The emergence of multi-drug resistant pathogens is an urgent health-related problem, and the appropriate use of antibiotics is imperative. It is often difficult to identify the causative bacteria in patients with aspiration pneumonia because tracheal aspirate contains contaminants of oral bacteria. We investigated the dynamics of microbiota in mechanically ventilated patients with aspiration pneumonia to develop a treatment strategy.Twenty-two intubated patients with aspiration pneumonia were recruited. Saliva and tracheal aspirate of the subjects were collected at three time points: (A) within 2 h after intubation, (B) just before administration of antibiotics, and (C) 48-72 h after administration of antibiotics. The microbiota in each specimen was analyzed by using the 16S rRNA gene clone library sequencing method. Bacterial floras of the samples were analyzed by principal component analysis.p < 0.001). The extent of decrease in anaerobes was fully dependent on the time difference between the sampling of tracheal aspirate (A) and (B)—in particular, over 3 h of mechanical ventilation. This indicates that the alterations of microbiota (involving the reduction of anaerobes in the lower respiratory tract) occurred during mechanical ventilation prior to the administration of antibiotics. After the administration of antibiotics, Enterobacter spp., Corynebacterium spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Granulicatera adiacens were predominantly detected in the tracheal aspirate (C).Principal component analysis based on the composition of genus revealed that although the changes of microbiota in the saliva from (A) to (B) were not clear, the composition of anaerobes in the tracheal aspirate (B) was lower than (A). In fact, the reduction of anaerobes, not in the saliva but in the tracheal aspirate from (A) to (B), was confirmed by incident rate ratios estimated by a multilevel Poisson regression model  may occur during the mechanical ventilation in these patients. Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli were detected from patients with aspiration pneumonia , Corynebacterium spp. , Pseudomonas aeruginosa (99%), Klebsiella pneumoniae (99%), Staphylococcus aureus (99%), and Granulicatera adiacens (99%) were predominantly detected in the tracheal aspirate (C) (% indicates similarities with the type strain). There were no cases in which the predominant phylotype of the tracheal aspirate was an anaerobe in the results of clone library analyses.The culture results and phylotypes detected by clone library method in each case are shown in Table The results of PCA based on the detected phylotypes at the genus level are shown in Fig. p = 0.76). On the other hand, the proportion of anaerobes were significantly lowered between tracheal aspirate (A) to (B). Figure 2, tracheal diameter, vasopressor use, ICU stay, mechanical ventilation days, hospital stay and mortality, there was no significant relationship between the proportion of anaerobes and these clinical factors. However, the extent of decrease in anaerobes was fully dependent on the time difference between the sampling of tracheal aspirate (A) and (B). There was no significant decrease of anaerobes in the tracheal aspirate (B) collected within 2 h after collecting the tracheal aspirate (A) ; a significant decrease in anaerobes occurred in tracheal aspirate (B) collected 3 to 10 h after collecting tracheal aspirate (A)  and collected 11 h or longer after collecting tracheal aspirate (A) . It is noteworthy that most anaerobes were not detected in tracheal aspirate (B) which was collected 11 h or longer after collection (A) , it is suitable for evaluating bacterial flora and pathogenic bacteria. Because highly diverse microbial communities including anaerobes and indigenous oral bacteria harbor in aspiration pneumonia, 16S rRNA gene sequencing analysis is frequently used to evaluate the causative bacteria –22. The before administration of antibiotics in intubated patients with aspiration pneumonia. It is worthy of note that in the tracheal aspirate from (A) to (B), the proportion of anaerobes decreased significantly, and that some facultative anaerobes such as Staphylococcus, Klebsiella, and Enterobacter tended to increase  was significantly lower than those of the tracheal aspirate (A) (mean 1.07 × 109 cells/mL) , it is not conclusive that the number of bacteria of these facultative anaerobes has actually increased; caution is necessary for this interpretation. Although we cannot deny the possibility that other factors related to the subjects may have had an effect on the outcome, we can assume that the anaerobes in the tracheal aspirate were lowered mainly due to the effect of mechanical ventilation. Notable fluctuations of microbiota in the tracheal aspirate were significantly dependent on the mechanical ventilation times—in particular when it was over 3 h. Although further investigation is necessary to explain this phenomenon clearly, several assumptions can be made. There is a possibility that the normal lung microbiome was disrupted by mechanical ventilation, or that those fluctuations may be caused by oxygen concentration increase via mechanical ventilation (because obligate anaerobes cannot survive in the presence of oxygen). This phenomenon supports previous studies regarding anaerobic organisms as major pathogens in non-intubated patients with aspiration pneumonia [In this study, a total of 116 samples of saliva and tracheal aspirate from 22 mechanically ventilated patients with aspiration pneumonia were collected. We analyzed the changes in the microbiota during mechanical ventilation, and during antibiotic treatments. To our knowledge, this is the first report suggesting that microbiota in the lower respiratory tract may change dynamically during mechanical ventilation neumonia .2: 30%); therefore, it may be deduced that a high concentration of oxygen is not always necessary in decreasing anaerobes in the lower respiratory tract.There were two exception cases (case 5 and 12). In case 12, the duration of mechanical ventilation treatment (only 1 h) may not have been long enough to significantly affect the microbial flora Fig. . The lonLactobacillus is a gram positive rod, it is possible that Lactobacillus was detected in the culture but it could not be identified in the clinical laboratory of the hospital. In case 17, Granulicatella was difficult to culture because of its pyridoxal dependence and slow growth characteristics [Enterobacter asbriae, E. cloacae, Corynebacterium propinquum, C. acoolens, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Granulicatera adiacens were predominantly detected in the tracheal aspirate (C)  in many of the cases Table . Howevereristics ; it may C) Table . These bFrom the present findings, it can be deduced that antibiotics should be selected on the premise that dynamic changes in microbiota (involved in the reduction of anaerobes in the lower respiratory tract) may occur during mechanical ventilation in intubated patients with aspiration pneumonia. It was suggested that antibiotic treatment for anaerobes in these cases may not be appropriate. Additional pathogenic investigation should be conducted after over 3 h from initiating mechanical ventilation. However, because this is a single location observational study, and because the sample size was small, evaluation by larger scale interventional analysis is required for finding the most appropriate antibiotic regimen. The use of wide spectrum antibiotics (unnecessarily) leads to the emergence of drug resistant bacteria—which is now an urgent health-related problem. Needless to say, accurate identification of pathogenic bacteria and the justified usage of antibiotics are both imperative.Streptococcus spp. in the oral and in the respiratory tract, which most NGS cannot classify at the species level [This study has several limitations. First, the sample size was small, which is one of our major limitations. Because of the small sample size, the full dynamics of the microbiota during mechanical ventilation in aspiration pneumonia could not be evaluated. It is difficult to say that sufficient information has been obtained for treatment strategy from this study. Second, the use of the Sanger sequencing method for analysis of the bacterial 16S rRNA gene is another limitation. NGS (next generation sequencer) is commonly used for analysis of human microbiota . An impoes level  because es level . In thisThe microbiota of the lower respiratory tract changes dynamically during mechanical ventilation before administration of antibiotics in intubated patients with aspiration pneumonia. The potential of microbiota alteration should be considered for the proper antibiotic treatment in these patients.Additional file 1: Appendix S1. Appendix describing additional detail of the method and figure legends of additional figure.Additional file 2: Appendix S2. In-house database containing 5878 16S rDNA sequences of type strains.Additional file 3: Figure S1–1, S1–2. Proportions of bacterial flora in saliva and tracheal aspirate .Additional file 4: Figure S2–1, S2–2. Unsupervised hierarchical clustering of genus for samples of saliva and tracheal aspirate.Additional file 5:Table S1. Predominant Phylotypes Detected by Clone Library Method in 22 Subjects.Additional file 6:Figure S3. Eigenvectors of PC 1 and 2 by bacterial genera.Additional file 7:Figure S4. Dynamics of microbiota in saliva and tracheal aspirate.


Cassia obtusifolia via inhibition against the protein tyrosine phosphatases 1B (PTP1B) and α-glucosidase. Among them, anthraquinones emodin and alaternin exhibited the highest inhibitory activities on PTP1B and α-glucosidase, respectively. Moreover, we examined the effects of alaternin and emodin on stimulation of glucose uptake by insulin-resistant human HepG2 cells. The results showed that alaternin and emodin significantly increased the insulin-provoked glucose uptake. In addition, our kinetic study revealed that alaternin competitively inhibited PTP1B, and showed mixed-type inhibition against α-glucosidase. In order to confirm enzyme inhibition, we predicted the 3D structure of PTP1B using Autodock 4.2 to simulate the binding of alaternin. The docking simulation results demonstrated that four residues of PTP1B  interact with three hydroxyl groups of alaternin and that the binding energy was negative , indicating that the four hydrogen bonds stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, resulting in more effective PTP1B inhibition. The results of the present study clearly demonstrate that C. obtusifolia and its constituents have potential anti-diabetic activity and can be used as a functional food for the treatment of diabetes and associated complications.The present work aims to evaluate the anti-diabetic potentials of 16 anthraquinones, two naphthopyrone glycosides, and one naphthalene glycoside from  Type 2 diabetes (DM2) is characterized by resistance of insulin-sensitive tissues, such as muscles, liver and fat, to insulin action. Although the mechanism of the insulin resistance is unknown, it is tightly associated with obesity . ProteinCassia is a large tropical genus composed of about 600 species of herbs, shrubs, and trees. Species of Cassia are a rich source of naturally-occurring bioactive compounds anthraquinones. These plants are reported to have anti-diabetic, laxative, purgative, antimalarial, ulcer healing, hepatoprotective, nephroprotective, and antitumor activities, and they are also used in the treatment of skin infection and periodic fever throughout tropical and subtropical region -1-naphthyl}-l-alanyl)amino]pentyl}oxy)-2-naphthoic acid (compound 23) (cyan color) are illustrated in 23 were stably positioned in the same pocket of the PTP1B by Autodock 4.2 (http://autodock.scripps.edu/downloads). As illustrated in 23. Moreover, the binding energies of both compounds were negative , indicating that additional hydrogen bonding would stabilize the open form of the enzyme and potentiate tighter binding to the active site of PTP1B, resulting in more effective PTP1B inhibition.The molecular docking models of alaternin (magenta color) and 3- amino]-2-deoxy-d-glucose (2-NBDG) uptake assay was performed with insulin-resistant HepG2 cells. The positive control metformin at a concentration of 10 μM significantly increased insulin-stimulated glucose uptake in insulin-resistant HepG2 cells. Concentrations of 12.5, 25, and 50 μM alaternin, and 3.125, 6.25, and 12.5 μM emodin, significantly enhanced the insulin-stimulated uptake of 2-NBDG in insulin-resistant HepG2 cells compared to the control  and ursolic acid (3.37 ± 0.18 μg/mL), respectively. Although some anthraquinones, including chrysoobtusin, 8-O-methyl chrysophanol, and physcion, together with inactive obtusifolin, aurantioobtusin and 1-O-methylemodin from the EtOAc fraction of the methanolic extract of C. obtusifolia were recently reported to be α-glucosidase inhibitors -2-deoxy-d-glucose (2-NBDG) was purchased from Life Technologies . Human insulin was purchased from Eli Lilly . All other chemicals and solvents used were purchased from Merck and Sigma-Aldrich, unless otherwise stated.Yeast α-glucosidase, acarbose, ursolic acid, Cassia obtusifolia were purchased from Omni Herb Co. , and authenticated by Prof. J.-H. Lee . A voucher specimen (no. 20130302) was deposited in the laboratory of Prof. J.S. Choi .The raw seeds of 2Cl2 fraction (394 g); alaternin (50 mg), aleo-emodin (60 mg), emodin (170 mg), 2-hydroxyemodin 1-methyl ether (68 mg), questin (40 mg), chryso-obtusin-2-O-β-d-glucoside (50 mg), and cassiaside (476 mg) from the EtOAc fraction (23 g); glucoaurantio-obtusin (100 mg), cassitoroside (30 mg), toralactone gentiobioside (300 mg), chrysophanol triglucoside (60 mg), and chrysophanol tetraglucoside (60 mg) from the n-BuOH fraction (220 g) according to the method described by Jung et al. [1H- and 13C-NMR, as well as by comparison with published spectral data. The structures of emodin and alaternin are shown in Chrysophanol (168 mg), physcion (250 mg), obtusifolin 893.5 mg), obtusin (430.3 mg), aurantio-obtusin (670.2 mg), chryso-obtusin (350.7 mg), and gluco-obtusifolin (230.2 mg) were isolated from the CH3.5 mg, og et al.  and wereAc − As)/Ac × 100, where Ac is the absorbance of the control, and As is the absorbance of the sample.Enzyme inhibition assays were carried out spectrophotometrically using a procedure reported by Li et al. . A 60 μLp-nitrophenyl phosphate as the substrate (pNPP) in a procedure reported by Cui et al. [p-nitrophenyl produced after enzymatic dephosphorylation from pNPP was estimated by measuring the absorbance at 405 nm using microplate spectrophotometer (Molecular Devices). The non-enzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. The percent inhibition (%) was obtained by the following equation: % inhibition = (Ac − As)/Ac × 100, where Ac is the absorbance of the control, and As is the absorbance of the sample. Ursolic acid was used as a positive control.The PTP1B inhibitory activities were evaluated using i et al. . Each weKi) were determined by interpretation of the Dixon plots, where the value of the x-axis implies −Ki.In order to determine the inhibition mechanism, enzymatic inhibition at various concentrations of alaternin was evaluated by monitoring the effects of different concentrations of the substrates via Dixon plots . Dixon plots of inhibition of PTP1B by alaternin were obtained in the presence of different concentrations of substrate: 1.0, 0.5, and 0.25 mM of pNPP. The test concentrations of alaternin in the PTP1B kinetic analysis were 0, 0.8, and 4.0 µM. Dixon plots of α-glucosidase inhibition by alaternin were performed in the presence of different concentrations of substrate: 2.5, 1.25, and 0.625 mM of pNPG. The test concentrations of alaternin used in the α-glucosidase kinetic analysis were 0.97, 1.95, and 3.90 µM. Both enzymatic procedures consisted of the same, aforementioned PTP1B and α-glucosidase assay methods. The inhibition constants  were added and incubated for 24 h, and then they were incubated with 100 nM insulin for 30 min. After this incubation, the 2-NBDG uptake in insulin-resistant HepG2 cells was measured. The cells were incubated with 40 μM 2-NBDG for 30 min. To stop the response, cells were washed with ice-cold PBS and the fluorescence intensity of 2-NBDG was measured on a Synergy HT microplate reader  at 485 nm excitation and 528 nm emission. Six replicate wells were established and each experiment was repeated three times.The HepG2 (human hepatocarcinoma) cell line was purchased from the American Type Culture Collection . Cells were maintained in MEM containing 2.0 mM d method , with slt-test  and noted at p < 0.05. All results are presented as means ± SEM of triplicate samples.Statistical significance was analyzed via one-way ANOVA and Student’s Cassia compounds were selected, and their inhibitory potential against α-glucosidase and PTP1B were evaluated in order to evaluate their anti-diabetic potential. All of them, including anthraquinones, naphtopyrone, and naphthalene glycosides, were shown to be potent α-glucosidase and PTP1B inhibitors. In particular, alaternin was shown to possess the most potent inhibitory activity. Hence, it was hypothesized that the presence of these constituents of C. obtusifolia was directly attributed to the potent α-glucosidase and PTP1B inhibitory activity. Moreover, in this study, we show for the first time that two active anthraquinones, alaternin, and emodin, improve insulin sensitivity by increasing insulin-stimulated glucose uptake in HepG2 cells. These findings suggest that alaternin and emodin could regulate the insulin sensitivity in vitro, modulating glucose transport. Therefore Cassia constituents should be further explored for the development of novel therapeutic or preventive agents for the treatment of diabetes.In conclusion, 19


The members of Rho family of GTPases, RhoA and Rac1 regulate endothelial cytoskeleton dynamics and hence barrier integrity. The spatial activities of these GTPases are regulated by post-translational prenylation. In the present study, we investigated the effect of prenylation inhibition on the endothelial cytoskeleton and barrier properties. The study was carried out in human umbilical vein endothelial cells (HUVEC) and protein prenylation is manipulated with various pharmacological inhibitors. Inhibition of either complete prenylation using statins or specifically geranylgeranylation but not farnesylation has a biphasic effect on HUVEC cytoskeleton and permeability. Short-term treatment inhibits the spatial activity of RhoA/Rho kinase (Rock) to actin cytoskeleton resulting in adherens junctions (AJ) stabilization and ameliorates thrombin-induced barrier disruption whereas long-term inhibition results in collapse of endothelial cytoskeleton leading to increased basal permeability. These effects are reversed by supplementing the cells with geranylgeranyl but not farnesyl pyrophosphate. Moreover, long-term inhibition of protein prenylation results in basal hyper activation of RhoA/Rock signaling that is antagonized by a specific Rock inhibitor or an activation of cAMP signaling. In conclusion, inhibition of geranylgeranylation in endothelial cells (ECs) exerts biphasic effect on endothelial barrier properties. Short-term inhibition stabilizes AJs and hence barrier function whereas long-term treatment results in disruption of barrier properties. The integrity of the vascular endothelial (VE) barrier is maintained by the equilibrium of competing contractile and adhesive forces generated by the acto-myosin-based endothelial cell (EC) contractile machinery and adhesive molecules located at cell–cell and cell–matrix contacts, respectively . ECs areThe Rho family of GTPases, particularly RhoA and Rac1, are the major regulators of EC contraction, actin cytoskeleton dynamics, and AJs and hence play a pivotal role in the maintenance of EC barrier integrity. Overexpression of a constitutive active RhoA resulted in the loss of basal VE-cadherin and potentiated the effect of hypoxia-reoxygenation (H/R) whereas overexpression of dominant negative RhoA attenuated the agonist-induced endothelial hyperpermeability ,5. On thIn the present study we investigated the effects of short- and long-term inhibition of protein prenylation on endothelial AJs and barrier function using an in vitro cell culture model of human umbilical cord endothelial cells (HUVECs). We report here a biphasic effect of short- and long-term inhibition of protein prenylation on HUVEC permeability. Short-term treatment has EC barrier protective effects whereas long-term inhibition has barrier disrupting effects.In the first instance the effect of long-term treatment of statins on EC barrier properties was investigated. We used two chemically different statins and both caused a two-fold increase in permeability already at low concentrations (0.1–0.2 µM) and four-fold increase at medium concentrations (1–2 μM) A. In-conEC cytoskeleton dynamics, particularly the peripheral actin, plays a vital role in the maintenance of endothelial barrier function. Statins caused disruption of EC actin cytoskeleton in a time-dependent manner. Initially, statin treatment resulted in the loss of actin stress fibers running across the cell after 1 h of treatment, however, the peripheral actin was preserved at this stage. Later after 3 h, the peripheral actin started also disappearing and after 12 h only some random actin fibers at the cell periphery were visible A. The chStatins mainly target the HMG-CoA reductase and thus block the cholesterol and related byproducts biosynthesis. These byproducts are required for the prenylation (lipidation) of several cellular proteins particularly the members of small GTPase family such as RhoA and Rac1 and thus their spatial localization at the cellular membranes for proper functioning A. The smIn order to further investigate the effect of inhibition of prenylation of Rho GTPases, a pharmacological inhibitor of GGT was used. GGT inhibitor had a time-dependent biphasic effect on basal endothelial permeability. Within the first 3 h it reduced the permeability A and cauPreviously, the nonprenylated forms of GTPases were considered to be inactive, therefore, we investigated whether Rac1 or RhoA in the presence of prenylation inhibitors could still be activated. As demonstrated by pulldown assay A, under Since we observed hyperactivation of RhoA/Rock signaling in statin-treated ECs, it was investigated whether pharmacological inhibition of Rock and/or an activation of Rac1 using cAMP signaling as a maneuver could antagonize statin-induced hyperpermeability. As demonstrated in EC barrier integrity is regulated by the actin-myosin based contractile machinery and actin cytoskeleton-anchored AJs that consist of VE-cadherin and catenins directly connected to the actin cytoskeleton . TherefoAvailability of the prenyl moieties for the incorporation into GTPases is one of the rate limiting factors for protein prenylation and inhibitors of HMG-CoA reductase  are well-known and are largely employed drugs to inhibit protein prenylation to investigate the role of protein prenylation in cell biology ,18. BoisIn the present study, we do observe protective effects of statins on short-term treatment, however, prolonged treatment has a detrimental effect on endothelial barrier function due to effects on Rho GTPases and actin-cytoskeleton  as discuWhether the nonprenylated GTPases can be activated  is controversial. However, some previous studies have shown accumulation of more GTP bound RhoA and Rac1 on treatment with various prenylation inhibitors including statins ,30, thouActin stress fibers are regulated by the balanced activities of actin nucleation and severing proteins . Rock inIn conclusion, inhibition of geranylgeranylation in ECs exerts a biphasic effect on endothelial barrier properties. Short-term treatment preferentially affects RhoA prenylation preserving partial Rac1 spatial activity and thus stabilizes endothelial junctions and hence barrier function. However, long-term treatment results in inhibition of both RhoA and Rac1 spatial activities resulting in collapse of actin cytoskeleton and disruption of barrier properties. Other members of the Rho family are also geranylgeranylated, and since we did not investigate these members, we cannot exclude the role of these GTPases in the actin–cytoskeleton dynamic and endothelial barrier regulation.® protease inhibitor cocktail was from Roche ; ThinCert® polycarbonate membrane filters (6-well) were from Greiner Bio-One ; Benzonase®, simvastatin, and fluvastatin were from Merck-Millipore ; EC basal medium plus supplement pack was from PromoCell ; HRP-conjugated anti-mouse IgG and -rabbit IgG antibodies were from Santa Cruz biotechnology ; Farnesyl pyrophosphate, Geranylgeranyl pyrophosphate, human thrombin, and phalloidin-TRITC were from Sigma ; Pierce® ECL solution, and Rac1-pulldown assay kit were from Thermo Scientific ; farnesyl transferase inhibitor LB42708, geranylgeranyl transferase inhibitor GGTI-298 and Y27632 were from Tocris Bioscience . All other chemicals were of the best available quality, usually analytical grade.Anti VE-cadherin antibody was from Beckman Coulter ; anti phospho MLC (S18/T19), anti phospho MYPT1 (T850), anti RhoA, anti Rac1, and anti GAPDH antibodies were from Cell Signaling Technologies ; CompleteCardiovascular Research 1997; 35: 2–3). HUVEC were isolated from umbilical cords obtained from the gynecology department of the University hospital Giessen after approval from the ethics committee of the Medical Faculty of Justus Liebig Uiversity, Giessen (AZ 18/13 dated 28.01.2013) and informed consents from the patients. The cells were cultured as described previously [The study conforms to the principles outlined in the ‘’Declaration of Helsinki’’ . Agents were added as indicated. Stock solutions of fluvastatin and thrombin were prepared in water, simvastatin, GGTI-298, LB42708, in DMSO and farnesyl pyrophosphate and geranylgeranyl pyrophosphate in methanol. Appropriate volumes of these solutions were added to the cells yielding final solvent concentrations ≤ 0.1% (vol/vol). Where combination of drugs was used, inhibitors were added 30–60 min before adding the statins. The same final concentrations of water, methanol, and DMSO were included in all respective control experiments.The basal medium used in incubations was modified Tyrode’s solution , simvastatin (1 μM), fluvastatin (2 μM), LB42708 (5 μM), GGTI-298 (10 μM), farnesyl pyrophosphate (2 μM), and geranylgeranyl pyrophosphate (2 μM).Immunocytochemistry and confocal microscopy were performed as described previously . BrieflyThe permeability of trypan-blue-labelled albumin across HUVEC monolayers was analyzed as previously described .The impedance-based endothelial barrier properties were analyzed using ECIS system  according to the protocols/instructions provided by the manufacturer of the instrument.Western blotting was performed as described previously . Blots wg to remove nuclei and the supernatants were used as cell lysates, mixed with 4× Laemmli buffer (1:4) and separated by SDS-PAGE in 12.5% polyacrylamide gels.Prenylation processing of GTPases was assessed with modifications from the protocol originally described by Garcia et al. . The celRac1 pulldown assay was performed using a commercial kit  as described recently . The pulp values of ≤ 0.05 were considered statistically significant.The data are presented as means (±S.E.M) of 3–5 experiments from independent cell preparations. The comparison between multiple groups was performed by one-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls post-hoc test using Graphpad Prism 6 software . The


The vasopressin V2 receptor antagonist, using DILIsym®. Simulations were conducted that assessed the potential for lixivaptan and its three main metabolites to cause hepatotoxicity due to three injury mechanisms: bile acid accumulation, mitochondrial dysfunction, and oxidative stress generation. Results of these simulations were compared to previously published DILIsym results for tolvaptan.In prior studies, the potential for hepatotoxicity of tolvaptan was correctly predicted using DILIsym®, a quantitative systems toxicology (QST) mathematical model of drug-induced liver injury. In the current study, we evaluated lixivaptan, another proposed ADPKD treatment and vasopressin VNo ALT elevations were predicted to occur at the proposed clinical dose for lixivaptan, in contrast to previously published simulation results for tolvaptan. As such, lixivaptan was predicted to have a markedly lower risk of hepatotoxicity compared to tolvaptan with respect to the hepatotoxicity mechanisms represented in DILIsym.These results demonstrate the potential for using QST methods to differentiate drugs in the same class for their potential to cause hepatotoxicity.The online version of this article (10.1007/s11095-019-2726-0) contains supplementary material, which is available to authorized users. DILIsymtacapone , i.e. thtacapone , determitacapone .In this paper, a DILIsym representation was constructed for lixivaptan and its three major metabolites, WAY-138451, WAY-141624, and WAY-138758, and was used to predict the potential risk of hepatotoxicity and likely margin of safety for lixivaptan at doses intended for the treatment of ADPKD. The simulation results for lixivaptan were compared to the previously published results for tolvaptan with a particular focus on the predictions made about the relative safety of the two drugs based on the mechanisms of toxicity included in DILIsym.DILIsym version 6A was used for all simulations described in this paper. The Lixivaptan-Specific SimPops and the Lixivaptan-Sensitive SimCohorts have been provided as supplemental materials; they will allow users with DILIsym v6A to reproduce the results in this paper.Lixivaptan and its three main metabolites were represented with the Physiologically-Based Pharmacokinetic (PBPK) sub-model within DILIsym with the goal of reasonably approximating liver exposure upon oral administration. The DILIsym PBPK sub-model framework used for lixivaptan consists of a compartmental model of the body with compartments for blood, gut, liver, muscle, and other tissues and has been described in depth elsewhere ,7,10. ThA customized SimPops including clinically relevant variability in lixivaptan exposure was created for this project. Parameter values related strictly to pharmacokinetics that were expected to vary among individuals were determined and ranges were assigned to these parameters. Details regarding the construction of the lixivaptan-specific SimPops, including the parameters varied and the distributions used for these parameters, are given in Supplement 16 individuals out of the 285-individual custom SimPops were selected as part of a Lixivaptan-Sensitive SimCohorts™. These simulated individuals were identified by escalating the lixivaptan dose to a supratherapeutic level in order to observe signs of simulated toxicity, and then selecting the 8 individuals with the highest ALT values. These individuals were added to the four individuals with the highest liver bile acid concentrations and the four individuals with the lowest electron transport chain activity in the simulations. These 16 simulated individuals were used for the dose escalation simulations.In addition, bile acid transporter sensitivity analysis simulations were performed on the v4A_1_Multi16 SimCohorts. This SimCohorts is included in DILIsym version 6A and is comprised of 13 individuals who are the most susceptible to the various hepatotoxicity mechanisms in DILIsym, combined with 2 less-susceptible individuals and the baseline individual. As this SimCohorts contains the individuals most likely to respond to most drugs, it is generally used as a screening tool for sensitivity analyses; if no response is observed in this SimCohorts, it is unlikely that a response will appear in the full SimPops. In the case of this work, exposure variability in lixivaptan was not added to the v4A_1_Multi16 SimCohorts.max) were each decreased by approximately 30% in renal failure patients compared to subjects with normal renal function . In light of this, it was determined that the customized Lixivaptan-specific SimPops created based on normal healthy volunteer studies adequately captured the expected exposure variability in patients with renal impairment, and therefore would suffice for the prediction of toxicity in both normal healthy volunteers and ADPKD populations.In the tolvaptan study, two custom SimPops were constructed, one that included clinically relevant variability in tolvaptan exposure (similar to the Lixivaptan-specific SimPops) and one for individuals with renal impairment due to the wider range of exposure variability observed in those individuals . Howeverin vitro systems to describe the relationships between lixivaptan and its metabolites with various mechanisms of hepatotoxicity. For each of these molecules, bile acid transporter inhibition, mitochondrial toxicity, and oxidative stress generation were assessed. The results from these assays were translated into toxicity parameters that were used as inputs into DILIsym. The details of the in vitro assays and their results are described in detail in Supplement Lixivaptan simulations required collecting data from 100 mg BID for 60 days  ,12;200/100 mg split daily dosing for 12 weeks ;400 mg BID for 7 days The primary lixivaptan clinical protocols simulated throughout this analysis were as follows:The primary criterion for comparing lixivaptan simulation results to clinical results and to previously reported simulation results for tolvaptan  was the 50 values were measured for the inhibition of bile acid transporters by lixivaptan and its metabolites; as such, mode of inhibition was not determined experimentally. Mixed inhibition with α = 5 was used as an estimate of the mode of bile acid transporter inhibition. In order to investigate the importance of mode of bile acid transporter inhibition to DILIsym predictions of lixivaptan, the proposed clinical dosing regimen was also simulated in the full 285-individual lixivaptan-specific SimPops with efflux bile acid transporter inhibition treated as noncompetitive and uptake bile acid transporter inhibition treated as competitive. These conditions represent the worst-case scenario; that is, the modes of inhibition most likely to cause bile acid accumulation and thus toxicity. Other simulations were conducted in the v4A_1_Multi16 SimCohorts where efflux transporter mode of inhibition was varied between competitive, noncompetitive, and mixed inhibition with α = 5. Further simulations were also conducted without lixivaptan-mediated ROS generation in order to determine the contribution of this mechanism to predicted ALT elevations from lixivaptan treatment.The mode of bile acid transporter inhibition has been shown to be important for the prediction of bile acid-mediated DILI; noncompetitive inhibition is more potent than competitive inhibition, with mixed inhibition lying on a spectrum between these poles ,14. For In order to understand the potential margin of safety for lixivaptan treatment, dose escalation simulations were conducted using the 16-individual Lixivaptan-Sensitive SimCohorts only. This approach was adopted to magnify a potential safety finding. These simulations were performed in the presence and absence of lixivaptan-generated and WAY-138451-generated ROS.max for lixivaptan and its metabolites after 7 days of 100 mg BID dosing is shown in Table As part of the development program for hyponatremia, lixivaptan was dosed in about 1700 subjects across 36 clinical studies  of simulated individuals had an ALT >3X ULN, whereas no individuals out of 67 showed elevated ALT in the CK-LX1403 clinical trial. The simulation result is not out of the realm of possibility, but suggests that the DILIsym simulation for lixivaptan yields results that are likely conservative compared to the clinical experience. Of note, the ALT elevations seen at the 400 mg BID dose in the DILIsym simulation were mild and were not associated with signs of severe hepatocellular toxicity.Results from simulations intended to explore the mechanisms of simulated ALT elevations with lixivaptan are reported in Table Results from simulated dose escalation studies performed on the Lixivaptan-Sensitive SimCohorts, which includes a broad range of exposure-related and susceptibility-related variability, are listed in Table 0–7 day greater than 300 μg·h/mL; the results demonstrate a clear relationship between simulated exposure and simulated response.Of particular interest in the Lixivaptan-Specific SimPops simulation results for the supratherapeutic 400 mg BID dose is the relationship between exposure and ALT elevation, as shown in Fig. The DILIsym simulations presented here suggest that the second-generation vasopressin V2 receptor antagonist lixivaptan is not likely to cause liver injury due to the mechanisms included in DILIsym v6A. DILIsym successfully recapitulated the observed clinical data regarding the safety of lixivaptan given at 100 mg BID for 60 days. Furthermore, simulation results for lixivaptan predicted no ALT elevations at the proposed clinical dose of 200/100 mg BID for ADPKD. This result compares favorably to the results previously published for tolvaptan at the FDA-approved dose of 90/30 mg split daily dosing for ADPKD ; however, it also suggests that DILIsym’s predictions for lixivaptan tend to overstate the clinical toxicity and therefore are to be considered conservative. This should provide added confidence in the results suggesting that lixivaptan is not likely to cause liver injury.Interestingly, mechanistic analyses suggested that lixivaptan’s primary toxicity mechanism of relevance (at simulated supratherapeutic doses) is likely to be ROS generation, whereas simulated tolvaptan toxicity was predicted to be a product of both bile acid accumulation and mitochondrial ETC inhibition. This further suggests substantial qualitative differences between lixivaptan and tolvaptan in terms of their likely liver effects. Such differences could potentially play a meaningful role in the differential clinical safety profile of the two drugs. First, drugs that have the potential to cause hepatocellular dysfunction through two or more concurrent mechanisms, like tolvaptan, have been associated with particularly pronounced liver injury . Second,The full SimPops simulation of lixivaptan was performed for 12 weeks in 285 simulated individuals, while the published simulations for tolvaptan were for 24 weeks in 229 simulated individuals . Based oAnother interesting point of comparison between lixivaptan and tolvaptan is the role of exposure in the simulated toxicity. For tolvaptan, exposure was not observed to be related to toxicity ; this waIt has been proposed that an adaptive immune-mediated attack plays a role in tolvaptan-mediated toxicity . DILIsymIt is also important to understand the potential effects of uncertainty in the model on the predictions generated here. In Table In sum, DILIsym simulation results suggest that lixivaptan is less likely to cause hepatotoxicity than tolvaptan at their respective therapeutic doses for the treatment of ADPKD. These results bode well for the continued development of lixivaptan as a safe therapy for the treatment of this disease. Finally, this work illustrates the applicability of QST modeling methods in comparing the toxicity potential of compounds within the same class. The difference in likely toxicity mechanisms between tolvaptan and lixivaptan demonstrates the fact that compounds within the same class can vary widely in terms of potential liability and potential mechanisms of toxicity.ESM 1(DOCX 639 kb)ESM 2(DOCX 1221 kb)ESM 3(MAT 8 kb)ESM 4(MAT 35 kb)ESM 5(MAT 77 kb)


Schistosoma japonicum protein p40 (rSjp40) can inhibit the activation of hepatic stellate cells, and based on this evidence we attempted to investigate whether or not BMP-7 is involved in rSjp40’s inhibition.Activation of hepatic stellate cells is the dominant pathogenic event during the process of liver fibrosis. Bone morphogenic protein (BMP)-7 has recently been identified as an anti-fibrotic factor and leads to phosphorylation of Smad1/5/8 in activated hepatic stellate cells. Its expression can be upregulated by the transcriptional activator, Y-Box protein-1 (YB1). Previous studies have found that the recombinant A human hepatic stellate cell line, the LX-2 cell line, was cultured and treated with rSjp40. The role of BMP-7 was analyzed by Western blot.Our findings testified that knockdown of BMP-7 impaired rSjp40-induced downregulation of α-SMA and phosphorylation of Smad1/5/8 in LX-2 cells. Furthermore, rSjp40 upregulated expression of BMP-7 at both mRNA and protein levels depending on YB1. Interestingly, YB1 was translocated from the cytoplasm to the nucleus upon treatment of rSjp40.These results suggest that rSjp40 inhibits the activation of hepatic stellate cells by promoting nuclear translocation of YB1 and inducing BMP-7/Smad1/5/8 pathway, which provide a new clue to guide ongoing research into the anti-fibrosis of rSjp40. Liver fibrosis is a pathological process due to most chronic liver injuries like viral hepatitis, alcohol abuse, metabolic disease, and parasite infection. Both quantitative and qualitative changes in the composition of liver extracellular matrix (ECM) occur during the development of liver fibrosis. The total ECM increases 3–5-fold, accompanied by the shift in the type of ECM from the normal low density basement membrane-like matrix to interstitial type matrix containing types I and III collagens . Accordivia binding to Smad4, thus, being translocated to the nucleus to suppress the accumulation of Smad3 by TGF-β1 stimulation  with 1% PMSF  and phosphatase inhibitor complex III (1 mM) . Samples were separated by 10% SDS-PAGE and then transferred from the gels to polyvinylidene difluoride (PVDF) membranes. After blocked in 5% nonfat milk, the membranes were incubated with the indicated primary and secondary antibodies. Protein bands were visualized with ECL system .GAPDH, forward (5′-GAC AAG CTT CCC GTT CTC AG-3′) and reverse (5′-GAG TCA ACG GAT TTG GTC GT-3′); BMP-7, forward (5′-GGC TGG CAG GAC TGG ATC AT-3′) and reverse (5′-ACC AGC GTC TGC ACG ATG GC-3′).Total RNA extraction, reverse transcription and quantitative real time PCR analysis were performed as previously described . Gene-spLX-2 cells left untreated or treated with rSjp40 (5 μg/ml) for 48 h were harvested and separated using the Nuclear and Cytoplasmic Protein Extraction kit  according to the manufacturer’s instructions.LX-2 cells were seeded on 24-well plates and left untreated or treated with rSjp40 (5 μg/ml) on the following day. Forty-eight hours after treatment, cells were fixed by 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. Then cells were incubated with the primary Ab against YB1 at 4 °C overnight, followed by AlexaFluor 594 secondary Ab  for 2 h at room temperature. The cells were also stained with Hoechst 33342  for 15 min and observed by fluorescent microscopy.P-value < 0.05 was considered significant.All experiments were analyzed by the Student’s t-test. A t(4) = 3.081, P = 0.0185). As shown in Fig. t(4) = 2.409, P = 0.0368), which was consistent with our previous results [t(4) = 2.353, P = 0.0391). These results suggest that BMP-7 plays an important role in rSjp40’s inhibition on HSC activation.To investigate whether BMP-7 is involved in rSjp40’s inhibition on HSC activation, we used BMP-7 specific siRNA to knockdown the expression of BMP-7 in LX-2 cells (t-test:  results –22. Howet(4) = 3.136, P = 0.0175). However, knockdown of BMP-7 markedly inhibited rSjp40-triggered phosphorylation of Smad1/5/8 in LX-2 cells (t-test: t(4) = 6.767, P = 0.0012)  = 2.194, P = 0.0466) (t-test: t(4) = 8.671, P = 0.0005)  = 17.61, P < 0.0001)  Fig. a. These t(4) = 2.513, P = 0.0329) (t-test: t(4) = 6.150, P = 0.0018)  = 3.648, P = 0.0109)  = 4.553, P = 0.0052), whereas cytoplasmic level of YB1 was decreased (t-test: t(4) = 6.503, P = 0.0014), with total level of YB1 unchanged  = 3.276, P = 0.0153) (Fig. via an unknown mechanism.In this study, we found that rSjp40 increased the phosphorylation level of Smad1/5/8 and decreased the expression of α-SMA in LX-2 cells. Interestingly, knockdown of BMP-7 inhibited rSjP40-triggered phosphorylation of Smad1/5/8 and downregulation of α-SMA. Based on the evidence that the anti-fibrotic actions of BMP-7 are due to its ability to counteract TGF-β1 signaling through increasing the phosphorylation level of Smad1/5/8 , 25, 26,YB1 can be shuttled between the cytoplasm and the nucleus. Many compounds such as oxymatrine and Hsc025 can induce the nuclear translocation of YB1 , 28. In 4 in mice [via acting on different targets [According to previous reports, YB1 is also a negative regulator in liver fibrosis. One direct evidence is that overexpression of YB1 improved liver fibrosis induced by CCl in mice . Adding  targets , 32, whi targets –35. In oIn conclusion, these present findings demonstrated that BMP-7 is required for rSjp40’s inhibition on HSC activation. Upon rSjp40 treatment, YB1 was translocated to the nucleus to promote BMP-7 expression, leading to phosphorylation of Smad1/5/8. Our studies provide a new clue to guide ongoing research into the anti-fibrosis of rSjp40.


The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling . We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint. Initiation of cell-activating signaling typically occurs within clusters of ITAM-coupled receptors such as FcγR  or the hegulated .Multiple mechanisms work together to tune the responsiveness of macrophages and other myeloid cells, including negative regulation by the phosphatases CD45 and CD148 , cytoskeUnlike Dectin-1 and FcγR signaling pathways in macrophages, which are typically triggered in the context of pathogen-induced µm-scale clusters of receptors , mast-ceAS)  . Csk is tivation . 3-IB-PPtivation  and the tivation , but it tivation . FinallyAS macrophages treated with the CskAS inhibitor (SFK activator) 3-IB-PP1  and CskAS-expressing, Cbl-b-deficient (CskASCbl-bKO) bone-marrow-derived macrophages (BMDMs). (For clarity we refer to BMDM subtypes using protein nomenclature.) Immunoblots of whole-cell lysates show the loss of expression of c-Cbl and Cbl-b in CskASc-CblKO and CskASCbl-bKO BMDMs, respectively, compared to CskAS BMDMs RNAs to knock down the expression of c-Cbl and Cbl-b. CskAS BMDMs were transfected with non-targeting control RNA (ctrl) or with siRNA constructs targeting Cbl or Cblb mRNA. Rested transfectants were then treated with 3-IB-PP1 to induce SFK activation. Immunoblots of siRNA-transfected BMDM lysates   and Fgr , which aemCskAS) , which semCskAS)  and Fyn emCskAS) . This hiAS or CskASLynKO BMDMs , c-Cbl, or Cbl-b plasmid DNA  or 8% (LynAY32F), levels indistinguishable from LynB (10%) (K40R) nor the predicted threonine phosphorylation site mutation (LynAT27A) significantly affected the rate of LynA degradation  . SubstitnB (10%) . Neitherradation .Y32A 3-fold more highly expressed than wild-type LynA, LynAK40R, or LynAT27A at steady state, resulting in 3- to 4-fold more protein remaining after 20 min treatment with 3-IB-PP1  we applied 3-IB-PP1 as a 15 s pulse treatment. At this early time point, all SFKs are expected to be activation-loop phosphorylated, with only 20% of LynA polyubiquitinated, as we have observed in wild-type BMDMs  its ability to induce the phosphorylation of LynA Y32 and (ii) its ability to phosphorylate and activate c-Cbl. To determine whether this effect could be mediated in trans by LynA and the other macrophage-expressed SFKs, we performed experiments in which LynAT410K (not degraded on its own during 3-IB-PP1 treatment) was coexpressed with kinase-active SFKs in Lck-deficient JCaM1.6 cells. LynAT410K degradation would indicate that other SFKs could induce both LynA Y32 phosphorylation in trans and c-Cbl activation  or wild-type His6V5-tagged LynA (as a positive control). Wild-type LynA and LynAY32A were cotransfected as untagged constructs, so they could be resolved independently of His6V5-tagged LynAT410K on LynA immunoblots (T410K activation loop (pLynAact), phosphorylation of Zap70  nor the myeloid-cell SFK targets (Syk and FAK/Pyk2) participate in LynA degradation, we turned our focus to the major SFKs in macrophages. We showed above that wild-type LynA is degraded in 3-IB-PP1-treated Jurkat cells cotransfected with c-Cbl and memCsktivation . The comunoblots . Cotransunoblots . CotransY32A, and LynB were all capable of inducing the degradation of LynAT410K in trans. Where depletion of LynAT410K transfected alone was undetectable after 5 min of 3-IB-PP1 treatment, cotransfected wild-type LynA, LynAY32A, and LynB induced depletion of LynAT410K by 20%, 37%, and 40%, respectively; wild-type LynA alone was depleted by 40% at the same time point  plasmid induced depletion of LynAT410K by 17% after a 5 min treatment with 3-IB-PP1     .AS mast cells and BMDMs and found that mast cells express very little c-Cbl (20% of the level in rested macrophages) and relatively high levels of Cbl-b (80% of the level in rested macrophages) (e.g. as a scaffold in PI3K activation), long-lived LynA potentiates a strong response to 3-IB-PP1. Thus mast cells bypass the LynA signaling checkpoint by maintaining low levels of c-Cbl and therefore high levels of activated LynA.We then used immunoblotting to compare the expression of c-Cbl, Cbl-b, and LynA in Cskophages) . Strikinophages) . Mast ceophages) , despiteophages) . These cophages) . We therAS bone marrow with a small activating (sa)RNA duplex . Exploiting the similarity between LynA and LynB to make a limited set of point mutations, we have discovered that, while activation-loop phosphorylation and SH3-domain interactions mediate slower-phase targeting by c-Cbl and other E3 ligases, rapid degradation of LynA is triggered by an interaction peculiar to its unique-domain insert region.c-Cbl is targeted to LynA via a noncanonical recognition site present in LynA but not the other SFKs. Although LynA and LynB have identical activation kinetics, lipidation sites, and activation-loop and SH3-domain sequences, LynA is degraded >10 fold more quickly than LynB in macrophages treated with the CskWe have discovered that phosphorylation of Y32 in the unique-region insert of LynA is required for its rapid degradation. This phosphorylation occurs within seconds of LynA activation in macrophages, and flags LynA for rapid, c-Cbl-mediated polyubiquitination. Phosphorylation of LynA Y32 had been observed in neuroblastoma cell lines upon receptor-tyrosine-kinase activation . In epid59, Fgr, and Lck) were unable to induce degradation of kinase dead LynAT410K despite an increase in their own activation-loop phosphorylation and their ability to phosphorylate the activation loop of LynAT410K. This suggests, perhaps surprisingly  cells more closely resembles that of wild-type LynA than LynB, some LynA interactions are lost  and have a relatively high basal threshold for inflammatory activation . Mast cells express almost no c-Cbl and maintain high levels of LynA at steady state and over time, consistent with permissive signaling in the absence of a large-scale ITAM receptor clustering event nucleated by multivalent receptor ligation. Appropriate regulation of immune receptor thresholds is critical for maintaining the function of innate immune cells; threshold dysregulation can lead to chronic feedback loops that drive inflammatory signaling in autoimmune disease and conversely tumor-supporting immunosuppressive signaling . ElucidaASCsk mice are hemizygous for the ASCsk BAC transgene on a -/-Csk background, as described previously (ASCblb-/-(Csk-/-)Csk mice were generated by crossing -/-Cblb female mice from M. Farrar (University of Minnesota) Csk mice, but we obtained three individuals by crossing -/-Cbl female mice from E. Peterson (University of Minnesota) with +/- and CskASCsk male mice from our colony and then crossing +/-Cbl+/-Cskmice with ASCbl+/-Csk mice. All mice were housed in specific pathogen-free conditions and genotyped using real-time PCR . All animal use complies with University of Minnesota (UMN) and National Institutes of Health (NIH) policy . UMN is accredited by AAALAC, experiments involving mice were approved by the UMN Institutional Animal Care and Use Committee . Animals are kept under supervision of a licensed doctor of veterinary medicine and supporting veterinary staff under strict NIH guidelines.C57BL/6-derived eviously . CskASCbnnesota)  with CskThe Jurkat T-cell strains Clone E6-1  and JCaMAS, c-Cbl, and LynB (to ensure a response to 3-IB-PP1) along with constructs encoding the unique regions of LynA, LynB and LynAY32A fused to eGFP. A construct containing eGFP alone was used as a control. 24 hr after transfection, cells were filtered to remove clumps of dead cells and resuspended in PBS containing Hoechst Stain (Thermo Fisher) and incubated for 30 min at 37°C in a 96-well plate. Cells were then stimulated with 3-IB-PP1 for 5 min before quenching with ice-cold PBS and collected by centrifuging for 5 min x 1500 rpm at 4°C. Cells were resuspended in ice-cold PBS and coverslipped on glass slides directly before imaging. Imaging was performed on a Leica DM600B epifluorescence microscope using DAPI and GFP channels. Exposure times for GFP were set to normalize the brightness of the GFP across the different transfection conditions, as cell-to-cell expression was variable. Images were pseudocolored and merged using ImageJ software (NIH) with the Fiji plugin  , and human FynB were gifts from C. Lowell  Site-directed mutagenesis  was used to prepare the point mutants referenced in the main text and to introduce stop codons removing epitope tags from Fgr, LynA, and LynAY32A. Sequences of all constructs were verified by Sanger sequencing .Plasmids containing c-terminally His Health) . Myc-tagBMDMs were prepared using standard methods . BrieflyCbl was adapted from a human siRNA construct . Cblb siRNA was designed using the Integrated DNA Technologies double-stranded siRNA design tools . Control double-stranded RNA from IDT was not predicted to be complementary to any sequence in the human or mouse transcriptome. The effective siRNA sequences from among those we tested are: Cbl guide sequence CCUACCAGGACAUUCAGAAAGCUUU (passenger sequence AAAGCUUUCUGAAUGUCCUGGUAGG), Cblb guide sequence (CUGACUUCUUGGUAUCUGAUAUATA (passenger sequence UAUAUAUCAGAUACCAAGAAGUCAGGU). Aliquots of 2 M BMDMs in 100 μl opti-MEM  were transfected with 1 μM siRNA via electroporation at 400 V for 10 ms using a BTX square-wave electroporator. Cells were then plated on 150 mm untreated cell culture dishes and rested in 10 ml DMEM10 for 30 min before adding 10 ml DMEM10 supplemented with sodium pyruvate and 10% CMG-14–12-cell-conditioned medium as described above. Transfections were pooled from several cuvettes to obtain enough cells for several stimulation conditions. After 24 h cells were resuspended in enzyme-free EDTA buffer and replated in untreated 6-well plates  at 1 M cells per well in unconditioned medium. Cells were used 48 hr after transfection.The siRNA sequence for knockdown of mouse Cbl was designed using an algorithm published previously (Cbl-targeting saRNA ‘CBL-Tr-NM_005188-Pr-30-Cp-0’ with guide sequence UCAAUUCUAGAUAAAGGCG (passenger sequence CGCCUUUAUCUAGAAUUGA) was ordered from IDT with 2’ O-methylated uracil tails (mUmU) to increase transfection efficiency. On the first day of transfection, aliquots of 105 mast cells were resuspended in 400 µl mast-cell medium in a 24-well plate and mixed with 100 µl saRNA + lipofectamine 2000 or 3000 for a final concentration of 5 nM saRNA. Cells were then rested overnight before a second transfection with 100 µl saRNA + lipofectamine. On day three the cells were transferred into fresh mast-cell medium and rested overnight. On the day of the experiment, mast-cell aliquots from the same saRNA condition were pooled, resuspended in 50 µl DMEM, and rested 2 hr before stimulation.An saRNA sequence designed to upregulate transcription of eviously . Cbl-tarBMDM stimulations have been described previously . After rImmunoblots were analyzed by densitometry using ImageStudio software (LI-COR). Images were background-subtracted, and bands of the appropriate molecular weight were demarcated and analyzed for each gel lane. Each value was corrected for the total lane protein content (REVERT TPS), with the exception of pErk1/2, which was corrected with reference to total Erk1/2. Data were further normalized to a control condition to show relative changes from a reference state: each-condition t = 0 or reference-condition t = 0 as indicated. For all figures, n values are biological replicates, reflecting independent experiments from different individual mice , different days and/or batches of cells, stimulus preparations, and experimental workflows. Statistical analysis was performed using Prism Software . Significance was assessed using one- or two-way ANOVA analysis with Tukey’s or Sidak’s correction for multiple comparisons, with analysis of the mean at each time point or condition. Unless specified otherwise, error bars represent standard error of the mean from at least three independent experiments. Asterisks reflect specified P-values. Figures were prepared using Graphpad Prism and Adobe Creative Cloud software . Where appropriate, images were optimized by applying brightness/contrast changes to the whole image. No gamma or other nonlinear correction was applied. Images were rotated for figure preparation only after densitometry analysis.ASCbl+/- Cskmice were rested or treated 15 s with 3-IB-PP1, washed with ice-cold PBS, and lysed in 1% Lauryl Maltoside Buffer containing 150 mM NaCl, 0.01% sodium azide, and protease and phosphatase inhibitors . After scraping the plates, cells and detergent were sonicated on a Diagenode Biorupter for 5 min with a 50% duty cycle. The lysate was then cleared by centrifugation for 15 min at 14,000 rpm at 4°C in a tabletop centrifuge. Lysates were precleared for 30 min at 4°C with Protein G Sepharose beads (Sigma-Aldrich) and normal rabbit serum . LynA-specific antibody , and subjected to in-gel digestion with TPCK-treated sequencing-grade trypsin  and STAGE Tip peptide cleanup as previously described  and loaded directly onto a 75 cm x 100 µm internal diameter fused silica PicoTip Emitter , packed in-house with ReproSil-Pur C18-AQ  heated to 55°C. Peptide elution was performed using a tripartite gradient, decreasing the fraction of Buffer A (0.1% formic acid in water) and increasing the fraction of Buffer B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min .m/z 380–1500 mass selection, an orbitrap resolution of 60,000 (at m/z 200), a target automatic gain control (AGC) value of 4e5, and maximum fill times of 50 ms. The PRM event employed an orbitrap resolution of 30,000 (at m/z 200), a target AGC value of 5e4 with a maximum ion injection time of 54 ms. The precursor ion of each targeted peptide was isolated using an isolation window of 1.6 m/z. Fragmentation was performed with a HCD collision energy of 30% and MS/MS scans were collected using a scan range from 100 to 1000 m/z. PRM data were collected in centroid mode. Data analysis was performed using manual integration using Thermo Xcalibur Qual Browser and Skyline (The column was mounted in a nanospray source in line with an Orbitrap Fusion mass spectrometer (Thermo Scientific). Spray voltage was 2.1 kV in positive mode, and the heated capillary was maintained at 275°C. The acquisition method combined two scan events corresponding to a full scan event and a parallel reaction monitoring (PRM) event targeting the singly-, doubly-, and triply-charged precursor ions of the three phosphorylated peptides without scheduling. The full scan event employed a  Skyline . Peak seReagents and protocols will be provided upon request. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is an interesting study that builds upon previous work by these authors that demonstrates a possible check-point role for LynA in macrophage activation caused by activation-induced degradation. This study demonstrates that this degradation is mediated by the E3 Ub ligase cCbl that targets the insert region on LynA and requires Tyr32. The data supporting the authors' conclusions are strong.The major weakness of the study is that cCbl has already been reported to cause Lyn degradation. Nevertheless, the current study has merit because it demonstrates that this role is specific for LynA and because the study presents these observations within an important biological context. Freedman and co-workers demonstrate a rapid negative feedback loop in a specific spliced form of the Src family tyrosine kinase LynA that is not seen with the alternatively spliced form, LynB, or with other members of the SFK family. This is physiologically significant as MAST cells that use LynB, but not LynA, do not exhibit this phenotype and have a more sustained and higher levels of ERK activation.Essential revisions:1) What is the meaning of "orthogonal" in the title?2) What is the function of Y32? Why does its phosphorylation enhance ubiquitination and degradation? It is suggested that this could be a binding site for cCBL as well as an activating phosphorylation? Was the binding of cCBL tested? This would be a logical mechanism of action. Does Y32 phosphorylation induce relocalization of Lyn into the cytoplasm from the membrane? That could also explain the mechanism.3) Does the Y32 get autophosphorylated in an in vitro kinase reaction? If so, is this phosphorylation in cis or trans? For example, is autophosphorylation concentration-dependent in vitro. Can the Y32 phosphorylation be reconstituted using kinase-inactive and phosphorylation site mutated proteins?4) What happens in mast cells if c-CBL is expressed? Would it result in loss of resting LynA? Would that attenuate ERK activation? Essential revisions:1) What is the meaning of "orthogonal" in the title?pY32-dependent recognition of LynA by c-Cbl is distinct from the known PXXP/SH3, PTB/pSFK activation loop, and kinase/substrate interactions mediating the slower degradation of all SFKs. We now convey this point in a less oblique manner in the revised title “Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells.” We have also revised the abstract and main text to emphasize the difference between canonical c-Cbl/SFK interactions and the preferential targeting of LynA for rapid polyubiquitination by c-Cbl.2) What is the function of Y32? Why does its phosphorylation enhance ubiquitination and degradation?In the original manuscript we reported that unique-region Y32 is the key residue marking LynA for rapid polyubiquitination and degradation. We speculated that this might be a site of phosphorylation, based on a prediction algorithm (NetPhos 2.0) and recent reports of this modification in tumor cells. However, we had no evidence that this site was actually phosphorylated in non-cancer cells or in hematopoietic cells, and thus the mechanism of Y32 in targeting LynA for rapid polyubiquitination was unclear. We have now performed quantitative, targeted LC-MS/MS analysis of LynA immunoprecipitates from resting and 3-IB-PP1-treated macrophages. The results of these experiments are reported in new Figure 4 and Figure 4—figure supplements 1-5. We can now unequivocally state that in primary macrophages LynA Y32 is a site of phosphorylation, induced upon SFK activation. Moreover, pY32 is massively enriched in polyubiquitinated LynA. We therefore conclude that activation of the SFKs leads to phosphorylation of LynA at position Y32, and that this is the mechanism by which LynA is targeted specifically for polyubiquitination and degradation.It is suggested that this could be a binding site for cCBL as well as an activating phosphorylation? Was the binding of cCBL tested? This would be a logical mechanism of action.These mass spectrometry experiments were quite labor intensive and occupied the entire revision time frame. We did perform co-immunoprecipitation experiments and found inducible association of c-Cbl with LynA, but we were unable to demonstrate that this association was due to Y32 phosphorylation. Like all SFKs, LynA interacts with c-Cbl via several binding interfaces, including the SH3 domain, the activation-loop peptide binding site, and the activation-loop phosphotyrosine. Additional cloning to isolate the contributions of each of these interfaces was not possible before resubmission.Does Y32 phosphorylation induce relocalization of Lyn into the cytoplasm from the membrane? That could also explain the mechanism.Y32A using epifluorescence microscopy. We were unable to get good staining of total LynA, but we were able to visualize eGFP-tagged unique-region constructs. As shown in new Figure 3—figure supplement 1. All three constructs were membrane localized in the presence and absence of 3-IB-PP1, even in the presence of a kinase-active LynB construct. While the LynA kinase domain may add some localization functionality, we can say that the Y32 mutation does not itself change the localization of the LynA unique region.We probed for localization differences mediated by the unique regions of LynA, LynB, and LynA3) Does the Y32 get autophosphorylated in an in vitro kinase reaction? If so, is this phosphorylation in cis or trans? For example, is autophosphorylation concentration-dependent in vitro.We used targeted mass spectrometry to detect phosphorylation of LynA Y32 with increasing doses of recombinant LynA (to probe cis vs. transautophosphorylation) or increasing doses of recombinant Hck (to probe transphosphorylation). Unfortunately, the data were uninterpretable because recombinant LynA purified from insect cells is already phosphorylated at position Y32. We are continuing to troubleshoot these experiments, but they are likely to take several more months and so unfortunately were not possible for this revision.Can the Y32 phosphorylation be reconstituted using kinase-inactive and phosphorylation site mutated proteins?T410K, which is not on its own degraded during 3-IB-PP1 treatment. LynA degradation can be specifically induced in trans by LynA, LynB, LynAY32A, and Hck56 (the shorter isoform of Hck). In contrast, Fyn, Fgr, Hck59, and Lck were unable to induce degradation of LynA. These data, presented in new Figure 7 and Figure 7—figure supplement 1-2, reveal SFK-specific regulation of LynA degradation, with LynA itself likely inducing its own degradation via Y32 autophosphorylation.We measured the ability of different Src family members to induce degradation of kinase-dead LynA4) What happens in mast cells if c-CBL is expressed? Would it result in loss of resting LynA? Would that attenuate ERK activation?We treated mast cells with small activating (sa)RNAs designed to increase transcription of c-Cbl mRNA. One of these constructs was found to increase c-Cbl expression an average of 2-fold, and we were able to detect an increase in LynA degradation and corresponding decrease in Syk and Erk phosphorylation in response to 3-IB-PP1 in c-Cbl overexpressing mast cells relative to mock-transfected cells . With this modest increase in c-Cbl expression, steady-state expression of LynA was not significantly affected.


Metabolic labeling of proteins with non-canonical amino acids (ncAAs) provides unique bioorthogonal chemical groups during de novo synthesis by taking advantage of both endogenous and heterologous protein synthesis machineries. Labeled proteins can then be selectively conjugated to fluorophores, affinity reagents, peptides, polymers, nanoparticles or surfaces for a wide variety of downstream applications in proteomics and biotechnology. In this review, we focus on techniques in which proteins are residue- and site-specifically labeled with ncAAs containing bioorthogonal handles. These ncAA-labeled proteins are: readily enriched from cells and tissues for identification via mass spectrometry-based proteomic analysis; selectively purified for downstream biotechnology applications; or labeled with fluorophores for in situ analysis. To facilitate the wider use of these techniques, we provide decision trees to help guide the design of future experiments. It is expected that the use of ncAA labeling will continue to expand into new application areas where spatial and temporal analysis of proteome dynamics and engineering new chemistries and new function into proteins are desired. Methods that allow for labeling of proteins co-translationally, i.e. as they are being synthesized, have wide ranging applications in engineering, biotechnology, and medicine. Incorporation of non-canonical amino acids (ncAAs) into proteins enables unique bioorthogonal chemistries, those that do not react with naturally occurring chemical functional groups, for conjugation. These conjugate substrates range from fluorophores, affinity reagents, and polymers to nanoparticle surfaces, enabling new advances in technology to study cellular systems and produce novel biocatalytic and therapeutic proteins. A key benefit of these techniques is the ability to enrich for labeled proteins of interest, whereas other labeling methods add or remove a mass (e.g. isotope labeling ) that caFirst coined by Sharpless and colleagues in 2001, click chemistries are a set of chemical reactions that are readily catalyzed in aqueous solutions at atmospheric pressure and biologically-compatible temperatures, with few toxic intermediates, and relatively fast reaction kinetics –5 and coHere, we focus on azide-alkyne cycloaddition as it is one of the most widely used, with broad availability of commercial reagents, moderately fast kinetics, and well-established protocols. Copper(I)-catalyzed azide-alkyne cycloaddition  . Th. Th67]. Overall, the site-specific approach provides significantly more control over the exact pre-defined location of ncAA incorporation into the protein as compared to other methods . It alsoResidue-specific methods have since been applied to identify de novo protein synthesis in a variety of contexts. Dieterich et al. introduced the bioorthogonal non-canonical amino acid tagging (BONCAT) strategy for selective analysis of de novo protein synthesis with high temporal resolution , 72. In Caenorhabditis elegans [Over the last decade, BONCAT has gained wide recognition because of its capability of tracking continuous changes in protein expression. It has been applied in mammalian cell cultures to study protein acylation , lysosom elegans , 81, and elegans .Until recently, it was presumed that BONCAT cannot be applied to in vivo labeling of the rodent proteome because mammalian cells would favor incorporating endogenous Met, rather than an analog, into newly expressed proteins . HoweverE. coli MetRS mutants that can charge the Met analog azidonorleucine (Anl) to Met sites [E. coli [Salmonella typhimurium [Yersinia enterocolitica and Yersinia pestis strains [Toxoplasma gondii [With the aim of probing proteomic changes in specific cell types, engineered aaRS technology was adopted to allow cell-selective labeling with ncAAs. This technique, pioneered by the Tirrell group, was first made possible by identifying et sites . Anl is [E. coli , 89, 90,himurium , Yersini strains , and Toxa gondii  in infecCaenorhabditis elegans as a model organism, Yuet et al. employed a phenylalanyltRNA synthetase mutant capable of incorporating the ncAA azidophenylalanine  and drosophila MetRS (dMetRS) mutants that can activate Anl [To further demonstrate the utility of this approach, other variants of aaRS have been evolved to enable cell-selective incorporation of ncAAs in mammalian cells and animals. Using vate Anl . The dMevate Anl , while tvate Anl  and to lvate Anl .More recently, Schuman and coworkers advanced the MetRS mutant technology to enable cell-selective labeling in live mammals for the first time . In thisCaenorhabditis elegans and Drosophila melanogaster [. In the meantime, residue-specific labeling will continue to be the predominant approach when using ncAAs for most proteomics applications. With the increasing variety of approaches to ncAA incorporation, it is important to identify which approaches are best suited to a given application. To help guide researchers find the optimal strategy to label proteins for a given proteomics application, a decision tree diagram is provided in Fig. While residue-specific ncAA labeling has been primarily used for proteomic applications due to the ease of use and incorporation throughout the proteome, site-specific labeling has the potential to also assist in this area , 98. Fornogaster , 100, hoTraditional approaches to bioconjugation in biotechnology often target reactive side chains of natural amino acids such as lysines, though this results in a complex mixture of products modified at different locations and to different extents, complicating protein separation and often reducing protein activity. For some applications, sufficient control is afforded by altering the pH of a conjugation reaction to enhance the reactivity of the N-terminal amino group , 102. AnAs uniquely reactive chemical moieties, ncAAs provide a tool for enhancing commercial and therapeutic applications of proteins in a bioorthogonal manner. ncAAs have been used to study protein stability and also generate proteins with improved stability . CharactIn some cases, residue-specific labeling provides adequate control of conjugation site to maintain sufficient protein activity. For example, Met replacement was used to functionalize a VLP which contained only one Met in each capsid monomer . For casmax of the optimally PEGylated hGH compared to hGH PEGylated at other sites, demonstrating the importance of site optimization and precise conjugation site targeting for pharmacokinetic properties [In many applications, including both the study of protein function and the design of enhanced proteins, it is desirable to incorporate the ncAA precisely at a pre-determined site. For example, conjugation site has been shown to have a significant effect on the stability and activity of antibody-drug conjugates , polymeroperties .In biocatalysis and enzyme production, site-specific incorporation of ncAA can be instrumental in preparation of robust, reusable proteins to improve industrial applicability. Deepankumar and coworkers immobilized transaminase to a chitosan substrate site-specifically to produce an immobilized enzyme which facilitated simple purification and maintained a specific activity nearly equal to that of the wild-type enzyme . The enhSite-specific ncAA incorporation also allows for close control over the number of sites which are modified by conjugation, which is an important aspect of conjugate optimization. For example, Wilding and coworkers recently demonstrated that dual PEGylation of T4 lysozyme at two site-specifically incorporated Azf Fig.  residuesGiven the varied, site-dependent effects of ncAA incorporation and conjugation, a major challenge with ncAA incorporation is understanding and predicting the impact that the mutation will have on the protein. However, recent progress has demonstrated the potential for molecular simulations to inform site selection , 128. FoWith the increasing variety of approaches to site-specific ncAA incorporation, it is important to identify which approaches are best suited to a given application. Figure To expand the potential of ncAA labeling for research and industrial applications, additional studies are necessary to address key limitations in the efficiency of ncAA incorporation and optimal modification site selection. It is generally recognized that one limitation of residue-specific ncAA labeling is that it commonly requires prior depletion of a natural amino acid to achieve high proteome labeling. This practice can disturb normal biological functions and hence adapting methods that enable high levels of ncAA incorporation in the presence of the canonical amino acid is an important advancement for applications in higher-order organisms , 85–87.Current challenges in obtaining the highest quality proteomic mapping lie in the optimization of the click chemistry reactions and enrichment protocols. Therefore, continued discovery of new click chemistries with faster kinetics and higher specificity will increase the potential for ncAAs in proteomics applications. In addition, development of techniques that allow cell- and tissue-specific labeling in mammalian systems with lower non-specific labeling and background noise will have a significant impact on resolving cellular proteomics maps with high resolution. This, combined with advances in engineering aaRS mutants that enable charging ncAAs at higher rates and promoters that can drive the expression of the mutant synthetase with high cell specificity, will enhance our understanding of the spatial and temporal aspects of proteome dynamics.E. coli by knocking out release factor components responsible for competition with nonsense suppression at amber stop codons to reduce premature termination [E. coli strain was produced [A major hurdle for biotechnology applications where stoichiometric labeling is desired is that ncAA incorporation efficiency for site-specific protein modification often varies by the incorporation site. Elucidating factors determining site-dependence will enable the more effective design of ncAA-modified proteins, for example, by targeting bases that flank suppressed codons . Additiomination , 131. Hoproduced , 131.Protein labeling, even site-specifically, can also have a dramatic effect on the properties of the protein in a manner that is highly dependent on modification site/sites. Currently, no complete set of parameters exists to identify sites amenable to labeling based on the primary, secondary, or tertiary structural context . This liAnother potential solution to improve ncAA incorporation into particular proteins of interest is in vitro or ‘cell-free’ protein synthesis where some of the factors limiting ncAA incorporation can be overcome. For example, multiple labs have removed the native tRNAs and then added a minimal set of in vitro synthesized tRNAs, essentially emancipating most codons for competition-free ncAA incorporation , 132. AdIn conclusion, ncAA labeling is a versatile tool that enables the identification of de novo protein synthesis and proteome dynamics and adds new functionality to proteins of interest. With the continued development of new technologies for ncAA incorporation, it is increasingly difficult to determine the best approach for a given application. To assist in the experimental design of new applications of ncAA labeling, decision tree diagrams are provided for proteomics and biotechnology applications in Figs.


Staphylococcus aureus. The dominant form is characterized by nonimmunologic features including skeletal, connective tissue, and pulmonary abnormalities in addition to recurrent infections and eczema. Omalizumab is a humanized recombinant monoclonal antibody against IgE. Several studies reported clinical improvement with omalizumab in patients with severe atopic eczema with high serum IgE level. We present the case of a 37-year-old male with HIES and cutaneous manifestations, treated with humanized recombinant monoclonal antibodies efalizumab and omalizumab. After therapy for 4 years, we observed diminished eczema and serum IgE levels.Hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by elevated levels of immunoglobulin E (IgE), eczematous dermatitis, cold abscesses, and recurrent infections of the lung and skin caused by Staphylococcus aureus. The diagnosis of hyper-IgE syndrome is syndromic and the most frequent laboratory meetings are eosinophilia (greater than 1500/ml) and elevated levels of IgE, which typically are above 2000 IU/ml. Leukocytes in blood and serum immunoglobulin levels IgM, IgG, and IgA are frequently normal, but they have been found in some cases of deficiencies immunoglobulins. There are two main forms of HIES: a form characterized by mutations in signal transducer and activator of transcription 3 gene (STAT3) with autosomal dominant inheritance (AD) (the Online Mendelian Inheritance in Man® (OMIM®) number registered in database is #147060) and a mutation in Dedicator of cytokinesis 8 (DOCK8) with autosomal recessive inheritance (AR), OMIM #243700, both in humans and mice. The dominant form is characterized by nonimmunologic features including skeletal abnormalities , connective tissue, and pulmonary abnormalities in addition to recurrent infections and eczema. In contrast, the recessive form lacks skeletal abnormalities and has marked viral infections caused by molluscum contagiosum and herpes simplex virus (HSV) and neurologic complications [ DOCK8 underlie AR-HIES, an estimated >100 patients worldwide have been identified. FOXP3 regulatory T cells that produce IL-10 play an essential role in regulating allergic inflammation. There are two types of FOXP3 regulatory T cells: the natural and induced. FOXP3 gene mutations produce immune deregulation with enteropathy, autoimmune diabetes, thyroiditis, food allergies, atopic dermatitis, and elevated levels of IgE. Cytokines produced by dendritic cells (DC) play a role in the induction and maintenance of T cell tolerance. IL-10 signaling-DC is needed for the generation of tolerogenic DC and induced regulatory T cells (iTreg) which maintain an appropriate balance Th1/Th2. In patients with HIES, signal transduction mediated by IL10-DCs is impaired, which predisposes to allergic manifestations. This generation of induced FOXP3 regulatory T cells is impaired in patients with HIES [Hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by elevated levels of immunoglobulin E (IgE), eczematous dermatitis, cold abscesses, and recurrent infections of the lung and skin caused byβ 5 pg/ml, IL-2R 1086 IU/ml, IL-6 11.9 pg/ml, and IL-8 8.66 pg/ml, showing increased levels of proinflammatory cytokines. He was sent to the service of clinical immunology and allergy, where he was diagnosed with HIES. To evaluate the pathway of IL-21, PBMCs of the patient were stimulated with rhIL-21 (militenyi) for 15 minutes and phosphorylation of STAT3 was evaluated by flow cytometry. The mAbs used were anti CD19-APC, anti-pSTAT3 (Y705)-PE, and isotype controls (mouse IgG1) from BD Pharmingen and Santa Cruz Biotechnology. Flow cytometry was performed on CYAN-ADP cytometer (Beckman Coulter). Cells were analyzed using Flow Jo 8.8 software (Tree Star). On September 2008, the clinical manifestations were dry skin and lichenified upper limbs and chest, generalized itching, peeling, and scratching scars, treated with hydroxyzine 10 mg every 8 hours, efalizumab, emollients, and general measures. On January 2009, the treatment was modified with monoclonal antibody omalizumab 300 mg every two weeks, with initial levels of total IgE 127,000 IU/ml. We made a new clinical assessment at 6 weeks; the patient presented clinical improvement with eczematous lesions on the chest and upper limbs and secondary hypochromic lesions and traces of scratching. Reassessing their total serum IgE levels in 439 UI/ml, we decided to increase omalizumab dose to 350 mg every two weeks. On May 2013, the patient presented skin lesions exacerbation with erythema, itching, eczema, and erythroderma. To 2013 the patient received 74 doses of omalizumab with stability of clinical manifestations. Eczema and total IgE levels were decreased even though with variations. The variations of CRP (C-reactive protein) do not correlate with the clinical manifestations of the patient and IgE levels  are a major cause of AD.We present the case of a patient with HIES and skin manifestations treated with humanized recombinant monoclonal antibody, omalizumab. HIES is a rare primary immunodeficiency, characterized by high levels of serum IgE, recurrent skin abscesses, eczema, and pneumonia. Inherited patterns with AD, AR have been reported, but most HIES cases are sporadic. Mutations in the signal transducer and activator of transcription 3 gene  is needed for the differentiation of subsets of T cells CD4 the Th17 cells . The patathogens . Our patIn the present case, dermatological findings led us to suspect HIES because of IgE levels and childhood onset-atopic dermatitis. Therefore, further investigations for HIES were done and the diagnosis was made by Grimbacher Criteria . Omalizu