Molnupiravir for the treatment of COVID-19.

Molnupiravir (MK-4482, EIDD-2801) is a promising broad-spectrum experimental antiviral developed by Merck & Co. It is a nucleoside analogue prodrug that undergoes rapid conversion into nucleoside triphosphate (NTP) by intracellular metabolic processes. NTP inhibits viral polymerase by acting as an alternative substrate. Molnupiravir was initially developed to treat influenza and Venezuelan equine encephalitis virus (VEEV) infection as it exerts its antiviral activity by inhibiting RNA-dependent RNA polymerase (RdRp). Currently, it is being developed for the treatment of SARS-CoV-2 infection. Molnupiravir has demonstrated potent in vitro antiviral activity against positive-sense RNA viruses including influenza viruses, SARS-CoV, SARS-CoV-2 and MERS-CoV with low cytotoxicity and a high resistance barrier. Molnupiravir has been evaluated in phase I, II and III trials where it has demonstrated good efficacy, dose-dependent pharmacokinetics and a sound safety profile. In an interim analysis of a phase III study, treatment with molnupiravir reduced the risk of hospitalization or death by 50% in patients with COVID-19; in the final analysis, the reduction was 30%. On the basis of positive results in clinical trials, molnupiravir has been authorized for emergency use by the U.K. Medicines and Healthcare products Regulatory Agency (MHRA) and the U.S. Food and Drug Administration (FDA) in adults with mild to moderate COVID-19.

A Mouse Model of Pseudomonas aeruginosa Pneumonia.

The rapid innate immune response to respiratory infection is essential to prevent the systemic dissemination of pathogens. This chapter outlines an experimental mouse model of respiratory infection by gram-negative Pseudomonas aeruginosa and analyses of leukocyte trafficking in the lungs. The reader will learn two methods to induce respiratory infection in mice that differ in whether the initial bolus is targeted within a specific lobe of the lung. We then describe a technique based on tissue digestion and flow cytometry that allows the investigator to distinguish leukocytes within different compartments of the lung, and discuss the advantages and limitations to such an approach.

Chitotriosidase Activity Is Counterproductive in a Mouse Model of Systemic Candidiasis.

Mammalian cells do not produce chitin, an insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), although chitin is a structural component of the cell wall of pathogenic microorganisms such as Candida albicans. Mammalian cells, including cells of the innate immune system elaborate chitinases, including chitotriosidase (Chit1), which may play a role in the anti-fungal immune response. In the current study, using knockout mice, we determined the role of Chit1 against systemic candidiasis. Chit1-deficient mice showed significant decrease in kidney fungal burden compared to mice expressing the functional enzyme. Using in vitro anti-candidal neutrophil functional assays, the introduction of the Chit1:chitin digestion end-product, chitobiose (N-acetyl-D-glucosamine dimer, GlcNAc2), decreased fungal-induced neutrophil swarming and Candida killing in vitro. Also, a role for the lectin-like binding site on the neutrophil integrin CR3 (Mac-1, CD11b/CD18) was found through physiological competitive interference by chitobiose. Furthermore, chitobiose treatment of wild type mice during systemic candidiasis resulted in the significant increase in fungal burden in the kidney. These data suggest a counterproductive role of Chit1 in mounting an efficient anti-fungal defense against systemic candidiasis.

The effect of talc particles on phagocytes in co-culture with ovarian cancer cells.

Talc and titanium dioxide are naturally occurring water-insoluble mined products usually available in the form of particulate matter. This study was prompted by epidemiological observations suggesting that perineal use of talc powder is associated with increased risk of ovarian cancer, particularly in a milieu with higher estrogen. We aimed to test the effects of talc vs. control particles on the ability of prototypical macrophage cell lines to curb the growth of ovarian cancer cells in culture in the presence of estrogen. We found that murine ovarian surface epithelial cells (MOSEC), a prototype of certain forms of ovarian cancer, were present in larger numbers after co-culture with macrophages treated to a combination of talc and estradiol than to either agent alone or vehicle. Control particles (titanium dioxide, concentrated urban air particulates or diesel exhaust particles) did not have this effect. Co-exposure of macrophages to talc and estradiol has led to increased production of reactive oxygen species and changes in expression of macrophage genes pertinent in cancer development and immunosurveillance. These findings suggest that in vitro exposure to talc, particularly in a high-estrogen environment, may compromise immunosurveillance functions of macrophages and prompt further studies to elucidate this mechanism.

An Electroencephalography Bioassay for Preclinical Testing of Analgesic Efficacy.

We present a multimodal method combining quantitative electroencephalography (EEG), behavior and pharmacology for pre-clinical screening of analgesic efficacy in vivo. The method consists of an objective and non-invasive approach for realtime assessment of spontaneous nociceptive states based on EEG recordings of theta power over primary somatosensory cortex in awake rats. Three drugs were chosen: (1) pregabalin, a CNS-acting calcium channel inhibitor; (2) EMA 401, a PNS-acting angiotensin II type 2 receptor inhibitor; and (3) minocycline, a CNS-acting glial inhibitor. Optimal doses were determined based on pharmacokinetic studies and/or published data. The effects of these drugs at single or multiple doses were tested on the attenuation of theta power and paw withdrawal latency (PWL) in a rat model of neuropathic pain. We report mostly parallel trends in the reversal of theta power and PWL in response to administration of pregabalin and EMA 401, but not minocycline. We also note divergent trends at non-optimal doses and following prolonged drug administration, suggesting that EEG theta power can be used to detect false positive and false negative outcomes of the withdrawal reflex behavior, and yielding novel insights into the analgesic effects of these drugs on spontaneous nociceptive states in rats.

Sub-paresthesia spinal cord stimulation reverses thermal hyperalgesia and modulates low frequency EEG in a rat model of neuropathic pain.

Paresthesia, a common feature of epidural spinal cord stimulation (SCS) for pain management, presents a challenge to the double-blind study design. Although sub-paresthesia SCS has been shown to be effective in alleviating pain, empirical criteria for sub-paresthesia SCS have not been established and its basic mechanisms of action at supraspinal levels are unknown. We tested our hypothesis that sub-paresthesia SCS attenuates behavioral signs of neuropathic pain in a rat model, and modulates pain-related theta (4-8 Hz) power of the electroencephalogram (EEG), a previously validated correlate of spontaneous pain in rodent models. Results show that sub-paresthesia SCS attenuates thermal hyperalgesia and power amplitude in the 3-4 Hz range, consistent with clinical data showing significant yet modest analgesic effects of sub-paresthesia SCS in humans. Therefore, we present evidence for anti-nociceptive effects of sub-paresthesia SCS in a rat model of neuropathic pain and further validate EEG theta power as a reliable 'biosignature' of spontaneous pain.

Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients.

BACKGROUND: Multi-organ failure occurs during critical illness and is mediated in part by destructive neutrophil-to-endothelial interactions. The ß2 integrin receptor, CR3 (complement receptor 3; Mac-1; CD11b/CD18), which binds endothelial intercellular adhesion molecule-1 (ICAM-1), plays a key role in promoting the adhesion of activated neutrophils to inflamed endothelia which, when prolonged and excessive, can cause vascular damage. Leukadherin-1 (LA-1) is a small molecule allosteric activator of CR3 and has been shown to promote adhesion of blood neutrophils to inflamed endothelium and restrict tissue infiltration. Therefore, LA-1 offers a novel mechanism of anti-inflammatory action by activation, rather than inhibition, of the neutrophil CR3 integrin. However, whether promotion of neutrophil-to-endothelial interaction by this novel therapeutic is of benefit or detriment to endothelial barrier function is not known. METHODS: Critically ill septic and trauma patients were prospectively enrolled from the surgical and the trauma ICU. Blood was collected from these patients and healthy volunteers. Neutrophils were isolated by dextran sedimentation and adhered to TNF-α (tumor necrosis factor-α)-activated human umbilical vein endothelial (HUVEC) monolayers in the presence or absence of fMLP (formylmethionine-leucine-phenylalanine) and/or LA-1. Electric cell-substrate impedance sensing (ECIS) and exposure of underlying collagen were used to quantify endothelial barrier function and permeability. RESULTS: Neutrophils from critically ill trauma and septic patients caused similar degrees of endothelial barrier disruption which exceeded that caused by cells obtained from healthy controls both kinetically and quantitatively. LA-1 protected barrier function in the absence and presence of fMLP which served as a secondary stimulant to cause maximal loss of barrier function. LA-1 protection was also observed by quantifying collagen exposure underlying endothelial cells challenged with fMLP-stimulated neutrophils. LA-1 treatment resulted in decreased migration dynamics of neutrophils crawling on an endothelial monolayer with reduced speed (µm/s = 0.25 ± 0.01 vs. 0.06 ± 0.01, p < 0.05), path length (µm = 199.5 ± 14.3 vs. 42.1 ± 13.0, p < 0.05), and displacement (µm = 65.2 ± 4.7 vs. 10.4 ± 1.3; p < 0.05). CONCLUSION: Neutrophils from patients with trauma or sepsis cause endothelial barrier disruption to a similar extent relative to each other. The CR3 agonist LA-1 protects endothelial barrier function from damage caused by neutrophils obtained from both populations of critically ill patients even when exposed to secondary stimulation.

Thalamic Bursts Down-regulate Cortical Theta and Nociceptive Behavior.

We tested the relation between pain behavior, theta (4-8 Hz) oscillations in somatosensory cortex and burst firing in thalamic neurons in vivo. Optically-induced thalamic bursts attenuated cortical theta and mechanical allodynia. It is proposed that thalamic bursts are an adaptive response to pain that de-synchronizes cortical theta and decreases sensory salience.

Electroencephalographic frontal synchrony and caudal asynchrony during painful hand immersion in cold water.

Recent studies in our laboratory showed that cortical theta oscillations correlate with pain in rodent models. In this study, we sought to validate our pre-clinical data using EEG recordings in humans during immersion of the hand in ice cold water, a moderately noxious stimulus. Power spectral analysis shows that an increase in pain score is associated with an increase in power amplitude within a frequency range of 6-7Hz at the frontal (Fz) electrode. These results are consistent with our previous pre-clinical animal studies and the clinical literature. We also report a novel reduction in power at the caudal (O1) electrode within a broader 3-30Hz rand and decreased coherence between Fz and C3, C4 electrodes within the theta (4-8Hz) and low beta (13-21Hz) bands, while coherence (an indirect measure of functional connectivity) between Fz and O1 increased within the theta and alpha (8-12Hz) bands. We argue that pain is associated with EEG frontal synchrony and caudal asynchrony, leading to the disruption of cortico-cortical connectivity.

Electroencephalographic signatures of pain and analgesia in rats.

Pain modulates rhythmic neuronal activity recorded by Electroencephalography (EEG) in humans. Our laboratory previously showed that rat models of acute and neuropathic pain manifest increased power in primary somatosensory cortex (S1) recorded by electrocorticography (ECoG). In this study, we hypothesized that pain increases EEG power and corticocortical coherence in different rat models of pain, whereas treatments with clinically effective analgesics reverse these changes. Our results show increased cortical power over S1 and prefrontal cortex (PFC) in awake, freely behaving rat models of acute, inflammatory and neuropathic pain. Coherence between PFC and S1 is increased at a late, but not early, time point during the development of neuropathic pain. Electroencephalography power is not affected by ibuprofen in the acute pain model. However, pregabalin and mexiletine reverse the changes in power and S1-PFC coherence in the inflammatory and neuropathic pain models. These data suggest that quantitative EEG might be a valuable predictor of pain and analgesia in rodents.

T-type calcium channel blocker Z944 restores cortical synchrony and thalamocortical connectivity in a rat model of neuropathic pain.

Oscillations are fundamental to communication between neuronal ensembles. We previously reported that pain in awake rats enhances synchrony in primary somatosensory cortex (S1) and attenuates coherence between S1 and ventral posterolateral (VPL) thalamus. Here, we asked whether similar changes occur in anesthetized rats and whether pain modulates phase-amplitude coupling between VPL and S1. We also hypothesized that the suppression of burst firing in VPL using Z944, a novel T-type calcium channel blocker, restores S1 synchrony and thalamocortical connectivity. Local field potentials were recorded from S1 and VPL in anesthetized rats 7 days after sciatic chronic constriction injury (CCI). In rats with CCI, low-frequency (4-12 Hz) synchrony in S1 was enhanced, whereas VPL-S1 coherence and theta-gamma phase-amplitude coupling were attenuated. Moreover, Granger causality showed decreased informational flow from VPL to S1. Systemic or intrathalamic delivery of Z944 to rats with CCI normalized these changes. Systemic Z944 also reversed thermal hyperalgesia and conditioned place preference. These data suggest that pain-induced cortical synchrony and thalamocortical disconnectivity are directly related to burst firing in VPL.

Cortical theta is increased while thalamocortical coherence is decreased in rat models of acute and chronic pain.

Thalamocortical oscillations are critical for sensory perception. Although pain is known to disrupt synchrony in thalamocortical oscillations, evidence in the literature is controversial. Thalamocortical coherence has been reported to be increased in patients with neurogenic pain but decreased in a rat model of central pain. Moreover, theta (4 to 8 Hz) oscillations in primary somatosensory (S1) cortex are speculated to predict pain in humans. To date, the link between pain and network oscillations in animal models has been understudied. Thus, we tested the hypothesis that pain disrupts thalamocortical coherence and S1 theta power in two rat models of pain. We recorded electrocorticography (ECoG) waveforms over S1 and local field potentials (LFP) within ventral posterolateral thalamus in freely behaving rats under spontaneous (stimulus-independent) pain conditions. Rats received intradermal capsaicin injection (Cap) in the hindpaw, followed hours later by chronic constriction injury (CCI) of the sciatic nerve lasting several days. Our results show that pain decreases coherence between LFP and ECoG waveforms in the 2- to 30-Hz range, and increases ECoG power in the theta range. These changes are short-lasting after Cap and longer-lasting after CCI. These data might be particularly relevant to preclinical correlates of spontaneous pain-like behavior, with potential implications to clinical biomarkers of ongoing pain.

High-frequency stimulation in the ventral posterolateral thalamus reverses electrophysiologic changes and hyperalgesia in a rat model of peripheral neuropathic pain.

Chronic neuropathic pain is associated with long-term changes at multiple levels of the neuroaxis, including in the brain, where electrical stimulation has been used to manage severe pain conditions. However, the clinical outcome of deep brain stimulation is often mixed, and the mechanisms are poorly understood. By means of electrophysiologic methods, we sought to characterize the changes in neuronal activity in the ventral posterolateral nucleus of the thalamus (VPL) in a rat model of peripheral neuropathic pain, and to reverse these changes with low-voltage, high-frequency stimulation (HFS) in the VPL. Extracellular single-unit neuronal activity was recorded in naive rats and in those with sciatic chronic constriction injury (CCI). Seven days after CCI, brush- and pinch-evoked firing, as well as spontaneous firing and afterdischarge, were significantly increased compared to naive rats. Spontaneous rhythmic oscillation in neuronal firing was also observed in rats with CCI. HFS decreased neuronal firing rates in rats with CCI up to ~50% except for spontaneous activity, whereas low-frequency stimulation had no effect. Compared to naive rats, burst firing properties (burst events, percentage of spikes in burst, and mean interburst time) were altered in rats with CCI, whereas these changes were reversed to near normal after HFS. Thermal hyperalgesia in rats with CCI was significantly attenuated by HFS. Therefore, this study demonstrates that electrical stimulation within the VPL can effectively modulate some nociceptive phenomena associated with peripheral neuropathic pain.

Minocycline injection in the ventral posterolateral thalamus reverses microglial reactivity and thermal hyperalgesia secondary to sciatic neuropathy.

We hypothesized that microglia in the ventral posterolateral (VPL) nucleus of the thalamus are reactive following peripheral nerve injury, and that inhibition of microglia by minocycline injection in the VPL attenuates thermal hyperalgesia. Our results show increased expression of OX-42 co-localized with phosphorylated p38MAPK (P-p38) in the VPL seven days after chronic constriction injury (CCI) of the sciatic nerve. However, astrocytic GFAP expression in the VPL is unchanged 7 and 14 days after CCI. Microinjection of minocycline into the VPL contralateral to CCI reverses thermal hyperalgesia, whereas vehicle injection has no effect on paw withdrawal latency. Minocycline abrogates the increased expression of OX-42 in the VPL after CCI. Therefore, peripheral nerve injury favors a hyperactive microglial phenotype in the VPL, suggesting remote neuroimmune signaling from the damaged nerve to the brain, concomitant with neuropathic behavior that is reversed by local intervention in the VPL to inhibit microglia.

The effect of PGG-beta-glucan on neutrophil chemotaxis in vivo.

The beta-glucans are long-chain polymers of glucose in beta-(1,3)(1,6) linkages, which comprise the fungal cell wall and stimulate cells of the innate immune system. Previous in vitro studies have shown the ability of beta-glucan to increase the chemotactic capacity of human neutrophils. The current study examined an in vivo correlate of that observation by testing the hypothesis that systemic beta-glucan treatment would result in enhanced migration of neutrophils into a site of inflammation and improve antimicrobial function. A model of acute inflammation was used in which polyvinyl alcohol sponges were implanted subcutaneously into the dorsum of rats. Animals treated with beta-glucan showed a 66 +/- 6% and 186 +/- 42% increase in wound cell number recovered 6 and 18 h postwounding, respectively. Increased migration did not correlate with increased chemoattractant content of wound fluid, alterations in neutrophil-induced loss of endothelial barrier function, or changes in neutrophil adhesion to endothelial cells. Systemic administration of SB203580 abrogated the enhanced migration by beta-glucan without altering normal cellular entry into the wound. Studies also showed a priming effect for chemotaxis and respiratory burst in circulating neutrophils isolated from beta-glucan-treated animals. Heightened neutrophil function took place without cytokine elicitation. Furthermore, beta-glucan treatment resulted in a 169 +/- 28% increase in neutrophil number and a 60 +/- 9% decrease in bacterial load in the bronchoalveolar lavage fluid of Escherichia coli pneumonic animals. Taken together, these findings demonstrate that beta-glucan directly affects the chemotactic capacity of circulating neutrophils through a p38 mitogen-activated protein kinase-dependent mechanism and potentiates antimicrobial host defense.

Differential effects of macrophage inflammatory chemokine-2 and keratinocyte-derived chemokine on hemorrhage-induced neutrophil priming for lung inflammation: assessment by adoptive cells transfer in mice.

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 +/- 5 mmHg and were then administered anti-MIP- 2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringer's lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; microU/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (increased MPO, increased % esterase+ cells in tissue) into the lung and local tissue inflammation (increased IL-6/MIP-2, decreased IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.

Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.

Acute lung injury (ALI) leading to respiratory distress is a common sequela of shock/trauma, however, modeling this process in mice with a single shock or septic event is inconsistent. One explanation is that hemorrhage is often just a "priming insult," thus, secondary stimuli may be required to "trigger" ALI. To test this we carried out studies in which we assessed the capacity of hemorrhage alone or hemorrhage followed by septic challenge (CLP) to induce ALI. Lung edema, bronchoalveolar lavage interleukin (IL)-6, alveolar congestion, as well as lung IL-6, macrophage inflammatory protein (MIP)-2, and myeloperoxidase (MPO) activity were all increased in mice subjected to CLP at 24 but not 72 hours following hemorrhage. This was associated with a marked increase in the susceptibility of these mice to septic mortality. Peripheral blood neutrophils derived from 24 hours post-hemorrhage, but not Sham animals, exhibited an ex vivo decrease in apoptotic frequency and an increase in respiratory burst capacity, consistent with in vivo "priming." Subsequently, we observed that adoptive transfer of neutrophils from hemorrhaged but not sham-hemorrhage animals to neutropenic recipients reproduce ALI when subsequently septically challenged, implying that this priming was mediated by neutrophils. We also found marked general increases in lung IL-6, MIP-2, and MPO in mice deficient for toll-like receptor (TLR-4) or the combined lack of TLR-4/FasL. However, the TLR-4 defect markedly attenuated neutrophil influx into the lung while not altering the change in local cytokine/chemokine expression. Alternatively, the combined loss of FasL and TLR-4 did not inhibit the increase in MPO and exacerbated lung IL-6/MIP-2 levels even further.