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Early and persistent activation of complement is considered to play a key role in the pathogenesis of COVID-19. Complement activation products orchestrate a proinflammatory environment that might be critical for the induction and maintenance of a severe inflammatory response to SARS-CoV-2 by recruiting cells of the cellular immune system to the sites of infection and shifting their state of activation towards an inflammatory phenotype. It precedes pathophysiological milestone events like the cytokine storm, progressive endothelial injury triggering microangiopathy, and further complement activation, and causes an acute respiratory distress syndrome (ARDS). To date, the application of antiviral drugs and corticosteroids have shown efficacy in the early stages of SARS-CoV-2 infection, but failed to ameliorate disease severity in patients who progressed to severe COVID-19 pathology. This report demonstrates that lectin pathway (LP) recognition molecules of the complement system, such as MBL, FCN-2 and CL-11, bind to SARS-CoV-2 S- and N-proteins, with subsequent activation of LP-mediated C3b and C4b deposition. In addition, our results confirm and underline that the N-protein of SARS-CoV-2 binds directly to the LP- effector enzyme MASP-2 and activates complement. Inhibition of the LP using an inhibitory monoclonal antibody against MASP-2 effectively blocks LP-mediated complement activation. FACS analyses using transfected HEK-293 cells expressing SARS-CoV-2 S protein confirm a robust LP-dependent C3b deposition on the cell surface which is inhibited by the MASP-2 inhibitory antibody. In light of our present results, and the encouraging performance of our clinical candidate MASP-2 inhibitor Narsoplimab in recently published clinical trials, we suggest that the targeting of MASP-2 provides an unsurpassed window of therapeutic efficacy for the treatment of severe COVID-19.

Peroxisome proliferator-activated receptor γ (PPARγ) is an intracellular transcription factor whose signaling activation by the selective agonist pioglitazone reduces alcohol drinking and alcohol-seeking behavior in rats. The present study utilized the two-bottle choice and operant self-administration procedures to investigate neuroanatomical substrates that mediate the effects of PPARγ agonism on alcohol drinking and seeking in msP rats. Bilateral infusions of pioglitazone (0, 5, and 10 μg/μl) in the rostromedial tegmental nucleus (RMTg) decreased voluntary alcohol drinking and alcohol self-administration. Microinjections of pioglitazone in the ventral tegmental area (VTA), central amygdala (CeA), and nucleus accumbens (NAc) shell had no such effect. Notably, water, food, and saccharin consumption was unaltered by either treatment. The yohimbine-induced reinstatement of alcohol seeking was prevented by infusions of pioglitazone (0, 2.5, 5, and 10 μg/μl) in the CeA, VTA, and RMTg but not in the NAc shell. These results emphasize the involvement of mesocorticolimbic circuitries in mediating the effects of PPARγ agonists on alcohol drinking and seeking. These results will facilitate future studies that investigate the pathophysiological role of PPARγ in alcohol use disorder and help clarify the mechanisms by which the activation of this receptor decreases the motivation for drinking.

RationalePeroxisome Proliferator Activator receptors (PPARs) are intracellular receptors that function as transcription factors, which regulate specific metabolic and inflammatory processes. PPARs are broadly distributed in the body and are also expressed in the central nervous system, especially in areas involved in addiction-related behavioral responses. Recent studies support a role of PPARs in alcoholism and pioglitazone: a PPARγ agonist used for treatment of type 2 diabetes showed efficacy in reducing alcohol drinking, stress-induced relapse, and alcohol withdrawal syndrome in rats.Objectives and MethodsIn the current work, we tested the pharmacological effects of pioglitazone on binge-like alcohol consumption using an intermittent two-bottle choice paradigm in Wistar rats and on the “drinking in the dark” (DID) model in mice with selective deletion of PPARγ in neurons.ResultsOur data show that repeated administration of pioglitazone (10, 30 mg/kg) reduces high voluntary alcohol consumption in Wistar rats. Pre-treatment with the selective PPARγ antagonist GW9662 (5 mg/kg) completely prevented the effect of pioglitazone, demonstrating that its action is specifically mediated by activation of PPARγ. In line with this result, repeated administration of pioglitazone (30 mg/kg) attenuated binge alcohol consumption in PPARγ(+/+) mice. Whereas in PPARγ(−/−) mice, which exhibit reduced alcohol consumption, pioglitazone had no effect. Of note, PPARγ(−/−) mice exhibited lower patterns of alcohol drinking without showing difference in sucrose (control) intake. Interestingly, PPARγ(−/−) mice displayed a higher sensitivity to the sedative and ataxic effect of alcohol compared with their wild-type counterpart.ConclusionsCollectively, these data suggest that PPARγ agonists, and specifically pioglitazone, could be potential therapeutics for the treatment of binge alcohol drinking.

A high incidence of secondary Klebsiella pneumoniae and Staphylococcus aureus infection were observed in patients with severe COVID-19. The cause of this predisposition to infection is unclear. Our data demonstrate consumption of complement in acute COVID-19 patients reflected by low levels of C3, C4, and loss of haemolytic activity. Given that the elimination of Gram-negative bacteria depends in part on complement-mediated lysis, we hypothesised that secondary hypocomplementaemia is rendering the antibody-dependent classical pathway activation inactive and compromises serum bactericidal activity (SBA). 217 patients with severe COVID-19 were studied. 142 patients suffered secondary bacterial infections. Klebsiella species were the most common Gram-negative organism, found in 58 patients, while S. aureus was the dominant Gram-positive organism found in 22 patients. Hypocomplementaemia was observed in patients with acute severe COVID-19 but not in convalescent survivors three months after discharge. Sera from patients with acute COVID-19 were unable to opsonise either K. pneumoniae or S. aureus and had impaired complement-mediated killing of Klebsiella. We conclude that hyperactivation of complement during acute COVID-19 leads to secondary hypocomplementaemia and predisposes to opportunistic infections.

Acute respiratory distress syndrome (ARDS) is a life-threatening disorder with a high rate of mortality. Complement activation in ARDS initiates a robust inflammatory reaction that can cause progressive endothelial injury in the lung. Here, we tested whether inhibition of the lectin pathway of complement could reduce the pathology and improve the outcomes in a murine model of LPS-induced lung injury that closely mimics ARDS in human. In vitro , LPS binds to murine and human collectin 11, human MBL and murine MBL-A, but not to C1q, the recognition subcomponent of the classical pathway. This binding initiates deposition of the complement activation products C3b, C4b and C5b-9 on LPS via the lectin pathway. HG-4, a monoclonal antibody that targets MASP-2, a key enzyme in the lectin pathway, inhibited lectin pathway functional activity in vitro , with an IC 50 of circa 10nM. Administration of HG4 (5mg/kg) in mice led to almost complete inhibition of the lectin pathway activation for 48hrs, and 50% inhibition at 60hrs post administration. Inhibition of the lectin pathway in mice prior to LPS-induced lung injury improved all pathological markers tested. HG4 reduces the protein concentration in bronchoalveolar lavage fluid (p<0.0001) and levels of myeloid peroxide (p<0.0001), LDH (p<0.0001), TNFα and IL6 (both p<0.0001). Lung injury was significantly reduced (p<0.001) and the survival time of the mice increased (p<0.01). From the previous findings we concluded that inhibition of the lectin pathway has the potential to prevent ARDS pathology.

Complement components are depleted during acute infection, and the proinflammatory anaphylatoxins C3a and C5a, which play a major role in the development of acute respiratory distress syndrome (ARDS), are released.1–4 Extensive complement activation may be prognostic of poor outcome – higher degrees of activation, especially of C3, being associated with fatality.4 Complement inhibitors are therefore considered promising therapeutics for tackling acute COVID-19 ARDS; potential targets include C3 and the C5a/C5aR axis as well as the lectin and terminal pathways.5,6 Analysis of post-mortem tissue from patients who succumbed to COVID-19 revealed severe endothelial damage characteristic of microangiopathies, with deposits of complement activation products, most notably the lectin pathway serine protease MASP-2.7,8 We have previously found evidence of a novel mechanism of lectin pathway activation, driven by a direct interaction between the SARS-CoV-2 nucleocapsid protein and the lectin pathway serine protease MASP-2.9 Narsoplimab is a fully humanised monoclonal antibody that targets MASP-2, blocking the activation of the lectin pathway (S1). TABLE 1 Clinical parameters for the subjects included in this study prior to treatment Clinical characteristics Narsoplimab-treated COVID-19 cohort (N = 9) Untreated COVID-19 cohort (N = 9) Median Range Median Range Age – years 62 41–79 74 50–79 Weight – kg 90 75–105 85 65–130 BMI – kg/m2 27.8 25.5–32.5 29 21–55 Sex Female 1/9 3/9 ARDS severity (Berlin criteria) Mild 2/9 Moderate 3/9 5/9 Severe 6/9 2/9 Laboratory findings Median Range Median Range PaO2:FiO2 ratio 140 110–250 180 80–284 White cell count – per mm3 8470 4600–21 520 7100 3140–14 590 Lymphocyte count – per mm3 550 350–1940 390 142–750 Platelet count – ×103 per mm3 247 154–313 223 102–444 Haemoglobin – g/dl 12.7 11.3–15.4 10.3 7.9–14.8 Other findings (reference ranges) C-reactive protein (.0–1.0 mg/dl) 14.5 1.9–17.9 9.3 .3–26.9 Lactate dehydrogenase (120/246 U/L) 443 312–582 346.5 190–494 Aspartate aminotransferase (13–40 U/L) 55 19–89 40 10–59 Alanine aminotransferase (7–40 U/L) 44 22–252 23 11–148 Creatinine (.3–1.3 mg/dl) .79 .48–1.13 1.85 .81–4.34 d-Dimer (<500 ng/ml) 1141 354–4471 1730 288–5020 Comorbidities Diabetes 2/9 3/9 Hypertension 5/9 6/9 Dyslipidaemia 1/9 3/9 Cardiovascular disease 0/9 5/9 Obesity (BMI ≥ 30 kg/m2) 2/8a 4/9 Overweight (BMI ≥ 25 kg/m2) 6/8a 8/9 Radiologic findings Bilateral interstitial abnormalities 9/9 9/9 Note: Data collected on day 0 of the study, immediately prior to administration of the first dose of narsoplimab in the treated cohort. [...]targeting the lectin pathway may suffice to reduce anaphylatoxin release below the threshold for maintaining ARDS and restore the bactericidal activity and opsonisation required for defence against secondary infection.

The purpose of this study was to evaluate the effect of OMS302 on intraoperative pupil diameter and early postoperative ocular pain when administered during intraocular lens replacement surgery. Four hundred and six patients (406 study eyes; 202 in the OMS302 group and 204 in the placebo group) were entered into this randomized, double-masked, placebo-controlled, multicenter Phase III study, which was conducted at 15 centers in the USA and the Netherlands. The patients received OMS302 (60.75 mM phenylephrine HCl and 11.25 mM ketorolac tromethamine) or placebo in irrigation solution during intraocular lens replacement. No other changes in procedure were required. Coprimary endpoints were change in pupil diameter over time from surgical baseline to end of procedure and patient-reported ocular pain during the first 12 hours postoperatively. Secondary endpoints included additional measures of pupil diameter and postoperative pain. OMS302 was superior to placebo in maintaining intraoperative mydriasis, preventing miosis, and reducing postoperative pain. The weighted mean (standard error) difference (OMS302 - placebo) in change in the area under the curve from baseline for pupil diameter was 0.590 ([0.049]; 95% confidence interval 0.494 to 0.686; P<0.0001). For ocular pain scores, the weighted mean (standard error) difference was -4.580 ([1.192]; 95% confidence interval -6.917 to 2.244; P=0.0002). All secondary efficacy results favored OMS302. Specifically, analyses supporting prevention of miosis (patients with ≥6 mm pupil diameter at completion of cortical clean-up and those with <6 mm diameter at any time during surgery) were significant for OMS302 (95.9% versus 77.0% and 9.2% versus 38.0%, respectively; P<0.0001 for each endpoint). OMS302 was well tolerated and not associated with any unexpected adverse events. OMS302 maintained mydriasis, prevented miosis, and reduced early postoperative pain when administered in irrigation solution during intraocular lens replacement, with a safety profile similar to that of placebo. OMS302 is preservative-free and bisulfite-free, and its administration does not require any modification to the surgical procedure.

Rationale Relapse to opioids is often driven by the avoidance of the aversive states of opioid withdrawal. We recently demonstrated that activation of peroxisome proliferator-activated receptor gamma (PPARγ) by pioglitazone reduces the motivation for heroin and attenuates its rewarding properties. However, the role of PPARγ in withdrawal and other forms of relapse to heroin is unknown. Objectives To further address this issue, we investigated the role of PPARγ on the development and expression of morphine withdrawal in mice and the effect of pioglitazone on several forms of heroin relapse in rats. Methods We induced physical dependence to morphine in mice by injecting morphine twice daily for 6 days. Withdrawal syndrome was precipitated on day 6 with an injection of naloxone. In addition, different groups of rats were trained to self-administer heroin and, after the extinction, the relapse was elicited by cues, priming, or stress. The effect of different doses of pioglitazone was tested on these different paradigms. Results Data show that chronic and acute administration of pioglitazone attenuates morphine withdrawal symptoms, and these effects are mediated by activation of PPARγ receptors. Activation of PPARγ by pioglitazone also abolishes yohimbine-induced reinstatement of heroin seeking and reduces heroin-induced reinstatement, while it does not affect cue-induced relapse. Conclusions These findings provide new insights on the role of PPARγ on opioid dependence and suggest that pioglitazone may be useful for the treatment of opioid withdrawal in opioid-addicted individuals.

PPARγ is one of the three isoforms identified for the peroxisome proliferator-activated receptors (PPARs) and is the receptor for the thiazolidinedione class of anti-diabetic medications including pioglitazone. PPARγ has been long studied for its role in adipogenesis and glucose metabolism, but the discovery of the localization in ventral tegmental area (VTA) neurons opens new vistas for a potential role in the regulation of reward processing and motivated behavior in drug addiction. Here, we demonstrate that activation of PPARγ by pioglitazone reduces the motivation for heroin and attenuates its rewarding properties. These effects are associated with a marked reduction of heroin-induced increase in phosphorylation of DARPP-32 protein in the nucleus accumbens (NAc) and with a marked and selective reduction of acute heroin-induced elevation of extracellular dopamine (DA) levels in the NAc shell, as measured by in vivo microdialysis. Through ex vivo electrophysiology in acute midbrain slices, we also show that stimulation of PPARγ attenuates opioid-induced excitation of VTA DA neurons via reduction of presynaptic GABA release from the rostromedial tegmental nucleus (RMTg). Consistent with this finding, site-specific microinjection of pioglitazone into the RMTg but not into the VTA reduced heroin taking. Our data illustrate that activation of PPARγ may represent a new pharmacotherapeutic option for the treatment of opioid addiction.