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A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.

Upon structure comparison between IL-1 beta and its antagonist IL-1ra, single or multiple residues along the IL-1 beta sequence were replaced with the corresponding amino acids present in the IL-1ra protein, in the attempt to identify sites important for receptor binding and for biologic activity on the two molecules. Ten of fifteen mutant proteins had activity comparable to that of wild-type IL-1 beta in three different biologic assays and in receptor binding, indicating that the introduced changes did not influence the functional structure of the protein. Conversely, three mutants (SMIL-9: 127/263 R/T-->W/Y; SMIL-10: 125/127/263/265 T/R/T/Q-->R/W/Y/E; SMIL-15:222/227 I/E-->S/S) showed an increased binding capacity for IL-1RI, not paralleled by increased agonist activity, indicating that the introduced IL-1ra residues could be involved in the nonagonist IL-1RI binding site. On the other hand, two mutants showed diminished binding capacity with concomitant decrease in biologic activity. Both mutants (SMIL-1, five substitutions in the loop 202-214; and SMIL-3, total replacement of the loop 164-173 with the IL-1ra stretch 52-55) included substitutions of residues allegedly important for agonist binding to IL-1RI. Mutant SMIL-3 showed the most profound reduction in binding capacity for IL-1RI (CDw121a) and a more than 1,000-fold reduced biologic activity both in vitro and in vivo, but it retained full capacity of binding to IL-1RII (CDw121b) and acted as a selective antagonist of IL-1RII. From these results the following conclusions can be drawn. IL-1 beta binds to IL-1RI and to IL-1RII through different sites, and the loop 164-173 appears as one of the areas involved in the selective interaction with IL-1RI. Agonist (IL-1 beta) and nonagonist (IL-1ra) binding to IL-1RI occur through distinct sites, with loops 164-173 and 202-214 of IL-1 beta identified as two of the sites selectively involved in agonist binding to the activating receptor.

24 workers (10 involved in the preparation and 14 in administration) exposed to cyclophosphamide (CP) and ifosfamide (IF) in two Italian hospitals were monitored. The extent of exposure was assessed by the analysis of air samples, wipe samples, pads and gloves. Urinary excretion at the beginning and at the end of the work shift was also measured by liquid-liquid extraction and analysis by HPLC-MS/MS. 3 out of 24 air samples resulted to be positive for CP or IF. In wipe samples, CP concentrations ranging from0.001 to 82.4 micrograms/dm2 in Hospital A (32 samples) and from 0.2 to 383.3 micrograms/dm2 in Hospital B (17 samples), were found. IF concentrations varied from0.001 to 90.9 micrograms/dm2 in Hospital A and from 0.01 to 141.5 micrograms/dm2 in Hospital B. Pads (from 11 to 13 for each operator) were contaminated with CP and IF especially on arms, legs and chest. The use of a plastic-backed liner on the working tray in the laminar flow hoods was demonstrated to compromise the containment properties of the hood. Urine samples were positive for CP in 50% of the workers (range: 0.1-2.1 micrograms/L), whereas IF was detected in 2 subjects only (range: 0.1-0.8 microgram/L). The results from this investigation demonstrated that vertical laminar airflow hoods, when incorrectly used, might represent a source of contamination and that higher risk may depend on lack of educational programmes and observance of preventive guidelines.

Given the enormous social and health impact of the pandemic triggered by severe acute respiratory syndrome 2 (SARS-CoV-2), the scientific community made a huge effort to provide an immediate response to the challenges posed by Coronavirus disease 2019 (COVID-19). One of the most important proteins of the virus is an enzyme, called 3CLpro or main protease, already identified as an important pharmacological target also in SARS and Middle East respiratory syndrome virus (MERS) viruses. This protein triggers the production of a whole series of enzymes necessary for the virus to carry out its replicating and infectious activities. Therefore, it is crucial to gain a deeper understanding of 3CLpro structure and function in order to effectively target this enzyme. All-atoms molecular dynamics (MD) simulations were performed to examine the different conformational behaviors of the monomeric and dimeric form of SARS-CoV-2 3CLpro apo structure, as revealed by microsecond time scale MD simulations. Our results also shed light on the conformational dynamics of the loop regions at the entry of the catalytic site. Studying, at atomic level, the characteristics of the active site and obtaining information on how the protein can interact with its substrates will allow the design of molecules able to block the enzymatic function crucial for the virus.

Preclinical studies in immature and adult rodents and clinical observations show that neuroinflammation and oxidative stress are rapid onset phenomena occurring in the brain during status epilepticus and persisting thereafter. Notably, both neuroinflammation and oxidative stress contribute to the acute and long-term sequelae of status epilepticus thus representing potential druggable targets. Antiinflammatory drugs that interfere with the IL-1β pathway, such as anakinra, can control benzodiazepine-refractory status epilepticus in animals, and there is recent proof-of-concept evidence for therapeutic effects in children with Febrile infection related epilepsy syndrome (FIRES). Inhibitors of monoacylglycerol lipase and P2X7 receptor antagonists are also promising antiinflammatory drug candidates for rapidly aborting de novo status epilepticus and provide neuroprotection. Antiinflammatory and antioxidant drugs administered to rodents during status epilepticus and transiently thereafter, prevent long-term sequelae such as cognitive deficits and seizure progression in animals developing epilepsy. Some drugs are already in medical use and are well-tolerated, therefore, they may be considered for treating status epilepticus and its neurological consequences. Finally, markers of neuroinflammation and oxidative stress are measureable in peripheral blood and by neuroimaging, which offers an opportunity for developing prognostic and predictive mechanistic biomarkers in people exposed to status epilepticus. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures.

BACKGROUND: Severe sleep disruption is a well-documented problem in mechanically ventilated, critically ill patients during their time in the intensive care unit (ICU), but little attention has been paid to the period when these patients become clinically stable and are transferred to a step-down unit (SDU). We monitored the 24-h sleep pattern in 2 groups of patients, one on mechanical ventilation and the other breathing spontaneously, admitted to our SDU to assess the presence of sleep abnormalities and their association with mechanical ventilation. METHODS: Twenty-two patients admitted to an SDU underwent 24-h polysomnography with monitoring of noise and light. RESULTS: One patient did not complete the study. At night, 10 patients showed reduced sleep efficiency, 6 had reduced percentage of REM sleep, and 3 had reduced percentage of slow wave sleep (SWS). Sleep amount and quality did not differ between patients breathing spontaneously and those on mechanical ventilation. Clinical severity (SAPS(II) score) was significantly correlated with daytime total sleep time and efficiency (r = 0.51 and 0.5, P < 0.05, respectively); higher pH was correlated with reduced sleep quantity and quality; and higher PaO(2) was correlated with increased SWS (r = 0.49; P = 0.02). CONCLUSIONS: Patients admitted to an SDU after discharge from an ICU still have a wide range of sleep abnormalities. These abnormalities are mainly associated with a high severity score and alkalosis. Mechanical ventilation does not appear to be a primary cause of sleep impairment.