Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive enzyme–inhibitor complex. This type of inhibition can involve competitive, mixed, non-competitive and uncompetitive inhibitors. While full reversible inhibitors show linear plots for reciprocal enzyme initial velocity versus inhibitor concentration, partial inhibitors produce hyperbolic plots. Similarly, dose–response curves show residual fractional activity of enzymes at high doses. This article reviews the theory and methods of analysis and discusses the significance of this type of reversible enzyme inhibition in metabolic processes, and its implications in pharmacology and toxicology. Reversible inhibition of enzymes is a very fast process. Establishment of the enzyme–inhibitor complex occurs within microseconds. With slow-binding inhibitors, establishment of the enzyme–inhibitor steady-state is much longer and can need minutes. To simplify, our topic is devoted to fast reversible inhibition of enzymes (rapid equilibrium assumption), which constitute the majority of encountered reversible inhibition processes. Several mechanisms account for rapid reversible inhibition of enzymes (E). Rapid reversible inhibition can be either competitive, non-competitive, mixed or uncompetitive. In most cases, the enzyme–inhibitor complex (EI) is non-productive. However, in certain cases, reversible inhibition is partial, the ternary complex enzyme–substrate–inhibitor (ESI) can be productive to some extent . The pheThe importance of enzyme PRI in metabolic processes has been recognized in early studies ,3. The pv) equation (Equation (1)) of the simplest Michaelis–Menten enzyme kinetic system (upper part of maxV) = catk[E], in which catk is the catalytic constant, [E] the enzyme concentration, and [S] the substrate concentration.Let us consider the rate (A reversible inhibitor (I) may bind to the free enzyme (E) and/or to the enzyme–substrate complex (ES). Four mechanisms account for fast reversible inhibition of enzymes: competitive inhibition, where inhibitor competes with substrate for the same site; non-competitive inhibition; mixed-type inhibition; and uncompetitive inhibition. Uncompetitive inhibitors bind exclusively to ES complexes. In general, the complex ESI is non-productive. However, in the case of PRI, the ESI complex displays some activity  is partially productive with a catalytic constant catβk < catk.Assuming a rapid equilibrium system, α ≥ 1 (except α ≤ 1 for uncompetitive PRI) indicates the change in sK and iK resulting from binding of I to the active site. iαK is sometime written iK’; the coefficient β, 0 < β < 1, indicates that ESI complex is productive. Then, in the case of competitive inhibition, α = ∞ and iK << iαK; for non-competitive inhibition, α = 1 and iK = iαK; for uncompetitive inhibition, iK = ∞ and iαK << iK. In the case of mixed-type inhibitions, competitive mixed-type, α > 1 and iK < iαK; uncompetitive mixed-type, α < 1 and iαK << i.KIn Assuming rapid equilibrium, the general scheme  can be dI], the maximum velocity at saturating substrate concentration tends to max,iV = catβk[E]. At first, Lineweaver–Burk plots (1/v vs. 1/[S]) and hyperbolic replots of slope and 1/v-axis intercept as a function of inhibitor concentration [I] can conveniently be used for diagnosis of PRI. 3D representations of Lineweaver–Burk, Hanes and Eadie-Hofstee plots at different inhibitor concentrations can be built /mK. More recently, a decrease in secondary plots of appKm/appkcat vs. [I] was found to be a valuable qualitative criterion for partial inhibition  = 0, y = 1 and at high [I], the residual fractional activity is i,miny. In Equation (3), IC50 is related to inhibition constant iK.v0 is the rate at given substrate concentration in the absence of an inhibitor.According to Copeland  , For full reversible inhibitors, relationships between  Prusoff . A recenity, see . In the i et al. . The autiv versus [I] at different substrate concentrations)  at different [I]) can be used for PRI diagnosis. It has also been proposed for determination of PRI parameters. This approach was extensively developed in Segel’s book [iv and slope versus [I] are hyperbolic for PRI, while they are linear for fully reversible inhibition. On the other hand, replots of 1/Δ intercepts (on x and y axes) and 1/Δ slope as a function of 1/[I] are linear for PRI and provide iK, α and β. .As mentioned above, Lineweaver–Burk plot analysis [E][S]. In the case of fully competitive inhibition where m,appK = mK(1+[I]/iK), i/vv0→0 at high[I]. Thus, the rate equation (Equation (2)) simplifies and in the case of PRI, at low [S], the hyperbolic plot of i/vv0 vs. competitor concentration does not reach 0 activity at high competing substrate concentration : Kinetic analysis based on the integration of the Michalis–Menten equation, i.e., analysis of the time course of reporter substrate degradation until substrate consumption, in the presence of an inhibitor, can also provide evidence for partial inhibition. The modern theoretical analysis of competing substrate progress curves kinetics was developed by Golicnik and Masson . Such anI] become high (>>iK), 1/[I]→0, and i/vv0 reaches an asymptotic value depending on β and iαK . In the case of multiple binding sites for inhibitors, causing PRI with coefficients β and δ, Equation (5a) becomes Equation (5b): iv/v0→δ⁄αγ at [I] >> ipK. This situation is described in β = 0.18) determined from the time course of hydrolysis of benzoylthiocholine (reporter substrate) in the presence of varying concentrations of a competing substrate, benzoylcholine (blind competing substrate). If the blind substrate were a fully competing substrate of benzoylthiocholine, BChE-catalyzed hydrolysis, iv/v0 should reach 0 at a high benzoylcholine concentration. The asymptotic limit (β) indicates partial inhibition. β, (0 < β < 1), can be simply determined from linear plots of fractional velocity (iv/(v0i− v)) as a function of the reciprocal of [I] at different [S] (Equations (6)–(9); β > 0, at 1/[I] = 0, the ordinate intercepts values are always positive, depending on [S], sK, α and β (Equation (6b)):More conveniently, the coefficient Then, equations for competitive, non-competitive and uncompetitive full and partial inhibitions are as follows: β = 1, α > 1):For partial competitive inhibition  at high [S].For partial uncompetitive inhibition: For full inhibition, intercepts on abscissa and ordinate are equal to 0 for all type of inhibitors.S instead of I. Thus, Yoshino  from Lineweaver–Burk plots) must be systematically built to avoid misdiagnosis of partial inhibition.Limited solubility of inhibitors beyond a certain concentration can be the cause of pseudo-partial inhibition. Thus, analytical measurement of inhibitor concentration in buffer solution, and the use of co-solvent to increase inhibitor solubility must be performed to eliminate this possible artefact. Allosteric inhibition and product inhibition  may also cause pseudo-partial inhibition. Thus, primary plots , Hill plots and secondary plots  and butyrylcholinesterase (BChE), important enzymes in the central and peripheral cholinergic system, deviate from the Michaelis–Menten model with positively charged substrates. In particular, AChE is inhibited by excess acetylcholine, the cholinergic system neurotransmitter, while the sister enzyme BChE is activated by excess substrate ,26. In cSphingobium japonicum UT26, it was shown that strong enzyme inhibition by substrate occurs. This inhibition was attributed to direct blockage and/or limited conformational flexibility of the product exit from the active site of the enzyme.To maintain constant glucose levels in the body, there is regulation of the activity of a multi-enzyme complex, the pyruvate dehydrogenase complex, which is inhibited at high concentrations of substrates: pyruvate, ADP, NAD and CoA . Many phReed et al.  listed mβ up to 0.24 [1–40 aggregation. These compounds are also partial non-competitive inhibitors of both ChEs [i/vv0) tends towards β (cf. Equations (2) and (3) and In addition to the previous example about inhibition of AChE by excess acetylcholine, we must consider the inhibition of ChEs by various toxic compounds. First, it is important to make a difference between the irreversible and reversible inhibitors of these enzymes. Irreversible inhibition of ChEs happens in acute poisoning by anti-cholinesterase compounds, e.g., carbamates and organophosphates, mostly pesticides and banned chemical warfare agents . These c to 0.24 . Recentloth ChEs . The part1/2 for the most sensitive birds [LD50 values. Thus, the increased sensitivity to diclofenac of the Gyps vulture compared to other related birds and chickens may be of pharmacogenomic origin. However, in other situations, increased toxicity due to partial inhibition of a detoxifying enzyme can have non-genetic origins, e.g., dysfunction of a metabolic chain, multiple antagonistic interactions on target enzymes.Trimetazidine is an effective therapeutic drug for the treatment of various forms of coronary heart diseases. The metabolic effects of trimetazidine, used in the clinic, increase the efficiency of cardiac work with partial inhibition of β-oxidation of fatty acids, allowing hydrolysis of ATP to be more efficiently converted into contractile heart work . Additiove birds . These pIC50 compared to orthosteric inhibitors  [β may limit the therapeutic interest of such inhibitors.On the contrary, PRI of enzymes may limit the action of inhibitors and therefore their pharmacological interest. For instance, melanin formation in skin and brain from naturally occurring monophenols depends on tyrosinase. Inhibitors of this enzyme have been developed to treat hyperpigmentation of the skin but could be of interest in treatment of neurodegenerative diseases. It was found that certain benzonitrile derivatives of mushroom benzonitriles are PRI of tyrosinase (to 0.42) . The higβ = 0.12) mixed-type inhibition of UDP-glucuronosyl transferase isoenzymes involved in paracetamol (acetaminophen) glucuronidation caused by dasabuvir, an anti-hepatitis C drug [PRI of enzymes involved in the metabolism of drugs, may affect clinical drug interactions. An example is given by the partial (s C drug . CorrelaThe typical antipsychotic drug risperidone, used for the treatment of schizophrenia, has been reported to cause hyperbolic inhibition of the uncompetitive type on D-amino acid oxidase with an inhibition constant in the low micromolar range . BecauseAnother potent practical therapeutic application could be to optimize the combination of synergistic drugs acting as enzyme inhibitors within large metabolic networks, and causing the minimal side effects. Current optimization approaches imply modulation of the dosage of different drugs for reducing the activity of target enzymes. Complex algorithms have been proposed for solving this problem . Alternaβ. The relevance of enzyme PRI in physiological processes is well documented where it is involved in multiple regulatory metabolic functions. Participation of PRI in epigenetic mechanisms is still poorly documented. In toxicology, PRI of target enzymes may dampen the acute action of toxicants and participate in defense mechanisms against poisonous natural and artificial xenobiotics. In pharmacology, enzyme inhibitors acting as PRI show certain therapeutic advantages over classical enzyme inhibitors in use in medicinal treatments. This is in particular the case with partially reversible uncompetitive inhibitors. Therefore, in the early stages of drug discovery [Partial inhibition of enzymes by ligands and an excess of natural or competing substrates is a quite common phenomenon. However, PRI has been insufficiently explored, analyzed, and exploited so far. Several simple graphical methods make it possible to detect PRI and to determine the inhibition parameters, in particular the coefficient iscovery , carefuliscovery  and drug


Retrospective review of all spinal fusions > 3 levels involving the thoracolumbar and/or sacroiliac at a single institution, by a single surgeon between 3/12/2020 and 8/13/2021 were reviewed. All screws that were secondarily navigated after identified as misdirected on intraoperative CT scan were included. Neuromonitoring reports were culled for mA threshold to triggered EMG response for all redirected screws. Intraoperative, post-de novo screw placement images (fluoroscopy scout and intraoperative CT) and post-redirection intraoperative scoliosis films and post-operative scoliosis films were independently reviewed by a senior neuroradiologist. Fifty redirected screws in the thoracic, lumbar, sacral, and ilium were identified as misdirected and redirected via navigation. The new trajectory of all screws was confirmed satisfactory by independent review between a senior neuroradiologist and neurosurgeon. Four screws could not be verified by post-operative imaging . All triggered EMG stimulated > 15 mA. No screws required return to the operating room for revision. No patients experienced a post-operative deficit. Redirection of misdirected thoracolumbar and sacroiliac screws can be performed using intraoperative CT and navigation as a means to detect and directly visualize appropriate placement. Pedicle screw and sacroiliac fixation are mainstay techniques in adult deformity spine correction. Accurate bony purchase bears consequence on the final integrity of the individual screw and construct as a whole . For thiWe retrospectively reviewed all spinal fusions > 3 levels involving the thoracolumbar and/or sacroiliac spinal segments at a single institution, by a single surgeon between 3/12/2020 and 8/13/2021. Operative reports were reviewed for (1) level and laterality of identified misdirected screws (2) removal and redirection of misdirected screws (2) intraoperative CT scan and fluoroscopy in the picture archiving and communication system (3) neuromonitoring reports. Reports in which screws were primarily navigated, intraoperative or post-operative imaging was missing, or associated neuromonitoring reports were missing were excluded. Our intraoperative imaging and navigation workflow is discussed below. Intraoperative, post-de novo screw placement images (fluoroscopy scout and intraoperative CT) and post-redirection intraoperative scoliosis films and post-operative scoliosis films were independently reviewed by a senior neuroradiologist. Neuromonitoring reports were culled for mA threshold to triggered EMG response for all redirected screws. Post-operative neurologic exam and any return to the operating room for instrumentation revision was evaluated.The patient is brought to the operating theater and inducted under anesthesia. Gardner-Wells tongs are placed in standard fashion and the patient is transferred prone to a Jackson table. Neuromonitoring leads are placed for triggered, motor-evoked, and somatosensory-evoked potentials in the upper and lower extremities. Incision then proceeds, followed by subperiosteal dissection, and exposure of appropriate landmarks for pedicle, S2A1, and iliac instrumentation is performed. Screws are placed in standard freehand fashion. A spinous process reference probe is then securely fastened to the base of the exposed levels in view of a reference array . The O-Arm  is then positioned around the patient and a scout image is performed to delineate the region to be scanned. A CT scan is then performed  from 19 procedures were retrospectively reviewed in operative reports. No cases with documented navigated, redirected screws were excluded for lack of imaging or intraoperative reports. Intraoperative CT scans were reviewed by an attending neurosurgeon and independently reviewed by a neuroradiologist for agreement in level and laterality of misdirection and post-navigation correction.Misdirected screws were classified as follows: 13 medial, 18 lateral, 4 inferior, 3 superior, 3 long, 2 inferolateral, 1 superolateral. The new trajectory of four redirected screws could not be satisfactorily evaluated by our neuroradiologist on post-redirection imaging . One screw appeared to be misdirected after navigation  with 21% loss for laterally directed screws in their thoracic cadaveric model . These fAdditional models have evaluated regained pull-out strength with screw redirection. Goda et al. human cadaveric study comparing primarily placed and secondarily redirected pedicle screws found at 24% loss of pull-out strength in the latter after a lateral breach . The perRobotics in Neurosurgery, delineating the foreseeable future of robotics in three main areas: telesurgical, supervisory controlled, and shared-control modes. This latter mode includes technologies in which the robot performs controlled functions, typically as a system registered to the patient, but under the direct control of the surgeon who will use the robot’s assistance for a specific goal [Methods of evaluating misplaced screws include probing for cortical breaches, neuromonitoring, and intraoperative imaging with technological adjuncts for live anatomic reconstructions . The latfic goal .Navigated assistance has gained popularity in spine surgery these new systems are coapted with known operative tools . Full integration of intraoperative CT assisted screw placement has been reported in large studies with early favorable results, with one prospective, post-marketing registry reporting a 1.8% need for revision in their series of 192 screw series . This prOther workflows emphasize technology as one of several safety checks for accurate screw placement. For example, Kassis et al. found that an intraoperative CT scan increased the sensitivity for detecting abnormal screw placement when preceded by an abnormal triggered EMG. This approach advocates for complementary, multimodal safety checks for navigated instrumentation .There is currently no standard, generalizable consensus on the overall benefit patient receiving de novo navigated instrumentation versus freehand. Discussion of the merits between one and the other is beyond the technical and philosophic scope of this paper. At our institution, the majority of fusion procedures > 3 levels are instrumented freehanded. The decision to use navigated assistance for redirection has emerged from (1) ease of synced workflow with an intraoperative CT scan and (2) belief in the benefit of visual-tactile synchrony gained with robotic assisted recannulation of a new tract. In essence, navigation is our “safety check” to complement other approaches when ensuring an accurate new cannulation corridor.Our results suggest the ongoing possibility of hybrid approaches between shared-control systems and skilled spine surgeons. We demonstrate the use of the navigation as safety check for accurate screw redirection, reporting no negative clinically outcomes or return to the OR and 97.8% accuracy based on post-redirection radiographic findings. There were no aberrancies in neuromonitoring. We also address navigation for non-pedicular approaches for iliac fixation with 100% accuracy, which has limited evaluation in the literature.Limitations of our study include lack of post-operative CT for true axial comparison of screw redirection. We sought to overcome this limitation via multimodal surrogates for accurate screw placement, including post-operative exam, neuromonitoring, and independent neuroradiologic evaluation of pre- and post-fluoroscopic and X-ray images of redirected screws. This study is also a retrospective case series without matched control and cannot be used to extrapolate comment regarding operative time or incidence of poor outcome in patients without navigated redirection.Redirection of suboptimally placed thoracolumbar and sacroiliac screws can be performed using intraoperative O-Arm and navigation as a means to detect and directly visualize appropriate placement. We report an overall satisfactory redirection of 97.8% screws in post-operative imaging and no return to the OR for revision. Additional studies are required to compare operative time between navigated and non-navigated screws.


Background: Spinal fusion is the most common surgical treatment for the management of degenerative spinal disease. However, complications such as screw loosening lead to painful pseudoarthrosis, and are a common reason for revision. Optimization of screw trajectories to increase implant resistance to mechanical loading is essential. A recent optimization method has shown potential for determining optimal screw position and size based on areas of high bone elastic modulus (E-modulus).Aim: The aim of this biomechanical study was to verify the optimization algorithm for pedicle screw placement in a cadaveric study and to quantify the effect of optimization. The pull-out strength of pedicle screws with an optimized trajectory was compared to that of a traditional trajectory.Methods: Twenty-five lumbar vertebrae were instrumented with pedicle screws .Results: An improvement in pull-out strength and pull-out strain energy of the optimized screw trajectory compared to the traditional screw trajectory was only observed for E-modulus values greater than 3500 MPa cm3. For values of 3500 MPa cm3 or less, optimization showed no clear benefit. The median screw length of the optimized pedicle screws was significantly smaller than the median screw length of the traditionally inserted pedicle screws, p < 0.001.Discussion: Optimization of the pedicle screw trajectory is feasible, but seems to apply only to vertebrae with very high E-modulus values. This is likely because screw trajectory optimization resulted in a reduction in screw length and therefore a reduction in the implant-bone interface. Future efforts to predict the optimal pedicle screw trajectory should include screw length as a critical component of potential stability. Spinal fusion, which typically consists of a combination of posterolateral pedicle screw instrumentation and rod construct , is the Computer-aided preoperative planning approaches have the potential to improve surgical outcomes by analyzing patient-specific three-dimensional (3D) key aspects. With rapid technological improvements in simulation models and intraoperative navigation in recent years, customized surgical plans can be created and implemented . A numbeThe aim of this biomechanical study was to verify the optimization algorithm for pedicle screw placement developed by Caprara et al.  in a cadThe study was approved by the local ethics committee. Twenty-five lumbar vertebrae  obtained from five fresh-frozen cadavers  were tested in this study. The median age was 60 years .The optimization algorithm used for screw positioning was described in detail in a previous study . In brieAn experienced spine surgeon used the MySpine planning software  to define the standard screw trajectory for the pedicles considered. The default positions were then used to place simplified screws in the 3D vertebral body models and initialize the GA method for all segmented vertebrae. The labeled entry points, pedicle points, and available screw lengths and diameters were used as input for the optimization to create the initial population for the genetic algorithm. Screw lengths varied from 25 to 60 mm (5 mm increments) and screw diameters varied from 5 to 7 mm (1 mm increments). The dimensions were selected from the MUST  pedicle screw system .Computed tomography (CT) scans  allowed assessment of vertebral bone integrity prior to screw insertion. Pedicle screws were inserted into both pedicles of each vertebral body with the help of a 3D-printed guide to ensure screw positioning according to planning. On one side, the pedicle screws were inserted with a traditional trajectory, i.e., parallel to the superior endplate and following the pedicle alignment . On the Screw length and diameter were based on the size of the vertebral body and pedicle, respectively. After preparation, the anterior parts of the lumbar vertebral bodies were mounted in appropriate trays using Polymethylmethacrylate . The boxes were made of polyethylene terephthalate (PET) for CT transparency.The setup for testing specimens follows that of another study . In a nuThe pull-out strength of each screw was defined as its resistance to axial loading and corresponded to the maximum of the experimentally recorded force-displacement curve. The strain energy was the area under the force-displacement curve from the beginning of the recording until the point at which pull-out strength was reached. Further, the estimated E-modulus within a screw resulted from its average value multiplied by the screw volume. Symmetry of anatomical and bone property aspects between the right and left sides of each vertebra was assumed. Therefore, the improvement in computed E-modulus and of the test results achieved through optimization resulted from the subtraction of the traditional value from the contralateral optimized value of the same vertebra. The diameter and length of the screws in the traditional trajectory group were compared to the screw sizes obtained with the optimization algorithm with a paired, non-parametric test (Wilcoxon signed-rank test). The significance level α was set to 0.05. Data were reported as median (25th percentile—75th percentile).3: the median pull-out strength improvement for this subset of screws was 399.0N (249.1–982.1N) and the median pull-out strain energy improvement was 3,034.3N mm .An improvement in pull-out strength and pull-out strain energy of the optimized screw trajectory compared to the traditional screw trajectory was only observed for values of average E-modulus times the screw volume greater than 3500  MPa cm3 or less, optimization showed no benefit. For E-modulus values within the screw ranging between 1,500 and 3,500 MPa cm3, both median pull-out strength and median pull-out energy were lower than traditionally inserted screws, at −106.8N (−485.2 to −7.5N) and −363.7N mm (−715.4 to −63.4N mm), respectively. There was also no advantage for values below 1,500, with median pull-out strength and median pull-out energy of −39.9N (−58.3–16.5N) and 45.5N mm (−44.5–66.5N mm), respectively, lower than the traditionally inserted screws.When the optimization resulted in values of 3500  MPa cmThe median pull-out strength was 698.7N  for pedicle screws with optimized trajectory. The median pull-out strength was 900.6N  for traditionally inserted pedicle screws.The median pull-out strain energy (N mm) was 1,244.8N mm (595.9–2962.5N mm) for pedicle screws with optimized trajectory. The median pull-out energy was 1076.5N mm (383.8–2512.4N mm) for traditionally inserted pedicle screws.The median screw diameter of the optimized screws was 7 mm (range: 5.8–7 mm). The median screw diameter of the traditionally inserted screws was 6 mm (range: 6–6.3 mm) .p < 0.001 (The median screw length of the optimized pedicle screws was 40 mm (range: 30–45 mm), which was significantly smaller than the median screw length of the traditionally inserted pedicle screws, which was 50 mm (45–55 mm),  < 0.001 .3). When the difference between optimized and traditionally inserted pedicle screws was less than 3500 MPa cm3, the optimized trajectory showed no real advantage.The main finding of the present study is that pedicle screw trajectory optimization is feasible. However, an increase in pull-out strength was only observed at a maximized difference in radiologically estimated E-modulus in screw volume  , but theThe results presented here should be considered as preliminary. It is intended that the algorithm will be further developed using screw length as an optimization criterion.In the present study, uniaxial pull-out tests were performed in order to draw conclusions about screw stability and thus the risk of screw loosening. However, these pull-out tests may not fully reflect the physiological loads to which the spine is subjected , so the In the present study, every effort was made to obtain the maximum number of lumbar specimens from a sample of cadavers with comparable age and sex characteristics. To minimize the impact of individual differences on the results of the studies, the design and placement of the screws were performed by the same team of researchers. It should also be noted that our results apply to the instrumentation material mentioned in the methods section, given that biomechanical properties such as fatigue resistance may differ between different instrumentation constructs .A level-dependent analysis was not performed. We assumed geometric and mechanical symmetry in the vertebral bodies to compare the performance between standard and optimal screw positions within each vertebra. In general, we observed that the within-subject  differences in bone density were significantly less important than the between-subject  differences.Furthermore, the clinical applicability of the optimized trajectory needs to be further evaluated by experienced spine surgeons. Depending on the existing pathology and the required intervention, the optimized trajectory may prove inappropriate by compromising structures that should be protected. In addition, the algorithm does not explicitly take screw length into account. Incorporating this into the algorithm by weighing the length of the screw more than it is presently done may improve the pull-out strength. This could be investigated in future studies.3), because the optimization of the screw trajectory resulted in a reduction of the screw length. In the future, more emphasis should be placed on screw length.Optimizing pedicle screw trajectory is possible. However, the pull-out strength could only be improved with very high optimizations (E-modulus times screw volume >3,500 MPa cm


Treatment of cystic fibrosis (CF) has been revolutionized by the use of cystic fibrosis transmembrane conductance regulator (CFTR) protein modulators such as elexacaftor/tezacaftor/ivacaftor (ETI) triple therapy. Prior studies support a role for type 2 (T2) inflammation in many people with CF (PwCF) and CF-asthma overlap syndrome (CFAOS) is considered a separate clinical entity. It is unknown whether initiation of ETI therapy impacts T2 inflammation in PwCF. We hypothesized that ETI initiation decreases T2 inflammation in PwCF.A single center retrospective chart review was conducted for adult PwCF. As markers of T2 inflammation, absolute eosinophil count (AEC) and total immunoglobulin E (IgE) data were collected longitudinally 12 months prior to ETI therapy initiation and 12 months following therapy initiation. Multivariable analyses adjusted for the age, gender, CFTR mutation, disease severity, inhaled steroid use, and microbiological colonization.There was a statistically significant reduction  in 12-month mean total IgE following ETI initiation; this change remained statistically significant in the multivariate model. The longitudinal analysis demonstrated no change in AEC following therapy initiation.This study demonstrates that there is a statistically significant percent reduction in mean total IgE but no change in AEC following ETI initiation. ETI may lead to decreased antigen and superantigen load in the airway as a result of improved mucociliary clearance and these changes may drive the decline in total IgE, without influencing the epigenetic drivers of eosinophilic inflammation. Further studies are warranted to determine the underlying mechanism of ETI impact on T2 inflammation and possible role for asthma immunomodulator therapy post ETI initiation in CFAOS. CFTR gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein, most commonly a deleted phenylalanine in the 508th position (F508del). CFTR is a chloride channel that regulates transport of sodium, chloride, and bicarbonate [Cystic fibrosis (CF) is one of the most common respiratory genetic diseases, affecting an estimated ~ 37,000 people in North America and ~ 162,000 people worldwide [Many studies have shown that CF patients commonly display evidence of type 2 (T2) inflammation  characteet. al. found a prevalence of peripheral eosinophilia of 45% in CF patients hospitalized for exacerbation, and eosinophilia was associated with significantly longer hospital stays [Several studies support this role for T2 inflammation in many patients with CF. Siedlecki, al stays . Our groal stays , 13. Intal stays . Expertsal stays . PreviouElexacaftor/tezacaftor/ivacaftor (ETI) modulator therapy has recently been approved by Food and Drug Administration and has become the main therapy prescribed for most people with CF. ETI has been shown to improve CFTR function, decrease sweat chloride concentrations, and improve both lung function and functional status as demonstrated in the PROMISE clinical trial. . While aThe aim of this study was to investigate the impact of CFTR modulator triple therapy on Absolute Eosinophil Count (AEC) and total IgE as markers of the type 2 inflammatory response in patients with CF.A single center retrospective chart review was conducted for adult cystic fibrosis patients (n = 108) seen at the University of Virginia Adult Cystic Fibrosis Clinic between 2018 and 2021. Inclusion criteria were a diagnosis of CF and initiation of ETI therapy and at least one measurement of IgE and/or AEC pre and post ETI initiation. Exclusion criteria were a history of transplant, IL-4 (dupilumab), IL-5  or IgE -targeting biologics, and insufficient AEC or total IgE data. This study was approved by our institution’s Institutional Review Board Human Subjects Research committee and followed procedures with ethical standards. (IRB-HSR 23337).AEC and IgE data were collected for each patient 12 months prior to initiation of ETI therapy and 12 months following initiation of therapy. AEC measurements from the center-associated lab were collected from the electronic health record (EHR). If more than one IgE or AEC measurements were available, the mean pre and post-ETI levels were calculated and included in the analysis. Given that infection results in activation of inflammatory responses, we excluded data collected during pulmonary exacerbation and/or use of IV antibiotics. AEC measurements were excluded if they were obtained within 7 days prior to determination of an exacerbation and/or initiation of IV antibiotic therapy or if they were within 14 days following exacerbation and/or completion of IV antibiotic therapy.1) and was categorized as normal (ppFEV1 > 90%), mild (70% < ppFEV1 ≤ 90%), moderate (40% < ppFEV1 ≤ 70%), and severe (ppFEV1 ≤ 40%). With regards to bacterial colonization history, data regarding prior Pseudomonas infection, MRSA infection, and other bacterial colonization were collected. Other bacteria were defined as Stenotrophomonas spp., Achromobacter spp., Acinetobacter spp., or Burkholderia spp. With regards to fungal colonization, data regarding prior Aspergillus spp., Exophiala spp., and Rasamsonia spp. colonization were collected. Colonization was defined as 2 positive cultures in a 6 months interval. The bacterial and fungal colonization was determined at the beginning of the study, for the following reasons: after ETI initiation and in the context of the pandemic, sputum collection declined; the program transitioned to telemedicine and remote sputum collection was challenging; in addition, the patient experienced a significant decrease in sputum production due to ETI. Inhaled steroid use was coded as yes or no, defined as use prior to ETI initiation.We were unable to exclude total IgE measurements taken during an exacerbation as total IgE measurements were obtained on a less frequent basis, often only once annually. As such, total IgE data were collected from the EHR including both data from the center associated lab and outside facilities, when available. Additional collected data included age, sex, disease severity, infection/colonization history, CFTR mutation information, and inhaled steroid use. Disease severity was defined based on percent predicted Forced Expiratory Volume in one second . A two-sided significance level of 0.05 was selected as the null hypothesis rejection criterion. Linear mixed-effect models were fit using the lme4  and lmerPseudomonas spp. colonization, MRSA colonization, other bacterial colonization, fungal colonization, and inhaled steroid use. A linear mixed-effect model was constructed to further investigate the mediating effect of initial disease severity on the ETI treatment effect. The model treated initial disease severity as a factor with four levels.To assess how log-transformed 12-month mean total IgE changed after ETI initiation, we fit a multivariable linear mixed-effect model with a random intercept of patient, regressing over an indicator variable for ETI initiation and adjusting for current age, gender, CFTR mutation, initial disease severity level, Pseudomonas colonization, or MRSA colonization were each examined individually with two linear mixed-effect models  that had a random intercept for patient. All models included an interaction between the presence of the pathogen and ETI initiation.The relationships between the change in mean total IgE after ETI and prior fungal colonization, Pseudomonas spp. colonization, MRSA colonization, other bacterial colonization, fungal colonization, and inhaled steroid use.To assess how log-transformed AEC changed after ETI initiation, we fit two linear mixed-effect models, a univariate and multivariable model, with a random intercept for patient. While AEC was collected longitudinally, it was still regressed over an indicator variable for ETI initiation because time since initiation was not meaningful. The multivariable model adjusted for age, gender, CFTR mutation, initial disease severity level, Baseline demographics and characteristics2.IgE and ETI therapySummaries of baseline demographics and patient characteristics are presented in Table 3.Post-ETI initiation change in IgE and lung functionThe estimated change in IgE after ETI initiation is plotted in Fig. 4.Post-ETI change in IgE and microbiological colonizationAfter determining that ETI initiation was associated with a change in 12-month mean total IgE, we investigated whether initial lung function impacted the magnitude of the change in total IgE after ETI. In this mixed model, the reduction of total IgE following ETI initiation remained statistically significant (p < 0.001). However, total IgE at baseline did not significantly differ by initial disease severity (p = 0.206), and the reduction in total IgE following ETI therapy did not significantly differ by initial disease severity (p = 0.890). These results showed that in our patients, the magnitude of the change in IgE after ETI does not seem to depend on initial disease severity.Pseudomonas colonization, or MRSA colonization alone modified the magnitude of the change in mean total IgE after ETI using subgroup analysis through univariate and multivariable models. Univariate and multivariable models produced similar results, so all values reported below are adjusted estimates.We also investigated whether fungus colonization, Pseudomonas colonization after starting ETI .As shown in Fig. ETI Fig. C. There 5.AEC and ETIIn summary, these results indicate that without considering the effects of the presence of other microorganisms, a reduction in mean total IgE was observed in the presence and not absence of a positive fungal culture, and in the absence and not presence of MSSA. The presence of Pseudomonas did not impact the IgE decline post ETI. The magnitude of reduction in mean total IgE after ETI initiation was only impacted by prior MRSA colonization.The estimated percentage change in AEC after ETI initiation is plotted in Fig. A large subset of patients with CF develop evidence of T2 inflammation as demonstrated by the presence of blood and airway eosinophilia, robust elevations in their total IgE, and evidence of allergen sensitization –12. WhenThe treatment of patients with CF has been revolutionized by the efficacy of CFTR modulator therapy. The mechanism behind the improvement in patient outcomes is a continued topic of investigation as our understanding of the role of CFTR function evolves. It is unclear whether institution of CFTR modulator therapy will impact a previously established T2 inflammatory state.To our surprise, multivariable analysis of AEC data in this study, after excluding measurements obtained during exacerbation and/or IV antibiotic use, demonstrated no change following therapy initiation. Eosinophilia is the quintessential defining feature of T2 inflammation , 21. As high state and it is well recognized that CF patients frequently display aspergillus-specific IgE and, indeed, this disease is often complicated by allergic bronchopulmonary aspergillosis [Staphylococci. While not an allergen, staph are copious producers of numerous superantigens that non-specifically  can broadly activate numerous families of T lymphocytes. This study showed that the total IgE declined post ETI in subjects previously colonized with MRSA, Pseudomonas and fungi. MSSA colonization was associated with no change in IgE pre and post ETI, while in the absence of MSSA, the IgE declined post ETI. CF has been associated with high expression of staph-derived superantigens in the airway [Staphylococcus aureus can also cause epigenetic changes, DNA methylation leading to a persistent inflammatory state [Staph) load secondary to ETI therapy initiation could explain the reduced total IgE production in the absence of ETI therapy having an effect on ameliorating the underlying T2high state.In contrast to AEC, however, this data demonstrated a ~ 20% reduction in 12-month mean total IgE following ETI triple therapy initiation after controlling for multiple factors including demographic data and inhaled steroid use in the absence of an apparent impact on the T2 signature, this impact on total IgE begs an alternative explanation. CFTR dysfunction results in impaired mucus production and clearance, thus promoting microbial colonization in the airway. These microbes and, in particular, fungi, can function as allergens, especially in the presence of a T2gillosis –24. The e airway , 26. Insry state . Immune This analysis serves as an important starting point to continue the evaluation of CFTR modulator therapy from an immunologic perspective. These results are intriguing and warrant further studies to elucidate the exact effect of ETI on T2 inflammation. Based on our current findings and previous studies that show a correlation between T2 inflammation based on AEC and IgE levels and frequency of exacerbations , we suspFurther studies to elucidate the impacts of possible confounding variables in a larger patient population are warranted. Moreover, studies investigating additional markers of the T2 inflammatory response, especially those utilizing samples obtained from airway will prove valuable. Lastly, all of the patients included in the study initiated ETI therapy during adulthood. Given that early initiation of ETI during childhood is increasingly common, children may have had less infectious history, reduced antibiotic use, and less airway injury and thus may not have had an epigenetically established type 2 inflammatory signature prior to ETI initiation. Further studies analyzing the impact of ETI on T2 inflammatory markers in children could therefore be valuable in investigating the role of CFTR modulator therapy on T2 inflammation. MSSA colonization occurs usually early in life, while Pseudomonas, fungus and MRSA occur later. This may possibly lead to epigenetic changes in airway inflammation related to MSSA colonization and persistent T2 inflammation in this subpopulation.There are important considerations and limitations regarding this study. There are inherent limitations in performing a retrospective analysis due to limited data measurement frequency, varying frequency of clinical assessment of exacerbation status, and possible unknown microbiological colonization history and allergen sensitization prior to subject care establishment at our center. As per standard of care, at our program, when the sputum provided is an adequate amount, fungal, bacterial and AFB cultures are performed from the same specimen. When the amount is small, only bacterial cultures are performed, and when the patient cannot produce sputum, throat swabs are performed. Based on registry data, the ETI lead to a decrease in bacterial and fungal colonization, but in the absence of adequate sputum or bronchoalveolar lavage specimen, this is hard to prove. A prospective study of influence of bacterial and fungal colonization on T-2 inflammation post ETI may be informative. T2 inflammatory markers are impacted by numerous confounders including exposure to other allergens, systemic and topical corticosteroid use, and others. A future prospective study is essential using more robust markers of T2 inflammation including serum cytokines, periostin, and fractional exhaled nitric oxide. Retrospective collection of systemic steroid bursts used in the study group was not possible.This study demonstrates that there is a statistically significant percent reduction in mean total IgE but no change in AEC following ETI initiation. ETI may lead to decreased antigen and superantigen load in the airway as a result of improved mucociliary clearance and these changes may drive the decline in total IgE, without influencing the epigenetic drivers of eosinophilic inflammation. Further studies are warranted to determine the underlying mechanism of ETI impact on T2 inflammation and possible role for asthma immunomodulator therapy post ETI initiation in CFAOS.


Trypanosoma brucei rhodesiense, the parasite causing the acute lethal form of Human African Trypanosomiasis. Starting from the dipeptide nitrile CD24, the further introduction of a fluorine atom in the meta position of the phenyl ring spanning in the P3 site and the switch of the P2 leucine with a phenylalanine led to CD34, a synthetic inhibitor that shows a nanomolar binding affinity towards rhodesain (Ki = 27 nM) and an improved target selectivity with respect to the parent dipeptide nitrile CD24. In the present work, following the Chou and Talalay method, we carried out a combination study of CD34 with curcumin, a nutraceutical obtained from Curcuma longa L. Starting from an affected fraction (fa) of rhodesain inhibition of 0.5 , we observed an initial moderate synergistic action, which became a synergism for fa values ranging from 0.6 to 0.7 . Interestingly, at 80–90% inhibition of rhodesain proteolytic activity, we observed a strong synergism, resulting in 100% enzyme inhibition. Overall, in addition to the improved target selectivity of CD34 with respect to CD24, the combination of CD34 + curcumin resulted in an increased synergistic action with respect to CD24 + curcumin, thus suggesting that it is desirable to use CD34 and curcumin in combination. Rhodesain is the main cysteine protease of  T. brucei gambiense, typical of western and central Africa and responsible for the chronic form of HAT, and T. b. rhodesiense, widespread in eastern and southern Africa and able to induce the acute form of HAT, which has a higher mortality rate 1/m ,35. CD34 and curcumin, we used the Chou–Talalay method to evaluate the multiple drug effects 1/m 1/m ,35. CI > 1, CI = 1, and CI < 1 correspond to antagonistic, additive, and synergistic effects, respectively [The Chou–Talalay method was then applied to evaluate multiple drug effects ,35. Geneectively ,35. In mectively  is: veryCI for mutually non-exclusive drugs that act independently was calculated as follows: IC50)1 and (IC50)2 were obtained by dose-response curves, and D1 and D2 are the concentrations able to induce a specific percentage of rhodesain inhibition, as determined by the Median Effect Equation. Then, considering that CD34, in analogy to CD24 , acts asThe determination of the activity of the inhibition against human Cathepsin L (hCatL) was performed in fluorescence-based assays . The proThe assay buffer contained 50 mM Tris(hydroxymethyl)aminomethane, 5 mM EDTA, 200 mM NaCl, pH = 6.5, and 0.005% Brij 35 to avoid aggregation and false-positive results. The enzyme buffer contained 2 mM DTT rather than Brij 35. Inhibitor solutions were prepared from stocks of DMSO. An initial screening at an inhibitor concentration of 20 µM was performed to identify ligands (CD24 and CD34) with an inhibition of hCatL higher than 80%.CD34 and CD24 were then tested separately by Tian continuous assays, twice in duplicate in 96-well plates, for a total volume of 200 µL. An equivalent amount of DMSO was used as negative control. In more detail, we used 0.001 µM, 0.01 µM, 0.05 µM, 0.1 µM, 0.5 µM, 1 µM, and 10 µM for CD34 and CD24. The residual enzyme activities were determined continuously over a period of 10 min at room temperature. 50 values. The inhibitory constants (Ki) were calculated according to the Cheng–Prusoff Equation (1), with KM = 6.5 µM for Cbz-Phe-Arg-AMC. The percentage of enzyme inhibition was plotted against the inhibitor concentrations to obtain the experimentally determined ICP. pastoris was performed as described in [E. coli Rosetta2 DE3 pLysS (Novagen), the T. b. rhodesiense rhodesain sequence (uniprot-ID.: Q95 PM0), followed by a TEV cleavage site and a hexahistidine-tag, was cloned between the NdeI and BamHI restriction sites of a pET-11a vector by Genscript using codon optimization. The plasmid was amplified in XL-10 cells. A GFP sequence was additionally introduced between the TEV site and the 6xHis-tag by Gibson assembly following extensive construct and purification optimization. The rhodesain plasmid and the GFP gene were amplified in a PCR using the primers 5′-CACCACCATCACCACCACTAAGG-3′ and 5′-GCTACCTTGAAAATACAGATTCTCCGGG-3′ for the vector and 5′-CTGTATTTTCAAGGTAGCGTGAGCAAGGGCGAGGAGC-3′ and 5′-GGTGGTGATGGTGGTGCTTGTACAGCTCGTCCATGCCG-3′ for the insert. The expression of rhodesain from ribed in . For purp < 0.05 with respect to the percentage of rhodesain inhibition of curcumin, CD34, and curcumin + CD34. The analyses were performed with GraphPad Prism 5.0.3. Results were expressed as the arithmetic mean ± standard deviation (SD).The statistical analysis of the data was performed using the one-way test (ANOVA) with Dunnett’s multiple comparison test, considering significant differences of CD24 compared to CD34 , CD34 showed increased target selectivity, which is fundamental considering the high degree of homology between rhodesain and human cathepsin L, both belonging to the papain family. Besides the slightly higher activity of CI with those obtained for the combination CD24 + curcumin [50, followed by a slight-to-moderate synergism at 60–70% and a synergistic action from 80% to 100% rhodesain inhibition. If we compare the obtained values of curcumin  . Considering that both CD24, could further enhance the strength of these charge–transfer contacts. Overall, both the improvement in the strength of interactions with rhodesain and presumably the different disposition in the catalytic site of CD34, with regard to CD24, could favor the interaction with curcumin, which is believed to bind to an allosteric site close to the catalytic site. In contrast, the presence of the fluoro-substituted aromatic nucleus at the P3 site, in agreement with our previous studies , should