated disease symptoms. A chemokine array indicated that cordycepin treatment reversed the high levels of CCL6, PARRES2, IL-16, CXCL10, and CCL12 in the brain and spinal cord of EAE mice, consistent with the RNA-seq data. Moreover, cordycepin suppressed the release of neuroinflammatory cytokines by activated microglial cells, macrophages, Th17 cells, Tc1 cells, and Th1 cells in vitro. Furthermore, cordycepin treatment exerted therapeutic effects on attenuating the disease severity in the early disease onset stage and late disease progression stage. Our study suggests that cordycepin treatment may not only prevent the occurrence of MS by inhibiting DC activation and migration but also potentially ameliorates the progression of MS by reducing neuroinflammation, which may provide insights into the development of new approaches for the treatment of MS.

Plasma metabolomics study reveals the critical metabolic signatures for benzene-induced hematotoxicity.

Metabolomics has been used to explore the molecular mechanism and screen biomarkers. However, the critical metabolic signatures associated with benzene-induced hematotoxicity remain elusive. Here, we performed a plasma metabolomics study in 86 benzene-exposed workers and 76 healthy controls, followed by a validation analysis in mice, to investigate the dynamical change of the metabolic profile. We found that 8 fatty acids were significantly altered in both benzene-exposed worker and benzene-exposed animal models. These metabolites were significantly associated with S-phenylmercapturic acid and WBC, and they mediated the benzene-induced WBC decline. Furthermore, in vivo results confirm that fatty acid levels were dynamically altered, characterized by a decrease at 15 days and then sharp increases at 30 and 45 days. Following these identified fatty acids, the potential metabolic pathways were investigated. Fatty acids, as precursors for fatty acid oxidation, may disturb the balance of fatty acid biosynthesis and degradation. Our results reveal that fatty acid metabolism was strongly reprogrammed after benzene exposure. This abnormal change of fatty acids might be the key metabolic signature associated with benzene-induced hematotoxicity.

Engineered Liposomes Protect Immortalized Immune Cells from Cytolysins Secreted by Group A and Group G Streptococci.

The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate the host cell plasma membrane. Recently developed liposomal nanotraps, mimicking the outer layer of the targeted cell membranes, serve as decoys for exotoxins, thus diverting them from attacking host cells. In this study, we develop a liposomal nanotrap formulation that is capable of protecting immortalized immune cells from the whole palette of cytolysins secreted by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis-important human pathogens that can cause life-threatening bacteremia. We show that the mixture of cholesterol-containing liposomes with liposomes composed exclusively of phospholipids is protective against the combined action of all streptococcal exotoxins. Our findings pave the way for further development of liposomal antivirulence therapy in order to provide more efficient treatment of bacterial infections, including those caused by antibiotic resistant pathogens.

Metabolic Implications of Immune Checkpoint Proteins in Cancer.

Expression of immune checkpoint proteins restrict immunosurveillance in the tumor microenvironment; thus, FDA-approved checkpoint inhibitor drugs, specifically PD-1/PD-L1 and CTLA-4 inhibitors, promote a cytotoxic antitumor immune response. Aside from inflammatory signaling, immune checkpoint proteins invoke metabolic reprogramming that affects immune cell function, autonomous cancer cell bioenergetics, and patient response. Therefore, this review will focus on the metabolic alterations in immune and cancer cells regulated by currently approved immune checkpoint target proteins and the effect of costimulatory receptor signaling on immunometabolism. Additionally, we explore how diet and the microbiome impact immune checkpoint blockade therapy response. The metabolic reprogramming caused by targeting these proteins is essential in understanding immune-related adverse events and therapeutic resistance. This can provide valuable information for potential biomarkers or combination therapy strategies targeting metabolic pathways with immune checkpoint blockade to enhance patient response.

Mercury immunotoxicity in the brown watersnake (Nerodia taxispilota): An in vitro study.

Mercury (Hg) is a heavy metal that enters the environment through natural and anthropogenic means. Once in the environment, Hg can biomagnify in food webs and is known to cause immunotoxic effects to wildlife. Compared with other vertebrates, knowledge of the reptilian immune system is lacking, especially in snakes. Further, even less is known about the impact of environmental contaminants on snake immunity. This gap in knowledge is largely due to an absence of established immune-based assays or specific reagents for these species. In this study, brown watersnakes (Nerodia taxispilota; n = 23) were captured on the Savannah River (Augusta, Georgia, USA), weighed, measured, bled, and released. Peripheral blood leukocytes (24 h old) were enriched and evaluated with an established mammalian in vitro lymphocyte proliferation assay. Enriched leukocytes were then exposed to mercury chloride (HgCl2 ) at 3.75, 37.5, and 75 µM. Total mercury (THg) in whole blood was also quantified. Snake peripheral blood leukocyte enrichment yielded >90% lymphocytes with viabilities averaging >70%. Exposure to HgCl2 resulted in significant dose-dependent suppression of proliferative responses relative to spontaneous proliferation at 37.5 and 75 µM (both p ≤ 0.01) but not 3.75 µM (p = 0.99). Mean ± 1 SE concentration of THg in whole blood was 0.127 ± 0.027 mg/kg (wet weight). Based on the in vitro findings with HgCl2 , snakes in systems with heavy Hg pollution may be at risk of immunosuppression, but N. taxispilota at the site in this study appear to be at low risk.

Is there a rational basis for cannabinoids research and development in ocular pain therapy? A systematic review of preclinical evidence.

BACKGROUND: Purpose of the present systematic review is to investigate preclinical evidence in favor of the working hypothesis of efficacy of cannabinoids in ocular pain treatment. METHODS: Literature search includes the most relevant repositories for medical scientific literature from inception until November, 24 2021. Data collection and selection of retrieved records adhere to PRISMA criteria. RESULTS: In agreement with a priori established protocol the search retrieved 2471 records leaving 479 results after duplicates removal. Eleven records result from title and abstract screening to meet the inclusion criteria; only 4 results are eligible for inclusion in the qualitative synthesis impeding meta-analysis. The qualitative analysis highlights the antinociceptive and anti-inflammatory efficacy of Δ8-tetrahydrocannabinol, cannabidiol and its derivative HU-308 and of new racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229. Moreover, CB2R agonists RO6871304 and RO6871085 and CB2R ligand HU910 provide evidence of anti-inflammatory efficacy. CB2 agonist HU308 reduces of 241% uveitis-induced leukocyte adhesion and changes lipidome profile. Methodological and design issues raise concern of risk of bias and the amount of studies is too small for generalization. Furthermore, the ocular pain model used can resemble only inflammatory but not neuropathic pain. CONCLUSIONS: The role of the endocannabinoid system in ocular pain is underinvestigated, since only two studies assessing the effects of cannabinoid receptors modulators on pain behavior and other two on pain-related inflammatory processes are found. Preclinical studies investigating the efficacy of cannabinoids in ocular inflammatory and neuropathic pain models are needed to pave the way for clinical translation.

Methylmercury-induced hair cell loss requires hydrogen peroxide production and leukocytes in zebrafish embryos.

Hearing impairment and deafness is frequently observed as one of the neurological signs in patients with Minamata disease caused by methylmercury (MeHg) poisoning. Loss of hair cells in humans and animals is a consequence of MeHg poisoning. However, it is still not clear how MeHg causes hearing deficits. We employed the hair cells of the lateral line system of zebrafish embryos as a model to explore this question. We exposed transgenic zebrafish embryos to MeHg (30-360 µg/L) at the different stages, and scored the numbers of hair cells. We find that MeHg-induced reduction of hair cells is in a concentration dependent manner. By employing antisense morpholino against to pu.1, we confirm that loss of hair cells involves the action of leukocytes. Moreover, hair cell loss is attenuated by co-treating MeHg-exposed embryos with pharmacological inhibitors of NADPH oxidases named diphenyleneiodonium (DPI) and VAS2870. In situ gene expression analysis showed that genes encoding the SQSTM1-Keap1-Nrf2 systems involved in combating oxidative stress and immune responses are highly expressed in the lateral line organs of embryos exposed to MeHg. This suggests that induction of hydrogen peroxide (H2O2) is the primary effect of MeHg on the hair cells. Genes induced by MeHg are also involved in regeneration of the hair cells. These features are likely related to the capacity of the zebrafish to regenerate the lost hair cells.