We have developed and characterized a model of isoproterenol (ISO)-induced myocardial necrosis, identifying three stages of cardiac damage: a pre-infarction (0–12 h), infarction (24 h), and post-infarction period (48–96 h). Using this model, we have previously found alterations in calcium homeostasis and their relationship with oxidant stress in mitochondria, which showed deficient oxygen consumption and coupled ATP synthesis. Therefore, the present study was aimed at assessing the mitochondrial ability to transport and oxidize cytoplasmic reducing equivalents (NADH), correlating the kinetic parameters of the malate-aspartate shuttle, oxidant stress, and mitochondrial functionality. Our results showed only discreet effects during the cardiotoxic ISO action on the endogenous malate-aspartate shuttle activity, suggesting that endogenous mitochondrial NADH oxidation capacity (Nohl dehydrogenase) was not affected by the cellular stress. On the contrary, the reconstituted system showed significant enhancement in maximal capacity of the malate-aspartate shuttle activity only at later times (post-infarction period), probably as a compensatory part of cardiomyocytes’ response to the metabolic and functional consequences of the infarcted tissue. Therefore, these findings support the notion that heart damage associated with myocardial infarction suffers a set of sequential biochemical and metabolic modifications within cardiomyocytes, where mitochondrial activity, controlling the redox state, could play a relevant role. These biochemical pathways involve the reduction of the substrate in the cytosol that are permeable to the mitochondrial inner membrane. These substrates are then oxidized by the mitochondrion to yield a substrate that can return to the cytosol for a new reduction. In the heart, the principal system is the malate-aspartate shuttle (MAS) since the glycerol-phosphate shuttle is almost undetectable [Tissues and organs with high oxidative performance, such as the myocardium, are highly dependent on adequate mitochondrial activity. Sufficient ATP production requires a coordinated flux of metabolites from the cytosol to within the mitochondrion to contribute reducing equivalents, like nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide  is one of the most popular experimental models to generate myocardial infarction. This chemical process has been used successfully in rodents revealing histological injuries in subendocardial regions, localized in the apical cardiac zone. In the first hours, the heart damage is accompanied by oxidative stress and inflammatory response with the onset of infarction 24 h after drug administration . The infThe present study aimed to characterize the MAS activity in the hearts of male Wistar rats subjected to treatment with a single dose of ISO. The experiments distinguished the endogenous and the reconstituted MAS activity and were performed at three different stages of the cardiac damage: (1) early phase before 24 h ; (2) myocardial infarction (at 24 h); (3) remodeling phase following the infarcted crisis . For a better biochemical context, our experiments included a characterization of the cardiac pro-oxidant status and mitochondrial performance determination.Enzymes such as malate dehydrogenase  and aspartate aminotransferase , coenzymes, 2-oxoglutarate, aspartic and glutamic acids, as well as the ethoxyformic anhydride  were obtained from Sigma-Aldrich Chemical Co. . The fluorescence probe 2′,7′-dichlorodihydrofluorescein di-acetate (H2DCF-DA), used to signal reactive oxygen species (ROS), was obtained from Molecular Probes, Co, . All other reagents were purchased from Merck .n = 40), weighing 250–280 g of body weight (b.w.) and fed ad-libitum, were injected subcutaneously (s.c.) with (−)-isoproterenol hydrochloride (ISO) at a dose of 70 mg/kg b.w, between 09:00 and 10:00 h (n = 25), whereas control rats (n = 15) received an equivalent volume of saline solution. Animals were euthanized after 3 and 6 h (pre-infarction period), 24 h (infarction), and 48 h and 96 h (post-infarction period) of ISO administration [Male Wistar rats . Glutamate and malate  were used as substrates for site I at state 4. Mitochondrial state 3 was initiated by adding ADP . 2PO4, measured at 375 nm and finally calculated by its absorption coefficient, according to Levine et al. [The ROS levels in mitochondrial preparations were estimated through the method described by Viarengo et al. , using te et al. .2PO4, 20 mmol/L Trisma-HCl, 5 mmol/L MgCl2 phosphate buffer (pH 7.2). The reaction was initiated with the addition of 5 mmol/L NADH and its oxidation was monitored spectrophotometrically at 340 nm [Endogenous NADH oxidation by intact mitochondria, assumed to be performed by a heart mitochondrial NADH dehydrogenase described by Nohl , was asst 340 nm . The reconstitution experiments were carried out with the addition of the “missing” cytosolic components of the MAS , keepingg for 10 min, and the supernatant at 90,000× g for 30 min. Finally, the pellet was sedimented at 105,000 g for 1 h, repeating twice the last step, each time for 30 min; the final pellet was suspended in a solution containing 10 mmol/L TRIS-sodium acetate, and 250 mmol/L sucrose [To evaluate the maximal capacity of heart mitochondria to oxidize NADH in the absence of membrane barriers, we prepared sealed particles from rat heart mitochondria. For this, NADH-oxidizing enzyme units were obtained after sonication of heart mitochondria suspended in the ice-cooled isolation buffer, described above. The suspension was sonicated 5 times in a Corex tube for 20 s at 25 W at 15 s intervals. The sonicated suspension was sequentially centrifuged at 9600×  sucrose . t-test. Comparisons among groups were evaluated using one-way analysis of variance (ANOVA) and Bonferroni’s t-test with a significance level of p < 0.05.Data are presented as mean ± standard error (SE). The significance of the differences was assessed by Student’s Using a single administration of ISO, infarct-like damage in the apex region of the left ventricle developed, occurring 12–24 h after ISO administration to rats. The lesion was defined by histological criteria, continuous telemetric electrocardiogram (ECG) recordings, and the increase in serum marker enzymes, specific for myocardial damage. Shortly after ISO treatment, there was an increase in heart rate and a lowering of blood pressure, resulting possibly in functional ischemia (pre-infarction period). Ultrastructural changes and mitochondrial swelling were evident and functional alterations in isolated mitochondria, such as decreases in oxygen consumption, respiratory quotient, ATP synthesis, and membrane potential, were noticed starting at 6 h after ISO administration (pre-infarction period) and lasted until 72 h later (post-infarction period). Mitochondrial proteins decreased after 3 h of treatment, reaching almost a 50% diminution, which was maintained during the whole study. An energy imbalance, reflected by a decrease in energy charge and the creatine phosphate/creatine ratio, was observed at the pre-infarct stage, and also ATP and total adenine nucleotides diminished clearly at this period, reaching a maximum at the onset of infarction and were accompanied by damage to the myocardial function, drastically decreasing left ventricular pressure and shortening the atrioventricular interval (22 h to 25 h after ISO treatment). During post-infarction, a partial recovery of energy charge, creatine phosphate/creatine ratio, membrane potential, and myocardial function occurred, but not of mitochondrial oxygen consumption, rate of ATP synthesis, total adenine nucleotides, or mitochondrial proteins [The level of ROS reacting with H2DCF-DA increased during the pre-infarction period (6 h after ISO treatment), returned to the control level (24 h), and increased again at the post-infarction period (48 h to 96 h post-ISO treatment) A. In con−1·mg−1 of protein -type kinetics were calculated for the endogenous (“Nohl” NADH dehydrogenase) in control preparations . In cont protein A. In the protein A. With t+ ratio, without transport and permeability of substrates interfering factors. Under these conditions, we obtained a Km for NADH oxidation of 2.8 ± 0.3 µmol/L, and a Vmax of 129.0 ± 14.3 nmol·min−1·mg−1 of protein , there were no significant changes in the M-M kinetics parameters of the endogenous NADH oxidation in the ISO-treated animals A,B. At t−1·mg−1 vs. 47.5 ± 2.3 nmol·min−1·mg−1 of protein, in controls, p < 0.01; −1·mg−1 against 47.5 ± 2.3 nmol·min−1·mg−1 of protein, in controls, p < 0.01; p < 0.01; p < 0.05; −1·mg−1 vs. 47.5 ± 2.3 nmol·min−1·mg−1 of protein, in controls, p < 0.05; −1·mg−1 vs. 47.5 ± 2.3 nmol·min−1·mg−1 of protein, in controls, p < 0.01; When we recorded mitochondrial NADH oxidation after reconstituting the MAS, we found significant changes in these parameters . During Cardiotoxicity related to isoproterenol treatment has been used in multiple studies to evaluate protective actions of a variety of pharmacological and nutritional remedies, for review see . HoweverIn the past, our group used the ISO-induced cardiac apical infarction as an experimental model to explore metabolic and cellular adaptations in three different stages of heart damage: pre-infarction, infarction, and post-infarction. Particular sequential alterations, as well as adaptations, were shown in the cardiac energy status and intracellular calcium handling ,7. Each In this report and using a similar experimental protocol, we extended our previous metabolic studies to characterize the mitochondrial capacity to consume oxygen, the pro-oxidant status, and the activity of the malate-aspartate shuttle. Our experiments sampled the whole heart to perform the metabolic determinations; hence, the findings represent global cellular alterations associated with the apical necrosis that characterizes this pathological model.Despite knowing that heart damage by ISO is accompanied by enhanced oxidative stress, structural instability, mitochondrial loss, and ionic imbalance , no repo+ ratio with metabolic consequences [The MAS plays a strategic role in the cytoplasmic redox state by allowing the mitochondrial oxidation of the NADH generated by glycolysis and other reactions, such as phosphoglycerate dehydrogenase, required for the synthesis of important metabolites, for example, serine and glycine . The MASequences . This coequences .Our results showed only discreet effects during the cardiotoxic ISO action on the endogenous cardiac NADH dehydrogenase activity, since only after 2 and 4 days of treatment (in the post-infarction stage), was observed an enhancement and a tendency to increase the maximal NADH oxidation . This reMAS inhibition by pharmacological treatment is capable to offer cardioprotection in a model of 40-min global no-flow ischemia experimental model .These findings support the notion that heart damage associated with myocardial infarction should be understood as a set of sequential biochemical and metabolic modifications within cardiomyocytes. Even though that each stage around the infarction event is characterized by its own and particular set of alterations , to understand better the global process it is essential to consider a special role for mitochondrial activity and redox state control.


Airway hyperresponsiveness (AHR) represents a central pathophysiological hallmark of asthma, with airway smooth muscle (ASM) being the effector tissue implicated in the onset of AHR. ASM also exerts pro-inflammatory and immunomodulatory actions, by secreting a wide range of cytokines and chemokines. In asthma pathogenesis, the overexpression of several type 2 inflammatory mediators including IgE, IL-4, IL-5, IL-13, and TSLP has been associated with ASM hyperreactivity, all of which can be targeted by humanized monoclonal antibodies (mAbs). Therefore, the aim of this review was to systematically assess evidence across the literature on mAbs for the treatment of asthma with respect to their impact on the ASM contractile tone. Omalizumab, mepolizumab, benralizumab, dupilumab, and tezepelumab were found to be effective in modulating the contractility of the ASM and preventing the AHR, but no available studies concerning the impact of reslizumab on the ASM were identified from the literature search. Omalizumab, dupilumab, and tezepelumab can directly modulate the ASM in asthma, by specifically blocking the interaction between IgE, IL-4, and TSLP, and their receptors are located on the surface of ASM cells. Conversely, mepolizumab and benralizumab have prevalently indirect impacts against AHR by targeting eosinophils and other immunomodulatory effector cells promoting inflammatory processes. AHR has been suggested as the main treatable trait towards precision medicine in patients suffering from eosinophilic asthma, therefore, well-designed head-to-head trials are needed to compare the efficacy of those mAbs that directly target ASM contractility specifically against the AHR in severe asthma, namely omalizumab, dupilumab, and tezepelumab. Asthma is a chronic, heterogeneous, inflammatory airway disorder consisting of a generally variable airflow limitation and several clinical symptoms including chest tightness, cough, wheezing, and shortness of breath . Airway Particularly, type 2 (T2)-high asthma is a complex endotype associated with high type 2 inflammatory markers including immunoglobulin E (IgE), interleukins (IL)-4, IL-5, and IL-13 , and it Although inhaled corticosteroids (ICSs) represent the mainstay of asthma management  and are Over the last decade, improved knowledge regarding the complex pathophysiology of asthma has led to the development of new treatment options , and theIn asthma pathogenesis, a number of inflammatory mediators of the T helper 2-dependent reaction play a pivotal role in a complex signaling environment that contributes to AHR: several experimental studies on isolated airway preparations and in vivo animal models have demonstrated an association between enhanced ASM hyperreactivity and overexpression of IgE , IL-4 1,18, IL-5The approved humanized monoclonal antibodies (mAbs) for the treatment of asthma are targeted against IgE , IL-5 (mepolizumab and reslizumab), IL-5 receptor α , and IL-4/IL-13 (dupilumab) ,25,26,27The question of this systematic review was to assess if current mAbs for the treatment of asthma may have an impact on ASM contractility and AHR.The protocol of this synthesis of the current literature has been submitted to the international prospective register of systematic reviews , and performed in agreement with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocol (PRISMA-P) , with thThe PICO  framework was applied to develop the literature search strategy and question, as previously reported . Namely,A comprehensive literature search was performed for in vitro, ex vivo, and clinical studies evaluating the impact of current mAbs for asthma on ASM contractility and AHR.The search was performed on ClinicalTrials.gov (accessed on 15 August 2021), Cochrane Central Register of Controlled Trials (CENTRAL), Embase, EU Clinical Trials Register, MEDLINE, Scopus, and Web of Science in order to provide relevant studies available with no time limit up to 5 July 2021.The research string was as follows: (“dupilumab”[Supplementary Concept] OR “dupilumab”[All Fields] OR (“tezepelumab”[Supplementary Concept] OR “tezepelumab”[All Fields]) OR (“mepolizumab”[Supplementary Concept] OR “mepolizumab”[All Fields]) OR  OR (“reslizumab”[Supplementary Concept] OR “reslizumab”[All Fields]) OR ) AND (((“airway”[All Fields] OR “airway s”[All Fields] OR “airways”[All Fields]) AND  OR “smooth muscle”[All Fields] OR (“smooth”[All Fields] AND “muscle”[All Fields]))) OR ((“airway”[All Fields] OR “airway s”[All Fields] OR “airways”[All Fields]) AND (“hyperresponsive”[All Fields] OR “hyperresponsiveness”[All Fields] OR “hyperresponsivity”[All Fields])) OR ((“isolate”[All Fields] OR “isolate s”[All Fields] OR “isolated”[All Fields] OR “isolates”[All Fields] OR “isolating”[All Fields] OR “isolation and purification”[MeSH Subheading] OR (“isolation”[All Fields] AND “purification”[All Fields]) OR “isolation and purification”[All Fields] OR “isolation”[All Fields] OR “isolations”[All Fields]) AND (“bronchi”[MeSH Terms] OR “bronchi”[All Fields] OR “bronchus”[All Fields])) OR ((“isolate”[All Fields] OR “isolate s”[All Fields] OR “isolated”[All Fields] OR “isolates”[All Fields] OR “isolating”[All Fields] OR “isolation and purification”[MeSH Subheading] OR (“isolation”[All Fields] AND “purification”[All Fields]) OR “isolation and purification”[All Fields] OR “isolation”[All Fields] OR “isolations”[All Fields]) AND (“airway”[All Fields] OR “airway s”[All Fields] OR “airways”[All Fields]))). Citations of previously published relevant reviews were examined to select further pertinent studies (if any) .Two reviewers independently checked the relevant studies identified from the literature search. The studies were selected in agreement with PICO and any difference in opinion about eligibility was resolved by consensus.1), and outcome measurements to evaluate the impact on ASM.Data from included studies were extracted in agreement with Data Extraction for Complex Meta-anALysis (DECiMAL) recommendations , and cheThe endpoint of this systematic review was to assess the impact of current mAbs for the treatment of asthma on ASM contractility and AHR.Data from original papers were extracted and reported via qualitative synthesis.Of the 81 potentially relevant records identified in the initial search, 16 studies were deemed eligible for qualitative analysis . OverallOmalizumab is a humanized mAb that blocks the interaction between IgE and high-affinity receptor FcεRI on inflammatory cells, including mast cells and basophils. Omalizumab is the first mAb approved by the European Medicines Agency (EMA) and FDA for the treatment of patients ≥6 years old with persistent severe allergic asthma, high levels of blood IgE, and at least a sensitization to a perennial allergen .p < 0.05) reduction when administered at 0.5 μg/mL and 1.0 μg/mL for IL-6, and at 1 μg/mL for IL-8. Omalizumab also suppressed the IgE-induced synthesis of IL-6 and IL-8 messenger RNAs (mRNAs), with a significant (p < 0.05) effect achieved when administered at 1.0 μg/mL ) than that on the day after the first challenge, with respect to baseline (0.13 mg/mL)  vs. 7 [25%]) ) .The evidence resulting from this systematic review indicates that omalizumab, dupilumab, and tezepelumab may modulate AHR via direct action on ASMCs and indirect influence on eosinophilic inflammation and parasympathetic activity; conversely, mepolizumab and benralizumab seem to prevent AHR prevalently by reducing airway inflammation and vagal firing .ASMCs are capable of expressing the heterodimeric TSLP receptor (TSLPR), consisting of the IL-7 receptor-α chain (IL-7Rα) and TSLPR subunit , the hetEosinophils, which are central to the pathogenesis of allergic and non-allergic asthma ,55, exprTherefore, the anti-IgE omalizumab, the anti-IL-4/IL-13 dupilumab, and the anti-TSLP tezepelumab are supposed to have the ability to directly modulate the ASM contractility and AHR in asthma, by specifically blocking the interaction between the ligands and FcεRI, IL-4Rα, and TSLPR respectively, and indirectly by targeting eosinophils and further inflammatory cells, thus leading to a reduction in the inflammatory cascade.Conversely, mepolizumab and reslizumab, which selectively block circulating IL-5, and benralizumab, which prevents the adhesion of IL-5 to the IL-5Rα chain, may indirectly modulate the contractile properties of ASMCs by targeting eosinophils and other immunomodulatory effector cells responsible for promoting inflammatory processes of asthma.The direct action of omalizumab, dupilumab, and tezepelumab on the ASM is mediated by complex intracellular signaling pathways . OmalizuDupilumab prevents IL-4 and IL-13 signaling by blocking the IL-4Rα chain common to the IL-4R and IL-13R complexes . Recent Tezepelumab selectively blocks human TSLP from interacting with the heterodimeric TSLPR complex . Compare2 muscarinic acetylcholine (ACh) receptor (mAChR) present on postganglionic parasympathetic nerves, resulting in an excessive ACh release [3 mAChRs expressed on post-junctional ASMCs stimulates PLC to produce diacylglycerol (DAG), which in turn activates phosphokinase C to promote ASM contraction [Only recently, an ex vivo study provided evidence that targeting the IL-5/IL-5R pathway is effective against AHR in passively sensitized human medium bronchi . Passive release . It has  release . Indeed, release , while E release ,88 The itraction .The present systematic review supports evidence for the direct efficacy of omalizumab, dupilumab, and tezepelumab, and the indirect beneficial effect of mepolizumab and benralizumab in modulating the contractility of the ASM and preventing AHR. Omalizumab generally improved AHR in vitro and in vivo, although surprisingly, several RCTs ,36,41 coIn vitro and ex vivo evidence indicates that dupilumab effectively reduced the AHR to His , whereasInterestingly, several studies conducted in vitro on ASMCs, in experimental animal models of asthma or in vivo in humans confirmed the beneficial role of omalizumab ,93,94,95AHR has been suggested to be a main treatable trait towards precision medicine in patients suffering from eosinophilic asthma ,97, and Among the currently available mAbs, omalizumab has the greatest amount of data concerning the impact on ASM contractility, as it was first, and for a long time the only available add-on biologic therapy for the treatment of severe asthma . To the The limitations of this systematic review are related to the intrinsic characteristics of the included studies: in vitro and ex vivo studies are based on models of asthma, and RCTs have been generally carried out in small and selected populations of asthmatic patients.In conclusion, the development of biological therapies has led to a significant step forward in the treatment of severe asthma; nevertheless, to date little is still known about the real clinical impact of mAbs against AHR. Future research of currently available and upcoming mAbs for the treatment of severe asthma should address this clinical issue as an important feature of long-term disease management.


Several decades after the bis(diethyl etherate) of tungsten(IV) chloride was first mentioned in the literature, it has now at last been structurally characterized by single-crystal X-ray diffraction analysis, finally confirming its composition. In particular, with regard to tungsten, many complexes of the type WX4(solvent)2 are known in the literature and are even found in textbooks nowadays, as, for instance, WCl4(py)2 2 2. Although this compound has been ‘floating around’ in the literature for many decades, surprisingly little analytical data concerning it have been reported, begging the question if it even truly exists.A hexacoordinated metal centre with an octahedral coordination environment is a very common motif in transition metal chemistry. This can involve neutral, as well as anionic, ligands or a combination of both. The latter can, et al. (19754(thf)2. This, in turn, was synthesized from tungsten(VI) chloride, WCl6, by treatment with diethylzinc in diethyl ether, whereas the authors proposed that the tungsten(IV) chloride bis(diethyl ether) adduct, WCl4(Et2O)2, was formed initially, and was then transformed into the isolatable bis(tetrahydrofuran) adduct. Unfortunately, no analytical data for the bis(diethyl ether) complex was provided. At roughly the same time, Chisholm, Cotton and co-workers investigated the use of WCl4(Et2O)2 as a possible precursor in the preparation of tungsten–tungsten triple-bond complexes 2 was explicitly mentioned as a bis(diethyl etherate), it is worth pointing out that it was prepared in situ (by the same route as described above), with a 100% yield assumed, and was used without characterization. Therefore, although WCl4(Et2O)x had already proven to be an interesting synthon, the exact nature and structure of the complex remained unclear and its composition, suggested to be a bis(diethyl ether) adduct, was based simply upon chemical intuition and the logical assumption of an octahedral complex with hexacoordinated tungsten x by elemental analysis to elucidate its exact composition. In contrast to previous articles, they reported that the mono(diethyl etherate), WCl4(Et2O), was obtained from diethyl ether solutions of WCl6, which was based on elemental analysis suggesting not two but one equivalent of C4H10O [WCl4(Et2O)2: C 20.3, H 4.3%; WCl4(Et2O): C 12.0, H 2.5%; reported by Castellani & Gallazzi , due to the relatively weak complexation of the diethyl ether ligands. In fact, Castellani & Gallazzi pointed out correctly that other complexes, such as WCl4(thf)2 and WCl4(py)2, always feature two complexed solvent molecules.Almost a decade later, in 1985, Castellani & Gallazzi 1985 revisiteazzi 1985: C 11.5,4(Et2O)2, and conduct a complete structural characterization by single-crystal X-ray diffraction and NMR spectroscopy , WCl4(Et2O)2, with trans-coordinated diethyl ether molecules in the axial positions 2 finally comes to an end, closing this gap in tungsten tetrachloride bis(solvent) complexes.It is puzzling that for decades this mystery remained unsolved, even though this complex was frequently used as a starting material in tungsten chemistry. Finally, in 2021, almost half a century after the complex first appeared in the literature, Jurca and co-workers were able to obtain a pure crystalline sample of the bis(diethyl ether) complex, WClns Fig. 1! With th4(Et2O), in 1985, as suggested? It seems not unlikely given the fact that diethyl ether is a rather weakly bonded ligand, and they reported black crystals instead of yellow ones and the elemental analysis was very much in line with a mono(diethyl etherate). Two interesting details are given by Jurca and co-workers here as well, namely, that ‘prolonged exposure to ambient conditions led to the appearance of dark spots on the crystal surface’ and that ‘the compound is unstable under vacuum’ , WCl


Tight relationships between sleep quality, cognition, and amyloid-β (Aβ) accumulation, a hallmark of Alzheimer’s disease (AD) neuropathology, have been shown. Sleep arousals become more prevalent with aging and are considered to reflect poorer sleep quality. However, heterogeneity in arousals has been suggested while their associations with Aβ and cognition are not established.We recorded undisturbed night-time sleep with EEG in 101 healthy individuals aged 50–70 years, devoid of cognitive and sleep disorders. We classified spontaneous arousals according to their association with muscular tone increase (M+/M–) and sleep stage transition (T+/T–). We assessed cortical Aβ burden over earliest affected regions via PET imaging and assessed cognition via neuropsychological testing.P = 0.0053, R²β* = 0.08). By contrast, more prevalent T–M+ arousals, upholding sleep continuity, were associated with lower Aβ burden , and better cognition, particularly over the attentional domain .Arousal types differed in their oscillatory composition in θ (4–8 Hz) and β (16–30 Hz) EEG bands. Furthermore, T+M– arousals, interrupting sleep continuity, were positively linked to Aβ burden , Actions de Recherche Concertées Fédération Wallonie-Bruxelles (SLEEPDEM 17/27-09), ULiège, and European Regional Development Fund (Radiomed Project). Sleep is central to health and cognition, and it deteriorates with aging . In addiSleep arousals, defined as transient accelerations in sleep electroencephalogram (EEG) rhythms, are usually considered as brain reactions to internal  or external  perturbations . AlthougImportantly, spontaneous arousals — i.e., not elicited by any identifiable internal or external stimuli — also constitute an authentic element of undisturbed sleep in healthy individuals. Their mechanisms, cerebral correlates, and functional consequences remain largely unknown , with soHere, we assessed whether different types of spontaneous arousals during sleep were differentially associated with Aβ cortical deposition and cognitive performance in a cohort of healthy individuals in late midlife. We were able to tease apart different types of arousals, based on their temporal relationships with increased muscular tone and sleep stage transitions. In line with the hypothesis that arousals perturb sleep, we anticipated that arousals fragmenting sleep structure would be associated with both worse cognitive performance and Aβ deposition in brain areas that are first affected by this AD-related neuropathological process.We recorded undisturbed sleep at habitual sleep times under EEG in 101 healthy individuals aged 50–70 years , following 1 week of regular sleep-wake schedule . In orde2,294.1 = 403.84, P < 0.0001, semipartial R² [R²β*] = 0.73). More importantly, it yielded a triple interaction between transition, EMG status, and frequency band , implying that arousals differ in their spectral composition based on the presence or absence of EMG changes and sleep stage transition  and transition , as well as in interactions between transition and frequency band , between EMG status and frequency band , and between EMG status and transition . Based on this first analysis, we therefore concluded that the factors of arousal heterogeneity eloquently define 4 types of arousals . We finally note that multiple post hoc comparisons within each band yielded significant differences across arousal types over the θ and β bands , and the cooccurrence of an EMG tone increase (M+) or not (M–) resulted in differences in their oscillatory properties. We computed individual relative power in the different EEG frequency bands defining an arousal . A generalized linear mixed model (GLMM) with relative power as a dependent variable (and adjusted for data distribution) first indicated that relative power changed across frequency bands  and EMG status , as well as an interaction between EMG status and transition , indicating that density of arousal types significantly varied. Interestingly, we did not find any significant main effect of early cortical Aβ burden , age , or sex . Critically, the GLMM yielded a significant triple interaction between early cortical Aβ burden, EMG status, and transition , implying that the association between arousals and early cortical Aβ burden depends on the concomitant change in muscular tone and sleep stage transition. The heterogeneity in spontaneous arousals was further reflected by the significant interactions between early cortical Aβ burden and EMG status . In line with our goal to consider very early AD-related neuropathological process, Aβ burden was quantified over the regions previously reported as the earliest cortical aggregation sites  — the fr1,95 = 14.15, P = 0.0003, R²β* = 0.13) and with a positive association between Aβ and T+M– arousals  — together with an expected main effect of age  . Critically, a post hoc contrast showed that the links between the 2 types of arousals and early cortical Aβ burden were significantly different . In addition, T–M+ and T+M– arousals were not correlated   and no mrrelated . Supplemrrelated . Resultsrrelated .P = 0.048, R²β* = 0.04), on top of the education effect, but no relation with T+M– arousal index (P = 0.25), age, or sex  and was not significant for the executive (P = 0.09) or memory domains (P = 0.91).We then tested whether cognition, as assessed in a global index through an extensive neuropsychological test battery, was differentially associated with the 2 arousal types showing opposite association with early cortical Aβ burden. In a GLMM, the association between global cognition and T–M+ arousal index was found to be significant  which was driven by the executive domain   and the different cognitive measures in separate exploratory GLMMs. We found no link between cognition and the number of awakenings , while aP =.007) . FurtherP =.007) .Brain dynamics that buttress cerebral functions entail stationary and nonstationary interactions between neuronal populations . Sleep sOur analyses show that the main characteristic differentiating the 2 types of arousals is whether or not they lead to a sleep stage transition. A second important criterion consisted of the concomitant increase in EMG tone. Aside from their different links with Aβ burden and cognition, T+M– and T–M+ arousals are not correlated with each other and differ in their spectral composition: T+M– bear a larger proportion of θ power, while T–M+ arousals are composed of a higher proportion of β power. The reason T–M– and T+M+ arousals are not significantly associated with Aβ and cognition is unclear and might reside in different prevalence or in diverging effects of sleep transitions and EMG bursts, which would hinder the relationship. Future studies are warranted to further investigate this issue.Two hypotheses can be put forward to explain the heterogeneity in arousals. On the one hand, all arousals, triggered by a common set of brain areas, might be part of a continuum in which each arousal is characterized by the intensity in its driving neural activity, its spectral composition, its associated muscular tone, and its probability of sleep stage transition. Alternatively, the 2 arousal types are distinct physiological events prompted by different triggering brain structures and propagation cerebral networks. Oddly enough, the origin of spontaneous arousals remains elusive. Recent functional MRI (fMRI) data show that subcortical regions  were activated during non-REM arousals, while cortical regions were deactivated . A recenThe cellular and molecular underpinnings of the distinct relationship between the 2 types of arousals, Aβ burden, and cognition are currently unknown. We can reasonably speculate that T+M– arousals have 2 potentially deleterious impacts. Firstly, they interrupt a sleep stage and consequently all its associated cellular phenomena, like plasticity . SecondlWe emphasize that (a) our cohort only comprised healthy individuals, devoid of SDB, and (b) we focused on spontaneous arousals, which are not generated in response to detectable endogenous or exogenous perturbation . Therefore, our findings probably do not apply to potentially more prevalent perturbation-induced arousals and their negative behavioral , 16 and Sleep arousals may connect the sleeper’s brain with the surrounding endogenous and exogenous relevant incoming information and contribute to elements of cortico-cortical information processing , 29, as In order to target early Aβ brain deposit , we recrParticipants were screened for sleep apnea/hypopnea syndrome during an in-lab night of sleep under polysomnography (PSG) preceding the one that was analyzed in the results section of this paper. This PSG included EEG , 2 bipolar electrooculograms (EOGs), 2 bipolar submental EMG electrodes, 2 bipolar electrocardiograms (ECGs), 2 sets of bipolar leg electrodes, thorax and abdominal belts, an oximeter, a nasal canula, and a snoring sensor. As is typically done in similar sleep studies , 32, volParticipants came to the lab for an adaptation night under PSG, after which those with sleep an AHI ≥ 15/hour were excluded from further participation. Volunteers were required to follow a regular sleep-wake schedule (±30 minutes) for 1 week based on their preferred bed and wake-up times before sleep EEG recording, in order to record their sleep in settings as close as possible to habitual conditions. Compliance was verified using sleep diaries and wrist actigraphy . Participants then joined the laboratory about 6.5 hours prior to habitual sleep time and were maintained in dim-light thereafter. Undisturbed habitual sleep was recorded with N7000 amplifiers  using 11 EEG derivations placed according to the 10–20 system , 2 bipolar EOGs, and 2 bipolar submental EMG electrodes. Recordings were sampled at 200 Hz and rereferenced to the mean of the 2 mastoids.Sleep stage scoring and arousal detection were carried out in separate steps by 2 independent algorithms. Sleep stage scoring was performed in 30-second windows using a validated algorithm  , 34. AutIn brief, arousal detection is performed over all electrodes on whole-night recordings split into 1-second epochs in 2 successive steps computed over the power in the broad-α (7–13 Hz), β (16–30 Hz), and lower-θ (3–7 Hz) frequency bands, excluding the σ band (11–16 Hz) — i.e., corresponding to frequency of sleep spindles — which cannot be considered as arousals. A fixed threshold is first applied to detect abnormal EEG activity relatively to the whole-night recording: any 1-second epoch with power in any of the 3 frequency bands higher than the whole-night median value in each frequency band is considered as a potential arousal. The second step adapts the threshold to account for the specific EEG background activity in a shorter time window. A specific threshold is computed for each 30-second window: all 1-second epochs without concomitant EMG tone increase are selected, as well as the first ten 1-second epochs without EMG increase before and after the 30-second window being evaluated; threshold of each frequency band consists in the median power over the selected 1-second epochs. Events composed of at least 3 consecutive 1-second epochs with changes in EEG frequencies higher than twice the local median and 1 median of the whole recording for that frequency band were considered as arousals. For detailed explanations on the method, see ref. 35.In order to evaluate the potential heterogeneity of arousals, we split them according to 2 criteria, which we considered as relevant in research settings, as well as clinical practice. The first criterion addressed whether arousals did trigger a sleep stage transition (T+)  or whether they did not (T–). The second criterion addressed their salience, reflected by the concomitant increase in EMG tone (M+) or its absence (M–).https://github.com/spm/spm12/, commit SPM12 r7219) on Fz electrode. Detrending was done over the 500 ms prior to the arousal event. Data were then averaged per arousal type prior to summing in the typical EEG bands that may compose an arousal . Given the variety of factors that impact total power   , and thuhttp://hmri.info) and SPM12  to obtain a quantitative MT map and segmented images , normalized to the standard MNI space using unified segmentation  to get a magnetization transfer–weighted (MT-weighted) contrast, based on multi-echo 3D fast low angle shot at 1 mm isotropic resolution  with fl . Participants received a single dose of the radioligand in the antecubital vein (target dose 185 MBq); images acquisition started 85 minutes after the injection and consisted of 4 frames of 5 minutes, followed by a 10-minute transmission scan using 68Ge line sources. Images were reconstructed using filtered back-projection algorithm including corrections for measured attenuation, dead time, random events, and scatter using standard software . Individual PET average images were produced using all frames and were then manually reoriented according to MT-weighted structural MRI volumes and coregistered to the individual space structural MT map. Flow-field deformation parameters obtained from DARTEL spatial normalization of the MT maps were applied to averaged coregistered PET images . From those 2 sessions, 3 domain-specific composites scores were computed for the memory, executive function, and attentional domains, and they consisted of the standardized sum of the standardized domain-specific scores, where higher values indicate better performance. A fourth global cognitive score consisted of the standardized sum of the domain-specific composite scores.The first session comprised (a) mnemonic similarity task (MST) ; (b) cathttp://amir.eng.uci.edu/MvCAT.php), and GLMMs were adapted accordingly as preconized by SAS statisticians. A subject was treated as a random factor (intercept); each model included sex and age as covariates, as well as education for models with cognitive score as dependent variables. Statistical significance threshold was set at P < 0.05 as no correction for multiple comparisons were required. The association between arousals and Aβ was tested in a single model, including arousal density as dependent variable together with transition  and EMG  arousal statuses and early Aβ burden as regressors (as well as sex and age). The association between arousal and cognition was tested in a single model including global cognition score as a dependent variable together with T+M– and T–M+ arousal density as regressors (as well as sex and age). Kenward-Roger correction was used to determine degrees of freedom. R²β* values were computed to estimate the effect sizes of significant fixed effects and statistical trends in all GLMMs  were adjusted for multiple testing using Tukey’s procedure. Cook’s distance was used to assess the potential presence of outliers driving the associations, and as values ranged below 0.4, no data point was excluded from the analyses .Statistical analyses were performed in SAS 9.4 (SAS Institute) using GLMMs. The distribution of dependent variables was determined by fitting all parametric probability distributions to data, using the “allfitdist” function in Matlab 2015 (ll GLMMs . P valueThe study was registered with EudraCT 2016-001436-35. All procedures were approved by the Hospital-Faculty Ethic Committee of ULiège. All participants signed an informed consent prior to participating in the study.Study concept and design were contributed by ES, PM, CP, C Bastin, FC, and GV. Data acquisition, analysis, and interpretation were contributed by DOC, MVE, JN, MG, EK, C. Berthomier, PB, MB, ES, MAB, C Bastin, FC, CP, PM, VM, and GV. DOC and GV drafted the first version of the manuscript. All authors revised the manuscript and had final responsibility for the decision to submit for publication. While all co–first authors tightly collaborated to acquire and analyze the data for an equivalent time and workload, each of the co–first authors had their own aspect of the data to deal with in priority. The order reflects these priorities.


Correct in vivo diagnosis of Alzheimer’s disease (AD) helps to avoid administration of disease-modifying treatments in non-AD patients, and allows the possible use of such treatments in clinically atypical AD patients. Cerebrospinal fluid (CSF) biomarkers offer a tool for AD diagnosis. A reduction in CSF β-amyloid (marker of amyloid plaque burden), although compatible with Alzheimer’s pathological change, may also be observed in other dementing disorders, including vascular cognitive disorders due to subcortical small-vessel disease, dementia with Lewy bodies and normal-pressure hydrocephalus. Thus, for the diagnosis of AD, an abnormal result of CSF β-amyloid may not be sufficient, and an increase in phospho-tau (marker of tangle pathology) is also required in order to confirm AD diagnosis in patients with a typical amnestic presentation and reveal underlying AD in patients with atypical or mixed and diagnostically confusing clinical presentations. Besides atrophy and neuronal as well as synaptic loss, there are two pathologic hallmarks of Alzheimer’s disease (AD): extracellular accumulation of amyloid beta peptide in the form of amyloid plaques and hyperphosphorylation of tau protein which polymerizes in the form of paired helical filaments, the key component of intraneuronal neurofibrillary tangles . From thOn June 2021, the anti-amyloid monoclonal antibody aducanumab  was appr42), which is a marker of amyloid pathology, inversely correlated with the amyloid plaque burden [P-181), which is a marker of tau protein hyperphosphorylation as well as tangle formation [T), which is considered as a non-specific marker of neuronal/axonal degeneration [40) can also be measured in the CSF, and it is useful in the form of the Aβ42/Aβ40 ratio, which seems to perform diagnostically better than Aβ42 alone [During the last two decades, various CSF biomarkers have been developed for the  diagnosis of AD ,26. Withe burden , phosphoormation  and totaneration . Amyloid42 alone . These b42 alone ,36,37,3842 (or low Aβ42/Aβ40) and an increase in either τP-181 or τT [P-181 and τT are increased, this “either” usually does not create diagnostic confusion. However, in the occasional patient with low Aβ42 (indicating the presence of amyloid plaque load) and increased τT , but normal τP-181 (suggestive of absence of tangle formation), diagnostic doubt arises. Thus, it has been suggested that in addition to low Aβ42 both τP-181 and τT should be increased in order to fulfill the AD profile [Initially it was suggested that the AD CSF biomarker profile is characterized by low Aβ81 or τT . Since t profile .42 (or Aβ42/Aβ40) and increased τP-181 is considered as compatible with an AD diagnosis, since this profile indicates the presence of both amyloid plaques and neurofibrillary tangles, whilst τT may be either increased or normal. The presence of atrophy in magnetic resonance imaging may also serve as a marker of nonspecific neurodegeneration, replacing τΤ. Thus, a profile of A+T+(N+) or A+T+(N−)  is compatible with AD [More recently, the so-called AT(N) system   has been with AD   profile (* for either + or −), the AD diagnosis is not confirmed and this profile is usually compatible with one of the frontotemporal lobar degeneration pathologies  [+T−(N*) profile has extended differential diagnosis.In cases without amyloidosis in the CSF, presenting the A of tau) . On the 42, which is reduced early in the asymptomatic stage, followed by an increase in τP-181, and it seems that most  AD patients enter the stage of MCI with both of the above biomarkers having become abnormal  [+T−(N−) is usually observed in the asymptomatic stage [+T−(N−) profile may experience subtle cognitive symptoms or even MCI [+T−(N−) profile is practically always observed in the pre-dementia and, usually, at the preclinical stages; since neurodegenerative changes remain limited, atrophy and increased τΤ are usually not evident yet. The above profile in the AT(N) system belongs to the so-called pathological change in Alzheimer’s, and is considered as a part of the Alzheimer’s continuum, but not synonymous to AD [In Alzheimer’s disease, CSF biomarker changes may become evident 10‒20 years prior to the symptomatic stage ,43,44. Uvel yet) ,46. Thusic stage . Howevereven MCI . This preven MCI . Thus, ius to AD .+T−(N+) profile points to significant neurodegeneration and, since there is no indication of tangle formation, another (non-AD) pathology is suspected to be present together with amyloid pathology. A typical neurodegenerative disorder with this profile may be dementia with Lewy bodies (DLB) [+T−N* profile always indicative of pathological change in Alzheimer’s (amyloid plaques)? In subcortical small-vessel disease (SSVD), CSF amyloid reduction is not uncommon [42 has also been observed in cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL), where the co-occurrence of AD pathology is extremely rare [+T−N* profile may not always be synonymous with amyloid pathology.On the other hand, the Aes (DLB) . Howeveruncommon ,37,49, auncommon ,51 and Cuncommon , whilst uncommon . One couuncommon  and NPH uncommon ; howeverely rare . Thus, tThere are some limitations in classical CSF biomarker determinations. Test results are affected by preanalytical factors, including CSF sampling and storage in addition to internationally accepted guidelines have been formulated for this reason . In addi42, and various factors may be responsible, including the APOE ε4 allele [42/Aβ40 ratio, which performs better than Aβ42 alone [Discordant biomarker results have been observed in different reference laboratories, especially for Aβ4 allele . The Aβ442 alone ,65 may r42 alone , but the42 alone .+T+(N)+ profile is simply compatible with the presence of AD, but gives no information for a possible additional synucleinopathy or, rarely, one of the frontotemporal pathologies. Furthermore, normal levels of all three CSF classical biomarkers may be observed not only in normal aging, but also in psychiatric disorders which may present with cognitive complaints [Classical CSF biomarkers can identify the presence of AD in patients with mixed conditions, such as cerebrovascular disease (evident in neuroimaging) or DLB  with a concomitant AD pathology ,37,38,54mplaints ,68 and implaints , which mmplaints , but resmplaints ,72. Anotmplaints ; much womplaints .Finally, one must keep in mind that CSF biomarkers are not stand-alone tools. As in most  areas of medicine, biochemical tests should be interpreted along with clinical, imaging and other laboratory data (and sometimes follow-up data) in order to reach the final diagnosis. 42 alone is not sufficient, since it may be compatible with pathological change in Alzheimer’s [42 (or Aβ42/Aβ40) and increased τP-181 should be present [P-181/Aβ42 ratio may provide good diagnostic accuracy for the differential diagnosis of AD from other disorders [According to the FDA approval, aducanumab targets parenchymal amyloid in Alzheimer’s disease and not in any condition with amyloid plaque burden . For CSF confirmation of an AD diagnosis, reduction in Aβheimer’s ; it may heimer’s  or vascuheimer’s ,56, whicheimer’s  and thus present . Even if present , other g present ,39 or sc present  are not isorders .Keeping the above limitations in mind, biomarkers are not useful only in research, but also in clinical practice, showing both added and prognostic value ,79. They42 and various forms of phospho-tau, might replace CSF and PET biomarkers in the future, since they are easily obtained, not only for initial diagnosis but also for repeated follow-up [The question arises as to whether amyloid PET alone is sufficient for the diagnosis of AD  and whether an additional tau PET would be required, with concerns regarding cost-effectiveness. CSF biomarkers are of much lower cost, but require a lumbar puncture which, being relatively invasive, is a source of concern in many patients and/or caregivers. Blood-based biomarkers, including Aβollow-up ; howeverollow-up .


In both studies, mean AUCτ,tp values for Ig20Gly were essentially equivalent to AUCτ with point estimates of geometric mean ratio (GMR) of AUCτ,tp/AUCτ near 1.0 and 90% CIs within 0.80–1.25. In contrast, for IVIG, 10%, mean AUCτ,tp values were lower than AUCτ by >20%, . Mean AUCτ,tp values calculated for 4 other SCIG products (based on mean IgG trough levels reported in the literature/labels) were also essentially equivalent to the reported AUCτ , while differences for IVIG products were >20%. In conclusion, steady-state serum IgG levels following weekly SCIG remain stable, allowing for reliable prediction of AUC over the dosing interval using trough IgG levels. These findings indicate that measuring steady-state serum IgG trough levels alone may be adequate for PK assessment of weekly SCIG.Patients with primary immunodeficiency diseases often require lifelong immunoglobulin (IG) therapy. Most clinical trials investigating IG therapies characterize serum immunoglobulin G (IgG) pharmacokinetic (PK) profiles by serially assessing serum IgG levels. This retrospective analysis evaluated whether steady-state serum IgG trough level measurement alone is adequate for PK assessment. Based on individual patient serum IgG trough levels from two pivotal trials (phase 2/3 European [NCT01412385] and North American [NCT01218438]) of weekly 20% subcutaneous IG , trough level-predicted IgG AUC (AUCThe online version contains supplementary material available at 10.1007/s10875-021-00990-z. Primary immunodeficiency diseases (PID) are a heterogeneous group of >400 disorders that are often characterized by absent or deficient antibody production, which can lead to frequent and severe infections , 2. PatiCmax) followed by distribution and elimination phases, the slower absorption of SCIG from the SC infusion site results in a more gradual and stable increase in IgG serum concentration for several days, with a lower Cmax than the peak achieved with IVIG infusions  Patients in the European study received IVIG, 10% for 13 weeks or SCIG, 16% for 12 weeks in period 1, and then received weekly SCIG, 20% at a dose of the period 1 dose adjusted to weekly equivalent for 52 weeks . In bothMean IgG trough levels were obtained from the published licensing studies for 6 other IG products . A description of the IG products evaluated is provided in Table For IVIG and SCIG administration of Kiovig/GammaGard, PK data were obtained from a multicenter, prospective, open-label North American study of patients with PID , 24. PatFor the facilitated SCIG, HyQvia/HYQVIA , PK data were obtained from a prospective, open-label, noncontrolled, multicenter US trial in patients with PID who had received IVIG treatment for ≥3 months, followed by HyQvia/HYQVIA  administered every 3–4 weeks for approximately 14–18 months , 25.For IVIG and SCIG administrations of Gamunex, PK data were obtained from an open-label, crossover North American trial in patients with PID who had previously received or were currently receiving IG replacement therapy. Patients received Gamunex 200–600 mg/kg IV every 3–4 weeks for at least 3 months, then were switched to weekly SC infusions at a dose adjustment coefficient (DAC) of 1.37 for up to 24 weeks , 26.Data for Hizentra were obtained from 2 studies conducted in Europe and the USA. In the European PK substudy, patients with PID who had previously received IVIG treatment  were switched to weekly subcutaneous (SC) treatment with Hizentra , 27. Aftτ,tp) was calculated based on serum IgG trough levels using the following formula:Ctrough,ss is the steady-state trough concentration and τ is the dosing interval.Trough-predicted AUC  of AUCτ,tp/AUCτ , were calculated. In addition, Bland-Altman plots of log-transformed AUCτ,tp and AUCτ values were generated to assess agreement of the methods at individual levels rather than as a summary over the full sample [AUCl sample .τ,tp was based on the published mean serum trough data, and the % difference between AUCτ,tp and AUCτ was determined using the following formula:For other SCIG or IVIG products, the calculation of AUCτ,tp and the % difference between AUCτ,tp and AUCτ.For completeness, for Ig20Gly, the mean trough data were also used to calculate AUCFor all studies included in this retrospective analysis, patients or their guardians provided written informed consent according to local consent procedures in accordance with the ethical standards of their respective institutional research committees and with the 1964 Helsinki declaration and its later amendments , 24–28.τ,tp for SCIG, 16% and SCIG, 20% were essentially equivalent to the reported AUCτ, with point estimates of GMR of AUCτ,tp versus AUCτ between 0.98 and 1.09, and all 90% CIs within the commonly used equivalence limit of 0.80–1.25  of AUCτ,tp versus AUCτ was 0.74 (0.70–0.78) and 0.77 (0.73–0.81) for the European study and North American study, respectively , except for HyQvia (33.0%). For the 2 products administered as both SCIG and IVIG therapy (Kiovig/GammaGard and Gamunex), the IV products resulted in lower mean values of AUCτ,tp and a greater difference between AUCτ,tp and AUCτ compared with the corresponding SC products, and greater mean Cmax values and lower mean Ctrough values than the SC products.AUCes Table . DiffereCmax and Ctrough for SCIG products were in the range of 1.9 to 23.7% (mostly within 15%) of each other. By comparison, the mean Cmax was more than double the Ctrough for all IVIG products. This minimal fluctuation between peak and trough concentrations after SC administration of a SCIG therapy at steady state has formed the foundation of this proposed trough-based PK assessment.This retrospective analysis sought to determine whether measurement of serum IgG trough levels alone is sufficient for steady-state PK assessment of SCIG therapies in patients who have PID. Given the PK characteristics of serum IgG after SC administration, i.e., long elimination half-life (in weeks) and prolonged SC absorption (in days), it is expected that the serum levels of IgG over a 1-week dosing interval at steady state would remain stable, with essentially no discernible peak in serum IgG levels. This is demonstrated by the data for Ig20Gly , 14 and τ,tp calculated based on steady-state trough levels for Ig20Gly administered weekly was found to be equivalent to the AUCτ reported in pivotal studies using a serial sampling method [τ,tp and AUCτ for Ig20Gly treatment was found to be as expected given a generally reported range of 15–20% for the coefficient of variation of IgG assays. Similarly, for other SCIG products, when AUCτ,tp was derived from mean serum trough IgG levels, it was within ± 10% of the reported AUCτ for each individual product. In contrast, the AUCτ,tp for IVIG and facilitated SCIG products was considerably lower than the reported AUCτ, and the agreement between AUCτ,tp and AUCτ was much lower for IVIG, 10%. These findings suggest that for SCIG therapies, steady-state serum IgG trough levels are representative of not only the exposure level at a single time point but also the total exposure over the dosing interval (AUCτ); therefore, the measurement of serum trough level alone at steady state may be adequate for PK assessment of weekly conventional SCIG treatment in patients with PID.AUCg method , 14. TheCmax and a consequently larger difference between Cmax and Ctrough, and therefore contributed to our findings for this product that trough-predicted AUCτ,tp is not equivalent to the reported AUCτ. Similarly, the larger difference between AUCτ,tp and the reported AUCτ observed for IVIG products is likely the result of their increased bioavailability relative to SCIG products, increasing Cmax, and the longer dosing interval, which allows for more IgG to be metabolized before the next dose is administered, lowering Cmin [HyQvia/HYQVIA is a facilitated SCIG product, which is co-administered with recombinant human hyaluronidase, an absorption enhancer . It is ling Cmin –8. GivenCtrough and AUCτ, are the most relevant PK assessment at steady state for SCIG products, and our proposed serum trough alone approach will cover both the single-point exposure and the total exposure. Therefore, the value of characterizing PK profiles of serum IgG over the 1-week dosing interval by serial sampling is very limited.Despite the fact that other PK parameters, such as volume of distribution, elimination half-life, and clearance, are occasionally reported for SCIG, these parameters are not truly accurately estimated from a steady-state PK profile over a 1-week dosing interval due to the prolonged absorption and long elimination half-life. In the authors’ opinion, the systemic exposure parameters, including One limitation of this analysis is that because this was a retrospective analysis, direct access to individual patient data was not available for all products; in these cases, the analysis was conducted only on the mean values reported in the published literature, and a Bland-Altman plot could not be provided. In addition, conclusions about the value of measuring serum IgG trough levels alone for PK assessment of SCIG products at steady state are limited in this manuscript to SCIG treatment received on a weekly basis, regardless of the dose level .τ) using serum IgG trough levels alone. Our findings appear to be generalizable across all conventional SCIG products. These results indicate that measuring steady-state serum IgG trough levels alone for PK assessment of weekly SCIG treatment is a reasonable and beneficial alternative to serial PK sampling during clinical development and beyond. For patients and investigators, the use of steady-state IgG trough levels for PK assessment offers multiple benefits, including more efficient clinical trial conduct through decreases in study costs and logistical complexity and, more importantly, a reduction in the patient burden of frequent blood sampling.In conclusion, steady-state serum IgG levels remain stable following weekly SCIG treatment in patients with PID, enabling reliable prediction of total exposure (AUCESM 1(DOCX 37 kb)