Case Report: Evolution of Humoral and Cellular Immunity in Two COVID-19 Breakthrough Infections After BNT162b2 Vaccine.

Background: SARS-CoV-2 breakthrough infections after complete vaccination are increasing whereas their determinants remain uncharacterized. Methods: We analyzed two cases of post-vaccination SARS-CoV-2 infections by α and ß variants, respectively. For each participant both humoral (binding and neutralizing antibodies) and cellular (activation markers and cytokine expression) immune responses were characterized longitudinally. Results: The first participant (P1) was infected by an α variant and displayed an extended and short period of viral excretion and symptom. Analysis of cellular and humoral response 72 h post-symptom onset revealed that P1 failed at developing neutralizing antibodies and a potent CD4 memory response (lack of SARS-CoV-2 specific CD4+IL-2+ cells) and CD8 effector response (CD8+IFNγ+ cells). The second participant (P2) developed post-vaccination SARS-CoV-2 infection by a ß variant, associated with a short period of viral excretion and symptoms. Despite displaying initially high levels and polyfunctional T cell responses, P2 lacked initial ß-directed neutralizing antibodies. Both participants developed and/or increased their neutralization activity and cellular responses against all variants, namely, ß and δ variants that lasts up to 3 months after breakthrough infection. Conclusions: An analysis of cellular and humoral response suggests two possible mechanisms of breakthrough infection: a poor immune response to vaccine and viral evasion to neutralizing antibodies.

Infection or a third dose of mRNA vaccine elicits neutralizing antibody responses against SARS-CoV-2 in kidney transplant recipients.

Transplant recipients, who receive therapeutic immunosuppression to prevent graft rejection, are characterized by high coronavirus disease 2019 (COVID-19)-related mortality and defective response to vaccines. We observed that previous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but not the standard two-dose regimen of vaccination, provided protection against symptomatic COVID-19 in kidney transplant recipients. We therefore compared the cellular and humoral immune responses of these two groups of patients. Neutralizing anti-receptor-binding domain (RBD) immunoglobulin G (IgG) antibodies were identified as the primary correlate of protection for transplant recipients. Analysis of virus-specific B and T cell responses suggested that the generation of neutralizing anti-RBD IgG may have depended on cognate T-B cell interactions that took place in germinal center, potentially acting as a limiting checkpoint. High-dose mycophenolate mofetil, an immunosuppressive drug, was associated with fewer antigen-specific B and T follicular helper (TFH) cells after vaccination; this was not observed in patients recently infected with SARS-CoV-2. Last, we observed that, in two independent prospective cohorts, administration of a third dose of SARS-CoV-2 mRNA vaccine restored neutralizing titers of anti-RBD IgG in about 40% of individuals who had not previously responded to two doses of vaccine. Together, these findings suggest that a third dose of SARS-CoV-2 mRNA vaccine improves the RBD-specific responses of transplant patients treated with immunosuppressive drugs.

Neurotoxic Potential of Deoxynivalenol in Murine Brain Cell Lines and Primary Hippocampal Cultures.

Chronic exposure to the mycotoxin deoxynivalenol (DON) from grain-based food and feed affects human and animal health. Known consequences include entereopathogenic and immunotoxic defects; however, the neurotoxic potential of DON has only come into focus more recently due to the observation of behavioural disorders in exposed farm animals. DON can cross the blood-brain barrier and interfere with the homeostasis/functioning of the nervous system, but the underlying mechanisms of action remain elusive. Here, we have investigated the impact of DON on mouse astrocyte and microglia cell lines, as well as on primary hippocampal cultures by analysing different toxicological endpoints. We found that DON has an impact on the viability of both glial cell types, as shown by a significant decrease of metabolic activity, and a notable cytotoxic effect, which was stronger in the microglia. In astrocytes, DON caused a G1 phase arrest in the cell cycle and a decrease of cyclic-adenosine monophosphate (cAMP) levels. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α was secreted in the microglia in response to DON exposure. Furthermore, the intermediate filaments of the astrocytic cytoskeleton were disturbed in primary hippocampal cultures, and the dendrite lengths of neurons were shortened. The combined results indicated DON's considerable potential to interfere with the brain cell physiology, which helps explain the observed in vivo neurotoxicological effects.

Heteromerization of Endogenous Mu and Delta Opioid Receptors Induces Ligand-Selective Co-Targeting to Lysosomes.

Increasing evidence indicates that native mu and delta opioid receptors can associate to form heteromers in discrete brain neuronal circuits. However, little is known about their signaling and trafficking. Using double-fluorescent knock-in mice, we investigated the impact of neuronal co-expression on the internalization profile of mu and delta opioid receptors in primary hippocampal cultures. We established ligand selective mu-delta co-internalization upon activation by 1-[[4-(acetylamino)phenyl]methyl]-4-(2-phenylethyl)-4-piperidinecarboxylic acid, ethyl ester (CYM51010), [d-Ala2, NMe-Phe4, Gly-ol5]enkephalin (DAMGO), and deltorphin II, but not (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80), morphine, or methadone. Co-internalization was driven by the delta opioid receptor, required an active conformation of both receptors, and led to sorting to the lysosomal compartment. Altogether, our data indicate that mu-delta co-expression, likely through heteromerization, alters the intracellular fate of the mu opioid receptor, which provides a way to fine-tune mu opioid receptor signaling. It also represents an interesting emerging concept for the development of novel therapeutic drugs and strategies.

Behavioural and metabolomic changes from chronic dietary exposure to low-level deoxynivalenol reveal impact on mouse well-being.

The mycotoxin deoxynivalenol (DON) has a high global prevalence in grain-based products. Biomarkers of exposure are detectable in most humans and farm animals. Considering the acute emetic and chronic anorexigenic toxicity of DON, maximum levels for food and feed have been implemented by food authorities. The tolerable daily intake (TDI) is 1 µg/kg body weight (bw)/day for the sum of DON and its main derivatives, which was based on the no-observed adverse-effect level (NOAEL) of 100 µg DON/kg bw/day for anorexic effects in rodents. Chronic exposure to a low-DON dose can, however, also cause inflammation and imbalanced neurotransmitter levels. In the present study, we therefore investigated the impact of a 2-week exposure at the NOAEL in mice by performing behavioural experiments, monitoring brain activation by c-Fos expression, and analysing changes in the metabolomes of brain and serum. We found that DON affected neuronal activity and innate behaviour in both male and female mice. Metabolite profiles were differentiable between control and treated mice. The behavioural changes evidenced at NOAEL reduce the safety margin to the established TDI and may be indicative of a risk for human health.

Morphine-dependent and abstinent mice are characterized by a broader distribution of the neurons co-expressing mu and delta opioid receptors.

Opiate addiction develops as a chronic relapsing disorder upon drug recreational use or following misuse of analgesic prescription. Mu opioid (MOP) receptors are the primary molecular target of opiates but increasing evidence support in vivo functional heteromerization with the delta opioid (DOP) receptor, which may be part of the neurobiological processes underlying opiate addiction. Here, we used double knock-in mice co-expressing fluorescent versions of the MOP and DOP receptors to examine the impact of chronic morphine administration on the distribution of neurons co-expressing the two receptors. Our data show that MOP/DOP neuronal co-expression is broader in morphine-dependent mice and is detected in novel brain areas located in circuits related to drug reward, motor activity, visceral control and emotional processing underlying withdrawal. After four weeks of abstinence, MOP/DOP neuronal co-expression is still detectable in a large number of these brain areas except in the motor circuit. Importantly, chronic morphine administration increased the proportion of MOP/DOP neurons in the brainstem of morphine-dependent and abstinent mice. These findings establish persistent changes in the abstinent state that may modulate relapse and opiate-induced hyperalgesia and also point to the therapeutic potential of MOP/DOP targeting. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.

Peripheral Delta Opioid Receptors Mediate Formoterol Anti-allodynic Effect in a Mouse Model of Neuropathic Pain.

Neuropathic pain is a challenging condition for which current therapies often remain unsatisfactory. Chronic administration of ß2 adrenergic agonists, including formoterol currently used to treat asthma and chronic obstructive pulmonary disease, alleviates mechanical allodynia in the sciatic nerve cuff model of neuropathic pain. The limited clinical data currently available also suggest that formoterol would be a suitable candidate for drug repurposing. The antiallodynic action of ß2 adrenergic agonists is known to require activation of the delta-opioid (DOP) receptor but better knowledge of the molecular mechanisms involved is necessary. Using a mouse line in which DOP receptors were selectively ablated in neurons expressing Nav1.8 sodium channels (DOP cKO), we showed that these DOP peripheral receptors were necessary for the antiallodynic action of the ß2 adrenergic agonist formoterol in the cuff model. Using a knock-in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP), we established in a previous study, that mechanical allodynia is associated with a smaller percentage of DOPeGFP positive small peptidergic sensory neurons in dorsal root ganglia (DRG), with a reduced density of DOPeGFP positive free nerve endings in the skin and with increased DOPeGFP expression at the cell surface. Here, we showed that the density of DOPeGFP positive free nerve endings in the skin is partially restored and no increase in DOPeGFP translocation to the plasma membrane is observed in mice in which mechanical pain is alleviated upon chronic oral administration of formoterol. This study, therefore, extends our previous results by confirming that changes in the mechanical threshold are associated with changes in peripheral DOP profile. It also highlights the common impact on DOP receptors between serotonin noradrenaline reuptake inhibitors such as duloxetine and the ß2 mimetic formoterol.

Three human aminoacyl-tRNA synthetases have distinct sub-mitochondrial localizations that are unaffected by disease-associated mutations.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key enzymes in the mitochondrial protein translation system and catalyze the charging of amino acids on their cognate tRNAs. Mutations in their nuclear genes are associated with pathologies having a broad spectrum of clinical phenotypes, but with no clear molecular mechanism(s). For example, mutations in the nuclear genes encoding mt-AspRS and mt-ArgRS are correlated with the moderate neurodegenerative disorder leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) and with the severe neurodevelopmental disorder pontocerebellar hypoplasia type 6 (PCH6), respectively. Previous studies have shown no or only minor impacts of these mutations on the canonical properties of these enzymes, indicating that the role of the mt-aaRSs in protein synthesis is mostly not affected by these mutations, but their effects on the mitochondrial localizations of aaRSs remain unclear. Here, we demonstrate that three human aaRSs, mt-AspRS, mt-ArgRS, and LysRS, each have a specific sub-mitochondrial distribution, with mt-ArgRS being exclusively localized in the membrane, LysRS exclusively in the soluble fraction, and mt-AspRS being present in both. Chemical treatments revealed that mt-AspRs is anchored in the mitochondrial membrane through electrostatic interactions, whereas mt-ArgRS uses hydrophobic interactions. We also report that novel mutations in mt-AspRS and mt-ArgRS genes from individuals with LBSL and PCH6, respectively, had no significant impact on the mitochondrial localizations of mt-AspRS and mt-ArgRS. The variable sub-mitochondrial locations for these three mt-aaRSs strongly suggest the existence of additional enzyme properties, requiring further investigation to unravel the mechanisms underlying the two neurodegenerative disorders.

Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain.

Peripheral delta opioid (DOP) receptors are essential for the antiallodynic effect of the tricyclic antidepressant nortriptyline. However, the population of DOP-expressing cells affected in neuropathic conditions or underlying the antiallodynic activity of antidepressants remains unknown. Using a mouse line in which DOP receptors were selectively ablated in cells expressing Nav1.8 sodium channels (DOP cKO), we established that these DOP peripheral receptors were mandatory for duloxetine to alleviate mechanical allodynia in a neuropathic pain model based on sciatic nerve cuffing. We then examined the impact of nerve cuffing and duloxetine treatment on DOP-positive populations using a knock-in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP). Eight weeks postsurgery, we observed a reduced proportion of DOPeGFP-positive small peptidergic sensory neurons (calcitonin gene-related peptide (CGRP) positive) in dorsal root ganglia and a lower density of DOPeGFP-positive free nerve endings in the skin. These changes were not present in nerve-injured mice chronically treated with oral duloxetine. In addition, increased DOPeGFP translocation to the plasma membrane was observed in neuropathic conditions but not in duloxetine-treated neuropathic mice, which may represent an additional level of control of the neuronal activity by DOP receptors. Our results therefore established a parallel between changes in the expression profile of peripheral DOP receptors and mechanical allodynia induced by sciatic nerve cuffing.