Mycobacterium abscessus infections are extremely limited. New or repurposed drugs are needed. The anti-M. abscessus activity of AR-12 (OSU-03012), reported to express broad-spectrum antimicrobial effects, was investigated in vitro and in vivo. Antimicrobial susceptibility testing was performed on 194 clinical isolates. Minimum bactericidal concentration and time-kill kinetics assays were conducted to distinguish the bactericidal versus bacteriostatic activity of AR-12.Therapeutic options for  Mycobacterium abscessus infections are extremely limited. New or repurposed drugs are needed. The anti-M. abscessus activity of AR-12 (OSU-03012), reported to express broad-spectrum antimicrobial effects, was investigated in vitro and in vivo. Antimicrobial susceptibility testing was performed on 194 clinical isolates. Minimum bactericidal concentration and time-kill kinetics assays were conducted to distinguish the bactericidal versus bacteriostatic activity of AR-12. Synergy between AR-12 and five clinically important antibiotics was determined using a checkerboard synergy assay. The activity of AR-12 against intracellular M. abscessus residing within macrophage was also evaluated. Finally, the potency of AR-12 in vivo was determined in a neutropenic mouse model that mimics pulmonary M. abscessus infection. AR-12 exhibited high anti-M. abscessus activity in vitro, with an MIC50 of 4 mg/liter (8.7 μM) and an MIC90 of 8 mg/liter (17.4 μM) for both subsp. abscessus and subsp. massiliense. AR-12 and amikacin exhibited comparable bactericidal activity against extracellular M. abscessus in culture. AR-12, however, exhibited significantly greater intracellular antibacterial activity than amikacin and caused a significant reduction in the bacterial load in the lungs of neutropenic mice infected with M. abscessus. No antagonism between AR-12 and clarithromycin, amikacin, imipenem, cefoxitin, or tigecycline was evident. In conclusion, AR-12 is active against M. abscessusin vitro and in vivo and does not antagonize the most frequently used anti-M. abscessus drugs. As such, AR-12 is a potential candidate to include in novel strategies to treat M. abscessus infections.Therapeutic options for  Mycobacterium abscessus is one of the most challenging etiological agents  are rapidly emerging opportunistic pathogens capable of causing severe lung infections in vulnerable individuals. Most countries report a dramatic increase in lung infections involving NTM in recent years . Among Nl agents . Infecti limited . Current limited –7. Furtho appear , 9. New in vitro and in vivo and is effective in eliminating certain multidrug-resistant (MDR) bacterial pathogens , a novel celecoxib derivative that lacks cyclooxygenase-2 inhibitor activity, exerts a biological effect by a variety of mechanisms that include repressing host cell chaperone machinery, inhibiting kinase pathways, and upregulating autophagy –16. AR-1–17–20–ogens 12–, 28–30. 12, a novM. abscessus activity of AR-12 was undertaken. AR-12 was active against M. abscessus growing in vitro and in vivo and proved especially effective in inhibiting the growth of intracellular organisms. Moreover, AR-12 did not act antagonistically toward the most frequently used anti-M. abscessus drugs. To our knowledge, this is the first report of the anti-M. abscessus activity of AR-12. As such, this work may provide a novel approach to treating M. abscessus infections.A detailed evaluation of the anti-M. abscessus isolates (148 subsp. abscessus and 46 subsp. massiliense) were collected. AR-12 displayed similar antimicrobial activity against both subspecies. The MICs ranged from 2 to 16 mg/liter, with an MIC50 and an MIC90 of 4 and 8 mg/liter (8.7 and 17.4 μM), respectively, emphasizing the broad sensitivity of M. abscessus to AR-12 , AR12 did not affect the MIC calculated subsequently for 16 clinical isolates or the M. abscessus subsp. abscessus reference strain (ATCC 19977) (unpublished data). Thus, it is unlikely that the regrowth shown here is due to increased antibiotic resistance. Rather, regrowth is more likely due to the loss of antibiotic potency with time in culture.To determine the potency of the killing kinetics of AR-12 against  control . TreatmeM. abscessus drugs (abscessus strain showed synergy (FICI = 0.5). No antagonism between drugs was observed.Indifference  was the most commonly observed interaction  of all the antimicrobial combinations of AR-12 and the most frequently used anti-M. abscessus subsp. abscessus (reference strain ATCC 19977) and subsp. massiliense  in primary peritoneal macrophages. Both AR-12 and amikacin at 0.5× and 1.0× MIC significantly inhibited the intracellular growth of subsp. abscessus relative to the untreated control; AR-12 demonstrated greater inhibition than amikacin  and amikacin (50 mg/kg) caused significant reductions of ∼3.7 log10 CFU and ∼3.0 log10 CFU, respectively, in lungs accessed at 14 days postinfection  has been reported , respectively. Pharmacokinetic studies in mice indicated that the peak serum concentration of oral AR-12 administered at 200 mg/kg exceeded 20 μM. At the end of a 28-day period, the mice exhibited no visible signs of toxicity. All the animals maintained stable body weights and lacked gross pathological abnormalities upon necropsy   . Similared data) , 24.50 of AR-12 for M. abscessusin vitro determined here, indicating that a safe and effective dose can be achieved to treat clinical M. abscessus infections. The best dose and formulation of AR-12 required to optimize therapeutic outcomes, however, remain to be determined.Data obtained in a phase I clinical trial, exploring the effectivity of AR-12 as a targeted anticancer drug, showed that an 8 μM or 3.7 mg/liter serum concentration was safely attained and well tolerated by patients administered 800 mg twice daily . This seFrancisella strains but inhibits the intracellular survival of Francisella indirectly by inducing host cell autophagy  found to be clinically important in treating M. abscessus infections according to guidelines  genes. Staphylococcus aureus ATCC 29213, Mycobacterium peregrinum (ATCC 700686), and M. abscessus ATCC 19977 reference strains were purchased from the American Type Culture Collection . M. abscessus was grown at 37°C on Middlebrook 7H10 (M7H10) agar plates supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.2% glycerol or with continuous shaking in Middlebrook 7H9 (M7H9) broth supplemented with 10% OADC and 0.05% Tween 80 (7H9sB). Suspensions of all isolates were stored at –80°C and cultured fresh before each assay. Middlebrook 7H9 broth, M7H10 agar, cation-adjusted Mueller-Hinton II broth (CAMHB), and OADC supplement were purchased from Becton, Dickinson, and Company .A total of 194 clinical AR-12 (OSU-03012) was purchased from MedChemExpress . Clarithromycin, amikacin, imipenem, cefoxitin, and tigecycline were purchased from Sigma-Aldrich . Stock solutions of AR-12 (128 μg/ml) were dissolved in 32% DMSO (4 μg/ml in 1%). Clarithromycin  was prepared in 100% DMSO, and other antibiotics at 2,048 μg/ml were prepared in deionized water. All antibiotics were stored as aliquots at –20°C and serially diluted just prior to experimental use.M. abscessus colonies were picked from M7H10 agar plates, grown to logarithmic phase in M7H9 broth, diluted to McFarland 0.5 with sterile saline, and adjusted to 1 × 105 to 5 × 105 CFU/ml using CAMHB. AR-12 was serially diluted 1:2 in a 96-well microtiter plate , and 100 μl of bacteria in suspended CAMH was added to each well. The plates were incubated at 37°C under aerobic conditions for 3 to 4 days until the control wells without AR-12 exhibited visible growth. Mycobacterium peregrinum (ATCC 700686) and Staphylococcus aureus (ATCC 29213) served as control reference strains. The MIC was defined as the lowest drug concentration at which no bacterial growth was observed by visual examination. All assays were repeated twice.Antimicrobial susceptibility testing was conducted in accordance with CLSI document M24-A2. In brief, individual M. abscessus subsp. abscessus and subsp. massiliense isolates were selected randomly for determination. After the antimicrobial susceptibility test at day 4 of AR-12 incubation, wells in which the drug concentrations were higher than the MIC were evenly resuspended. Portions (100 μl) of the contents from wells were serially diluted (10-fold) and plated to count the remaining CFU. The CFU were determined after 5 days of incubation at 37°C. The MBC values were defined as the minimum drug concentration with no colony growth. An antibiotic is considered bactericidal when the MBC/MIC ratio is ≤4; otherwise, it is considered bacteriostatic.The minimum bactericidal concentration (MBC) was determined in order to distinguish between the bacteriostatic and bactericidal activities of AR-12. abscessus ATCC 19977 and a clinical subsp. massiliense isolate (G137) to evaluate the potency of the killing kinetics of AR-12 against M. abscessus. Tubes (5 ml) of 7H9sB containing AR-12 or amikacin at 1× or 8× MIC were inoculated with 5 × 105 CFU of bacteria growing exponentially; the tubes were incubated for 7 days at 37°C with shaking (100 rpm). The bacteria were enumerated on days 0, 1, 2, 3, 5, and 7 by plating serial dilutions on lysogeny broth agar plates; CFU were enumerated after an additional 5 days incubation at 37°C. All assays were performed in duplicate.We used the subsp. in vitro using the broth microdilution chequerboard titration technique. Briefly, serial 2-fold dilutions of AR-12 were added to the columns of wells, and serial dilutions of the five antibiotics were added to the rows of wells in a 96-well microtiter plate, yielding a two-dimensional matrix composed of AR-12 in combination with various antibiotic concentrations. The 2-fold dilutions of AR-12 or each antibiotic alone and the medium without AR-12 or antibiotics were used as controls. Randomly selected M. abscessus subsp. abscessus and subsp. massiliense isolates (∼106 CFU/ml) were added to each well, and the plates were incubated for 3 to 4 days at 37°C. The final MIC of each drug combination was determined as described for antimicrobial susceptibility testing above. The results of synergy testing were interpreted based on the FICI determined according to the following formula: FICI =  + . Drug interactions exhibit three patterns: synergy (FICI ≤ 0.5), indifference (0.5 < FICI ≤ 4.0), and antagonism (FICI > 4.0).Synergy between AR-12 and clarithromycin, amikacin, imipenem, cefoxitin, and tigecycline was assessed Mycobacterium species, were obtained by previously described methods  to remove the extracellular organisms. Fresh RPMI 1640 supplemented with 10% human serum and then AR-12 or amikacin at 0.5× to 1× MIC was added; infected control cells were treated with culture medium alone. The cells were lysed with 0.05% sodium dodecyl sulfate at various times postinfection, and the numbers of CFU were quantified by plating serial dilutions of lysates on M7H10 agar plates. Cell viability was evaluated by trypan blue exclusion before and after infection and/or drug treatment at each time point. Peritoneal macrophages were 95% viable at 48 h postinfection; drug exposure did not affect cell viability (data not shown).Mouse peritoneal macrophages, frequently used in studies undertaken to examine the factors that affect intracellular replication of  methods –35. The 7 CFU of M. abscessus ATCC 19977. On day 3 postinfection, three mice were sacrificed, and the CFU/lung were determined to evaluate the model’s efficacy. The remaining 21 infected mice were divided randomly into three groups: group 1 , group 2 , and group 3 (administered 50 mg/kg amikacin intramuscularly for 2 weeks daily). The dosage of amikacin was selected based upon the report of Das et al. .Statistical differences between study groups were determined by a nonpaired Student


This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y12 receptor antagonists—cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y12 receptor antagonists.We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y The leading cause of death in Western countries, according to current World Health Organisation data, is cardiovascular disease that results primarily from arterial thrombosis dependent on blood platelet hyperactivity. Thromboembolic events can be prevented by anti-platelet therapy . However12 receptor. Its inhibition blocks the ADP-dependent platelet activation pathway [1), rendering the clot formation process ineffective. Additionally, the inhibition of the third platelet receptor from the P2 class, ATP-gated ion channel receptor P2X1, which activation does not directly induce the platelet aggregation but causes fast calcium mobilization and platelet shape change, was also suggested as a potential way of reducing thrombotic events [12 inhibitors are thienopyridines , the ATP analogue cangrelor  and the cyclopentyltriazolopyrimidine derivative ticagrelor [Currently, one of the major targets of antiplatelet drugs is the P2Y pathway —the enhac events . The moscagrelor .2 formation) is often combined with clopidogrel. This approach is however still burdened with a problem of drug resistance, especially in patients suffering from type 2 diabetes—a group with increased risk thromboembolic events [Efficient anti-platelet treatment is often hindered in clinical practice by reduced sensitivity to many anti-platelet agents and by high inter-individual variation in response to treatment, resulting in bleeding and a high risk of failure. That problem is usually managed by a combined therapy—administering two or more drugs affecting various platelet activation pathways. For example, acetylsalicylic acid , and a panel of nine AR agonists  and dual (simultaneous P2Y12 antagonization and AR agonization) models were used : PSB07712 receptor antagonists: cangrelor (C) or prasugrel metabolite R-138727 (PM), the experimental set up was one AR agonist + one P2Y12 antagonist. Each compound was used at its aggregation IC50 value [AR agonists were used in a combination with two types of P2Y50 value ,21: PSB0p < 0.005).Before measuring platelet activity parameters, the potential cytotoxic effects of AR agonists on blood platelets were determined using calcein assay as described previously . The cytRepresentative cytometric dot-plots and histograms for results described in the 12 antagonists used alone significantly reduced platelet activation. The AR agonists UK423,097, HE-NECA, NECA, MRE0094, 2-chloroadenosine, and CGS21680 demonstrated significant inhibition  as well as for the samples incubated with AR agonists: UK423,097, HE-NECA, NECA, MRE0094, CGS21680, and CV1808 A. The fo12 inhibitors ability to increase VASP phosphorylation in activated platelets was measured. No significant effects were observed for AR agonist, P2Y12 antagonist, or combinations thereof , or a high dosage . Their impact was tested on resting and activated (20 µM ADP) isolated platelets. The results were normalized for platelet count. Positive control of platelets treated with forskolin (5 µM) yielded high results of median 4935→  pmol/1 × 108 plt.Two AR agonists that were established to strongly inhibit platelet functions  and one less efficient AR agonist (PSB0777) were used in a combination with P2Y12 antagonist cangrelor significantly increased cAMP formation in both tested concentrations. A significant increase was also found for the AR agonists UK423,097 (both concentrations), HE-NECA (100 µM), and PSB0777 (both concentrations). AR agonists and P2Y12 antagonists increased cAMP formation, showing a strong antiplatelet effect, which further increased when use in combination: cangrelor + UK423,097 (both concentration pairs), cangrelor + HE-NECA (both concentration pairs), and cangrelor + PSB0777 (paring in high concentrations)  .12 antagonist cangrelor significantly increased cAMP formation in high concentration, as well as AR agonists: UK423,097 (both concentrations), HE-NECA (100 µM), and PSB0777 (100 µM). AR agonists and P2Y12 antagonists, when used in combination, deepened the antiplatelets effect compared to these drugs applied alone  were previously reported to have remarkable anti-platelet properties [2A and A2B adenosine receptors could be a beneficial supplement to current antithrombotic therapy, especially in the light of frequently observed, high inter-individual variability in response to platelet inhibitors.AR agonists are a re-emerging group of compounds with various functions which can be useful in the prevention and treatment of several human diseases ,24,25. Ioperties ,13,19,2112 antagonists  were ev12 antagonists (cangrelor or prasugrel metabolite) [12 antagonist and an AR agonist  leads to increased inhibition of platelet function than the P2Y12 antagonist alone, and that their antiplatelet effect was much more pronounced in individuals with poor response to P2Y12 inhibitors [12 antagonists by introducing measurements of a panel of platelet activation hallmarks.Our previous findings demonstrated the anti-aggregatory activity of some of the AR agonists presented here  examined in the absence or presence of the P2Yabolite) , and thahibitors . This st2A over A2B: UK423,097, HE-NECA , 2-chloroadenosine , MRE0094, regadenoson, PSB0777, and CGS21680. Other AR agonists investigated in this study  are not selective between A2A and A2B receptor subtypes. Our previous papers showed that A2B AR receptor selective agonist either did not show anti-platelet effect or had only minimal one, which could be explained by a slight cross-reactivity with A2A AR receptor [2A and A2B ARs sequence is identical in 59%, no definite proof of a selective A2B AR agonist having antiplatelet properties is available.In this work, AR agonists of different selectivity to AR receptors were used—the following agonists were found to be selective for Areceptor ,19. Even12 antagonists: cangrelor and prasugrel were utilized to assess whether AR agonists can enhance their antiplatelet effect. Prasugrel is a pro-drug and requires metabolization to its active components, therefore its most abundant stable active metabolite R-138727 was used [12 antagonists, as well as AR agonists, were used at IC50 values [50 = 1.3 µM) required to achieve the effective platelet inhibition most likely stems from the fact that only one of the prasugrel active metabolites is used, whereas in vivo prasugrel metabolization results in a number of active metabolites, all of which may exert an antiplatelet effect of their own. We aimed to reflect suboptimal dosages of both classes of compounds in order to demonstrate the combined effect. In addition, it allowed us to work with concentrations clinically achievable in the patients’ bloodstream.Two P2Ywas used . P2Y12 a0 values ,21. The 2A AR is known as the important receptor on blood platelets and a mediator of the adenosine-dependent inhibition of platelet aggregation [2A AR was also reported to reduce the P-selectin expression on the platelet cell surface when platelets were stimulated with the use of thromboxane A2 or ADP [12 inhibitors on those markers are strengthened by the addition of AR agonists. Our results, therefore, provide further proof that such dual experimental therapy could prevent excessive blood clotting.Aregation . The actregation . The act2 or ADP . In this12 antagonist. A summary of the obtained results is presented in This publication provides for the first time the evidence of antiplatelet activity of AR agonist CV1808, demonstrating that CV1808 inhibits exogenous fibrinogen binding, strengthens the antiplatelet effect of PM across all the investigated parameters, and potentiates the ability of cangrelor to limit calcium mobilization. UK423,097 was consistently able to inhibit platelet function across tested parameters, and also HE-NECA and NECA significantly and strongly decreased platelet activation as evidenced by most of the markers (GPIIb-IIIa activation being an exception). On the other hand, regadenoson and PSB0777 did not significantly affect any of the tested platelet activation markers, regadenoson also was unable to strengthen the antiplatelet effect of cangrelor. Overall, however, all the AR agonists studied in this work showed ability to potentiate anti-platelet effect of at least one P2Y2A receptor agonist does not predict a high anti-platelet effect of this compound. For example, we found a moderate antiplatelet effect for PSB0777 and CGS21680 which have been shown to have the high affinities to A2A receptor . Simila= 620 nM .R2 = 0.73), but in our study fibrinogen binding turned out to be a more sensitive marker for detecting the inhibition caused by AR agonists. It could be speculated that binding of exogenous fibrinogen could also be mediated via other platelet-fibrinogen interactions, such as fibrinogen binding to receptors GPVI, GPIb or even non-specific binding. Hence, this parameter could also detect the impact of AR agonists and P2Y12 inhibitors on other signalling pathways, but this issue needs further studies.The inter-individual variation in sensitivity to AR agonists was high, especially for weaker agonists, such as PSB0777 or CV1808, the coefficient of variation frequently exceeding 100%. Such a high value and relatively small sample size may account for no significant drop being observed in platelet function in some cases. It is noteworthy that P-selectin expression and GPIIb-IIIa activation methods were found to be less sensitive in detecting the effects of AR agonists in comparison with fibrinogen binding and calcium ion mobilization. P-selectin expression and GPIIb-IIIa activation indicate overall platelet activation and are standard markers of full platelet activation leading to thrombus formation. Additionally, GPIIb-IIIa is a key, final target for anti-platelet therapeutic intervention ,36. The Calcium mobilization is a very dynamic process, susceptible to rapid change in response to stimulus and therefore a good marker to detect changes in platelet signalling. It governs not only platelet activation, but also secretion and aggregation . It is t12 antagonists, we have selected two of the most potent AR agonists  and one representative of the less effective AR agonists (PSB0777) in combination with cangrelor. The compounds were tested in two concentrations: one being the IC50 value and the second one a high concentration selected to demonstrate a maximal inhibitory effect (100 µM for AR agonists and 1 µM for cangrelor); in resting and activated (20 µM ADP) platelets. It was demonstrated that all three tested AR agonists have the ability to increase cAMP formation in human platelets ex vivo . These results are in accordance with the data reported previously in in vitro cell culture studies using standard cell lines such as CHO or HEK-293 [cAMP is an important mediator since its increase leads to the activation of signalling pathway (mainly protein kinase A cascade) resulting in the inhibition of platelet function . To asse HEK-293 ,40. The  HEK-293 .12 antagonist increase cAMP formation in ADP-activated platelets. This suggests that such dual experimental therapy may be beneficial in pathologically upregulated platelets or in case of platelets with arrested or delayed cAMP formation rates. The observation that cAMP formation is increased by AR agonists and the fact that AR agonists enhanced the cAMP elevation caused by P2Y12 inhibitors confirms that this dual experimental therapy is effective at the pivotal point of platelet activation control.Interestingly, this work provides the first report suggesting that the AR agonists alone and in the combination with a P2Y12 receptor [12 inhibitors. Additionally, the method produced inconsistent readings and very high experimental background. In our opinion, therefore, the use of VASP phosphorylation detection kits dedicated to monitoring of P2Y12 function in clinical setting is not suitable to research purposes aiming to detect subtle changes in platelet signalling.The phosphorylation of vasodilator-stimulated phosphoprotein (VASP) is a marker of ADP-induced platelet activation through P2Yreceptor . We hypoThe majority of the results (cAMP measurement was an exception) presented in this manuscript were obtained using methods assessing platelet function in whole blood. Such the approach was chosen deliberately since it is known that endogenous adenosine undergoes rapid and excessive uptake and metabolism by erythrocytes . SynthetOur rationale to use methods based on flow cytometry resulted from a need to screen many experimental samples (at least 30 per experiment for each blood donor) providing an opportunity to quickly and reliably obtain a complete panel of data with minimal amount of human material (whole blood) used. Furthermore, flow cytometry analysis is especially valuable when working with blood platelets which are prone to artefactual activation. In the case of cAMP formation experiments, when platelet isolation was inevitable to perform a colorimetric assay, it requires the number of tested combinations to be limited.12 kit was purchased form BioCytex . Cell permeant calcium indicator Fluo-4 AM acetoxymethyl ester (Fluo-4 AM), probenecid (water soluble), pluronic acid (water solution) and thapsigargin were purchased from Thermo Fisher Scientific . Cyclic AMP Select ELISA Kit was purchased form Cayman Chemical. Phosphate buffered saline pH 7.4 (PBS) was obtained from Corning . Dimethyl sulfoxide (DMSO), adenosine diphosphate (ADP), and bovine serum albumin (BSA) were obtained from Sigma . All other chemicals, unless otherwise stated, were purchased from Avantor Performance Materials Poland S.A. .Adenosine receptor agonists were purchased from Sigma —NECA (CAS № 35920-39-9); Cayman —regadenoson (CAS № 313348-27-5), 2-chloroadenosine (CAS № 146-77-0); Tocris Bioscience —PSB0777 (CAS № 2122196-16-9), CGS21680 (CAS № 124431-80-7), CV1808 (CAS № 53296-10-9); Abcam —HE-NECA (CAS № 141018-30-6); Axon Medchem —UK 432097 (CAS № 380221-63-6)); and MyBioSource —MRE0094 (CAS № 131865-88-8)). Cangrelor (AR-C69931MX) was from Cayman Chemicals). Prasugrel metabolite (R-138727) was obtained from BOC Sciences . Calcein AM was obtained from Molecular Probes . Antibodies anti-human CD61/PerCP, CD61/PE, CD62P/PE, PAC-1/FITC, mouse IgG1/PE isotype control, mouse IgG1/FITC isotype control, Cellfix, and buffered sodium citrate was purchased from Becton-Dickinson . Fibrinogen from Human Plasma, Oregon Green 488 Conjugate, and Fluo-4, AM, cell permeant were purchased form Invitrogen . PLT VASP/P2Y50 value [The stock and working solutions of cangrelor and prasugrel metabolite were prepared in distilled water. The 100 mM stock solutions of AR agonists were prepared in DMSO, excluding PSB0777 which was dissolved in water. Stock solutions were then diluted with DMSO and PBS to working concentrations  into a vacuum tube containing 0.105 mol/L buffered sodium citrate  for experiment conducted using whole blood, or into a vacuum tube containing acid citrate dextrose (ACD)  for experiments requiring isolated platelets (cAMP level measurement). All individuals stated that they had not taken medications known to influence platelet function for at least two weeks prior to the study.Experiments were approved by the Ethics of Research in Human Experimentation Committee at the Medical University of Lodz, approval number (RNN/43/17/KE). Blood was collected from healthy donors who gave written consent  or 15 (prasugrel metabolite) minutes at 37 °C. Platelets were activated with 20 µM ADP for 5 min at RT. Samples were then diluted 10-fold with PBS, labelled with anti-CD61/PerCP, anti-CD62P/PE and PAC-1/FITC antibodies , and fixed with CellFix (prepared according to manufacturer instructions) for 1h at RT. Directly before measurement, the samples were diluted 1:1 with PBS and the assay was performed, gathering 10000 CD61/PerCP-positive events, using FACSCanto II flow cytometer (BD Bioscience). The percentage of marker-positive platelets (above isotype cut-off) was measured.Whole blood was preincubated with an AR agonist and/or a P2Y12 inhibitor for 3 (AR agonists and cangrelor) or 15 (prasugrel metabolite) minutes at 37 °C. Exogenous Oregon Green-labelled fibrinogen was added to the samples (3 µg/mL), which were subsequently activated with 20 µM ADP for 5 min at RT. Samples were then diluted 10-fold with PBS, labelled with anti-CD61/PE antibodies , and fixed with CellFix (prepared according to manufacturer instructions) for 1 h at RT. Directly before measurement, the samples were diluted 1:1 with PBS and the assay was performed, gathering 10,000 CD61/PE-positive events, using FACSCanto II flow cytometer (BD Bioscience). The percentage of marker-positive platelets was measured.Whole blood was preincubated with an AR agonist and/or a P2Y12 kit was used to monitor specific platelet ADP receptor antagonists according to manufacturer’s instructions; the analysis was performed on whole blood preincubated with an AR agonist and/or a P2Y12 inhibitor for 3 (AR agonists and cangrelor) or 15 (prasugrel metabolite) minutes at 37 °C. Afterwards, samples were supplemented with prostaglandin E1, activated with ADP, fixed, permeabilized and stained with anti VASP-P monoclonal antibodies and next with secondary polyclonal fluorescently labelled antibodies. The non-specific fluorescence was determined using negative isotypic control. Using FACSCanto II flow cytometer (Becton-Dickinson) 10,000 events identified as platelets (CD61-positive) were acquired, and their mean fluorescence intensity was measured using FACSCanto II flow cytometer (BD Bioscience).A PLT VASP/P2Y12 inhibitor for 3 (AR agonists and cangrelor) or 15 (prasugrel metabolite) minutes at 37 °C. Next, samples were diluted 10-fold in PBS containing 1 mM of CaCl2, and incubated with Fluo-4 AM  for 15 min at 37 °C, with pluronic acid  to facilitate the solubilisation of Fluo-4. To prevent the efflux of calcium indicator out from the cells, samples were supplemented with the inhibitor of organic-anions transporters, probenecid . Platelets were then labelled with anti-CD61/PerCp antibodies for 20 min, RT, and samples were diluted 10-fold in PBS containing 1 mM of MgCl2. Directly before the measurement platelets were stimulated with ADP  and after 10 s the end-point fluorescence intensity was measured on FACSCanto II flow cytometer (BD Bioscience).Whole blood was preincubated with an AR agonist and/or a P2Yg for 12 min to obtain platelet rich plasma (PRP) with 50 ng/mL PGE1 added, and subsequent centrifugation of PRP at 800× g for 15 min, again with 50 ng/mL PGE1 added, and re-suspension of platelet pellet with Tyrode’s buffer  with 0.3% bovine serum album and 0.5 mM papaverine. Platelet count in the suspension was measured using Sysmex XS-800i  automated morphology instrument. After at least 35 min from last PGE1 addition, platelets were incubated with AR agonist and/or P2Y12 antagonist for 3 min at 37 °C. If required, platelets were then incubated with 20 µM ADP for 5 min at RT. Subsequently lysis was performed using lysis buffer  in 1:1 v/v ration to the platelet volume for 10 min at RT, and suspension was centrifuged at 10,000× g for 3 min. Cyclic AMP was measured using a Cyclic AMP Select ELISA Kit  according to manufacturer’s instructions.Blood platelets were isolated by a two-step centrifugation at 37 °C: first 200× post hoc Bonferroni’s multiple comparisons test. Data departing from Gaussian distribution were assessed with the Friedman’s test with Dunn’s correction for multiple comparisons. The statistical analysis was performed using the following software packages: Statistica v.13 , and GraphPad Prism .The results are expressed as median with interquartile range. The Shapiro-Wilk test and Mauchley’s test were used to test the data distribution and sphericity of variances, respectively. Data with Gaussian distribution was analysed with one-way ANOVA for repeated measurements with the 12 receptor inhibitor; therefore, a dual experimental therapy involving combination of P2Y12 inhibitors and AR agonists appears to be a feasible solution to overcoming problems of drug resistance leading to dosage increase and resulting in severe side effects, and a way of combating inter-individual variation [12 receptor and the activation of AR receptors using novel agonists of AR receptors may prove to be a favourable strategy of preventing thrombotic events, and should therefore be further investigated .In summary, this work provides the comprehensive evidence of the antiplatelet potential of AR agonists, demonstrated on multiple levels of platelet activation process, from calcium flux inhibition, and cAMP formation increase to restriction of surface markers of platelet activation such as P-selectin expression and GPIIb-IIIa activation together with fibrinogen binding. All the AR agonists studied were able to strengthen the effect of at least one P2Yariation . The app


AR and AR-splice variant 7) in lysed whole blood, and (3) conducted shallow whole-genome plasma sequencing to detect AR amplification. Although 44–53% of patient samples were informative for each assay, a combination of all three approaches led to improved diagnostic sensitivity, providing tumor-related information in 89% of patients. Additionally, distinct resistance mechanisms co-occurred in two patients, further reinforcing the implementation of multi-analyte liquid biopsy approaches.Novel androgen receptor (AR) signaling inhibitors have improved the treatment of castration-resistant prostate cancer (CRPC). Nonetheless, the effect of these drugs is often time-limited and eventually most patients become resistant due to various AR alterations. Although liquid biopsy approaches are powerful tools for early detection of such therapy resistances, most assays investigate only a single resistance mechanism. In combination with the typically low abundance of circulating biomarkers, liquid biopsy assays are therefore informative only in a subset of patients. In this pilot study, we aimed to increase overall sensitivity for tumor-related information by combining three liquid biopsy approaches into a multi-analyte approach. In a cohort of 19 CRPC patients, we (1) enumerated and characterized circulating tumor cells (CTCs) by mRNA-based in situ padlock probe analysis, (2) used RT-qPCR to detect cancer-associated transcripts (e.g.,  AR gene amplification [AR gene mutations [Prostate cancer (PC) is one of the most common malignant cancers in men causing more than 350,000 deaths worldwide per year . As PC ification ,5, AR geutations ,7, and tutations ,10,11,12utations , liquid AR-V7, AR full length (AR-FL), and kallikrein-related peptidase 3 ) transcripts directly in tumor cells. Furthermore, we 2) developed an RT-qPCR-based assay using a pre-amplification step to assess AR-V7, AR-FL, and KLK3 expression in lysed whole blood samples from CRPC patients. Moreover, we 3) investigated AR copy-number gain by shallow whole-genome sequencing (sWGS) of plasma DNA. We aimed at identifying which of these liquid biopsy approaches, either single or in combination as a multi-analyte approach, are best suited to obtain tumor-related information.Here, we combined three liquid biopsy approaches to analyze matched CRPC samples. As outlined in AR-V7 detection and could be performed directly on cells attached to the surface of the CellCollector, as well as cells fixed on glass slides. Using this in situ technology, transcripts were visualized within the cells as bright fluorescent spot-like signals which are referred to as rolling circle products (RCPs) [KLK3 RCPs in the cells of healthy donors or PC-3 cells but identified some cells with one AR-V7 RCP/cell or one to two AR-FL RCPs/cells. These results demonstrate the high specificity of in situ padlock probes, as 99.23–99.29% of PBMCs did not show any false-positive signals for AR-V7, and 98.91–99.04% of PBMCs did not show any false-positive signals for AR-FL. In VCaP cells, we detected a median of 0 KLK3 RCPs/cell (interquartile range (IQR) 0–0), 2 AR-V7 RCPs/cell (IQR 1–4), and 9 AR-FL RCPs/cell (IQR 5–13). Although detectable in some VCaP cells, the sensitivity for KLK3 was very low. Based on the background of AR-V7 and AR-FL observed in healthy controls and PC-3 cells, we defined a cut-off for the identification of CTCs in patient samples. Only cells with ≥1 KLK3 RCPs/cell, ≥2 AR-V7 RCPs/cell, or ≥3 AR-FL RCPs/cell were rated as CTCs.In situ padlock probe analysis was performed as previously described . The asss (RCPs) . We evalAR-V7-expressing CTCs. The expression level of AR-V7 mRNA in CTCs reached up to 14 RCPs/CTC. Sixteen percent of the patients (3/19) were positive for AR-FL-expressing CTCs, with expression levels reaching up to 9 RCPs/CTC. Twenty-one percent of the patients (4/19) were positive for KLK3-expressing CTCs, with an expression level of 1 RCP/CTC. Most CTCs expressed only one of the investigated transcripts, but some CTCs co-expressed AR-V7 and AR-FL, AR-V7 and KLK3, or all three transcripts. As visualized in AR-V7 expression). Representative images of captured and in-situ-analyzed CTCs are shown in We then investigated the CTC status of 19 patients with CRPC. Using the cut-off described above, we identified CTCs in 53% of the patients (10/19), ranging from 1–10 CTCs/patient. Fifty-three percent of the patients (10/19) were positive for AR-V7, AR-FL, and KLK3 transcripts in whole blood of 16 patients using a modified approach from Todenhöfer et al. [GUSB) as an internal reference gene for normalization (ΔCq) and used the lowest ΔCq value of negative controls  as a cut-off for positive test results  and 38% of the patients (6/16) were positive for AR-FL and KLK3, respectively . In contrast, less patients were tested positive for AR-FL and KLK3 by in situ padlock probe analysis than by RT-qPCR (19% versus 31% for AR-FL and 25% versus 38% for KLK3). The concordance between in situ padlock probe and RT-qPCR assays, i.e., both assays showing the same result, was 38% for AR-V7 and 63% for AR-FL and KLK3. Of note, the patient who tested AR-V7-positive with the RT-qPCR approach had no detectable AR-V7-expressing CTCs. This resistant patient would have been missed, if only the in situ padlock probe analysis had been performed.A subgroup of 16 patients was analyzed by both in situ padlock probe and RT-qPCR, allowing for a direct comparison. More patients were tested positive for AR amplifications and to quantify tumor content in cell-free DNA using the probabilistic ichorCNA model [AR amplicons were identified in 50% of the patients (5/10), which is in range with previous findings in advanced PC [AR amplifications are depicted in In a subset of ten patients, we had access to corresponding plasma samples, which were analyzed by sWGS, i.e., plasma-Seq , to callNA model . Althouganced PC ,19. ReprAR amplifications could be identified by plasma-Seq in 50% of the patients (5/10). By combining these assays, tumor-related information was obtained in 89% of all patients (17/19). Interestingly, we observed AR amplification in circulating tumor DNA (ctDNA) co-occurring with CTC-AR-V7 expression in two of five patients. In total, we detected the resistance markers AR-V7 or AR amplification in 74% of the patients (14/19). Of note, in 86% of resistant patients (12/14), the resistance was detected by one of the assays exclusively. For example, in patient 5 only AR amplification was detected by plasma-Seq, while no AR-V7 expression was detected by in situ or RT-qPCR analysis. The results of our multi-analyte approach are summarized in The combination of in situ padlock probe analysis of CTCs, RT-qPCR gene expression assays, and plasma-Seq yielded more information than any of the assays alone. In total, PC-associated transcripts could be detected in 53% of the patients (10/19) by in situ padlock probe analysis and in 44% of the patients (7/16) by the RT-qPCR assay. n = 4), enzalutamide (n = 12), abiraterone and enzalutamide (n = 1), or enzalutamide and taxane (n = 1). After the sample collection for liquid biopsy analyses, the patients received the next line of therapy including treatment with abiraterone (n = 5), enzalutamide (n = 1), taxane (n = 5), abiraterone and taxane (n = 4), enzalutamide and taxane (n = 2), or others (n = 1). PSA levels in blood were measured at the time of sample collection and monitored for 12 weeks. Three patients showed decreased PSA levels within 12 weeks, but only one of them reached a PSA50 response. Fifteen patients showed no PSA response, with PSA levels increasing up to 10-fold from baseline reflecting androgen indifferent disease. For one patient, no data regarding treatment and PSA response was available. Poor PSA response is visualized in the waterfall plot in p > 0.05; p = 0.823). Furthermore, none of the identified biomarkers was significantly associated with altered overall survival ) for a more reliable identification of CTCs. Furthermore, we will use optimized in situ assays for all investigated transcripts [AR-V7, which explains the poor detection of KLK3 in the VCaP cell line and the high positivity of patients for AR-V7 in comparison to AR-FL and KLK3. Moreover, we will include the AdnaTest ProstateCancerPanel ARV7  as a “gold standard” for AR-V7 detection [Similarly, background positivity for nscripts . In thisIn our pilot study, in situ padlock probe single cell analysis revealed heterogeneous expression patterns of individual CTCs. CTC single-cell analyses are promising tools to monitor clonal evolution and the emergence or disappearance of resistant clones. Furthermore, CTC heterogeneity has been described as poor prognostic marker for response to pathway-specific drugs such as ARSIs. Non-pathway-specific taxane-based chemotherapy on the other hand is less affected by CTC heterogeneity . TherefoAR amplification and AR-V7 splice variant. Interestingly, we observed that AR amplification can co-occur with CTC-AR-V7 and CTC-AR-FL expression. In this context, it is not fully understood how AR splicing is regulated. One hypothesis suggests that high availability of AR mRNA might be the predominant factor for the generation of AR-V7 [AR gain might be a basic mechanism increasing AR-FL expression and, hence, the presence of AR-V7 mRNA. In contrast, a recent study describes dysregulation of splicing-regulatory genes as the driving force for AR-V7 expression [AR-FL-negative but AR-V7-positive CTCs. Furthermore, it is well-documented that both transcripts are co-expressed and that the translated proteins AR-V7 and AR-FL can co-localize and act as agonists inside the cell nucleus [AR-FL and AR-V7 might be a growth advantage for tumor cells. While we observed the co-occurrence of two resistance mechanisms, it was shown by others that AR gain and AR mutations conferring resistance are described to be inversely correlated [We investigated two common AR-dependent resistance mechanisms, namely of AR-V7 ,39,40. Opression  which co nucleus ,43. Therrrelated .AR amplification and AR mutations as well as AR-independent resistance mechanisms [AR mutations, next-generation sequencing also allows for the detection of AR-independent resistance mechanisms such as treatment-induced neuroendocrine small cell PC [AR-V7 has become one of the leading targets of PC research, as various studies suggested that a CTC-based AR-V7 status may serve as a biomarker for treatment selection in CRPC patients ,11,12,29chanisms . Our stu cell PC ,45. Dete cell PC .This project was designed as a pilot proof-of-principle study, therefore only a small number of patients were included. Moreover, not all three assays could be performed with all patients due to limited availability of blood samples. Indeed, a limitation of our multi-analyte approach is the need for two different types of blood collection tubes to enable optimal stabilization of RNA (PAXgene Blood RNA tube) and DNA (PAXgene Blood ccfDNA tube), adding up to a total volume of 12.5 mL of blood. Immediately after the collection of blood samples, the CellCollector for in vivo CTC isolation is inserted into the patients’ vein through the intravenous catheter. Thus, no additional blood sample or second venipuncture is needed for CTC analysis. The inclusion of the AdnaTest ProstateCancerPanel ARV7 and ctDNA-mutation analyses, which is planned for our next study, does not necessarily require additional blood samples as several studies have demonstrated the feasibility of ctDNA isolation and analysis from immunomagnetic CTC-depleted blood ,48.In the present study, we did not discover a potential correlation between liquid biopsy markers and change in PSA or overall survival. This may both be caused by the small case number and the nonstandardized treatment approaches of patients included, with therapeutic regimens individually adapted. Moreover, the current study aimed at improving the diagnostic sensitivity of liquid biopsy approaches for tumor-related information and was not designed to investigate the influence of liquid biopsy markers on patient outcome.A total of 19 CRPC patients with high tumor burden and disease progression were enrolled in the study at the Division of Clinical Oncology, Medical University Graz (Austria). The study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the ethical committee . A written informed consent was obtained from all patients and healthy donors. Blood samples were collected in PAXgene blood ccfDNA tubes (10 mL blood) , PAXgene blood RNA tubes (2.5 mL blood) , and VACUETTE blood collection tubes K3E K3EDTA (9 mL blood) . Plasma was extracted from PAXgene blood ccfDNA tubes . PAXgeneThe PC cell lines VCaP  and PC-3 (American Type Culture Collection (ATCC), Manassas, VA, USA) were used as positive and negative controls for in situ padlock probe assays. Cell lines were cultured and seeded on glass slides as described previously ,34. SeedAR-V7, AR-FL, and KLK3 in PBMCs, VCaP, and PC-3 cells fixed on glass slides and cells captured on the CellCollector was performed as described by El-Heliebi et al. [AR-V7, AR-FL, and KLK3 transcripts were counted manually. In contrast, cells on glass slides were analyzed semi-automatedly using the image analysis software CellProfiler [KLK3 RCPs/cell, ≥2 AR-V7 RCPs/cell or ≥3 AR-FL RCPs/cell were rated as CTCs.In situ padlock probe analysis for the detection of PC-related transcripts i et al. . In shorProfiler . SignalsAR-V7 and KLK3 transcripts in whole blood, RT-qPCRgene expression assays were performed [AR-FL assay and TaqMan probes for AR-V7, AR-FL, and KLK3 were provided by ViennaLab Diagnostics . To allow for normalization, GUSB was included in the gene expression assays as an internal reference [In parallel to the in situ approach, we aimed to detect the same tumor-related transcripts in whole blood. For the detection of erformed ,53. Primeference . PAXgeneGene-specific preamplification contained SsoAdvanced PreAmp Supermix , primers and TaqMan probes at a final concentration of 50 nM, and 5.00–6.25 µL cDNA in a total volume of 25 µL. Enzyme activation and initial denaturation at 95 °C for 3 min was followed by ten cycles at 95 °C for 15 s (denaturation) and 58 °C for 4 min , and finally cooling to 4 °C.qPCR was performed using the RealFast 2x Probe Mix  primers and TaqMan probes at a final concentration of 250 nM, and 1.8–4.5 µL of preamplified template in a total volume of 20 µL. Enzyme activation at 95 °C for 2 min was followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s.AR-V7, AR-FL, and KLK3 were normalized to GUSB, yielding the respective relative ΔCq values. Technical replicates were averaged (mean) and lowest mean ΔCq value among the negative control samples was set as threshold for positivity as previously described in Todenhöfer et al. [Sixteen patient samples and 16 samples of healthy (negative) controls were analyzed by probe-based gene expression assays. Cycle of quantification (Cq) values of AR focal amplifications, sWGS, i.e., plasma-Seq, was performed [AR amplifications were called as described previously in Ulz et al. [To detect erformed . Brieflyerformed . The fraerformed . IchorCNz et al. . Brieflyz et al. .p-values were provided. A p-value of <0.05 was considered statistically significant.Statistical analyses of patient data were performed with Stata SE Version 15.1 . Means and medians were reported with standard deviations (SDs) and IQR, respectively. Change in PSA was calculated taking the best PSA response within 12 weeks, multiplied by 100, divided by the PSA value at baseline. Subsequently, univariate linear regression models were calculated to assess a potential impact of liquid biopsy markers on PSA response. Univariate Cox regression models were calculated to assess an influence of liquid biopsy markers on patient overall survival. Hazard ratios (HR), 95% confidence intervals (95% CI) and AR-V7 with high sensitivity and in addition allowed for enumeration of CTCs and assessment of intrapatient CTC heterogeneity, thereby providing prognostic and potentially predictive information in 53% of the patients. On the other hand, the in situ padlock probe approach is technically more challenging than the whole-blood RT-qPCR approach and the detection of AR-V7 is limited to CTCs. (ii) The whole-blood RT-qPCR approach increased the cumulative overall sensitivity of the multi-analyte approach. However, sensitivity for AR-V7 was low and AR-FL and KLK3 transcripts alone are less relevant for treatment decisions. Therefore, sensitivity needs to be further improved for informative targets such as AR-V7. Nevertheless, this whole blood RT-qPCR approach is very attractive due to its low cost, little technical requirements, and the potential to detect tumor RNA not only from CTCs but other sources as well. (iii) Although ctDNA-based plasma-Seq cannot detect AR-V7 and other mRNA markers, inclusion of the plasma-Seq approach further increased overall sensitivity and provided additional information on therapy resistance. Based on AR amplification, which cannot be detected by the mRNA-based approaches, we identified three resistant patients that would have been overlooked otherwise. Moreover, the co-occurrence of two distinct resistance mechanisms that we observed in some patients may be clinically relevant. Overall, the combined multi-analyte liquid biopsy approach showed increased sensitivity, providing tumor-related information in 89% of CRPC patients. We recognize the limitations of our pilot study and will further improve this multi-analyte approach by optimizing our RT-qPCR and in situ padlock probe assays, and by adding mutation analyses and the AdnaTest ProstateCancerPanel ARV7. Moreover, it is crucial to collect specimens at several timepoints longitudinally in a future study to better understand the evolution of resistance mechanisms over time.In our pilot study we assessed a multi-analyte-based approach capable of detecting tumor-related information for its use in a future clinical study. Each liquid biopsy approach has its strengths and limitations summarized as follows: (i) The in situ padlock probe analysis detected Amin El-Heliebi, In vivo collection and localized quantification and profiling of circulating cells, proteins and nucleic acids. WO2017081049 A1. Patent pending.


To investigate the hyperreflective foci (HRF) as an inflammatory biomarker using optical coherence tomography angiography (OCTA) in neovascular age-related macular degeneration (AMD) patients after antivascular endothelial growth factor (anti-VEGF) treatment and its association with the retinal microcapillary density.  Twenty-five eyes from 25 patients with neovascular AMD were included in the study. All eyes were imaged with OCTA at baseline (M0) and after 3 consecutive injections  of anti-VEGF. The number of HRF in the superficial capillary plexus (SCP), deep capillary plexus (DCP), and outer retina was counted. The vascular density of the fovea, parafovea, and the whole macula, as well as the area of the foveal avascular zone (FAZ), was measured. P < 0.01) and outer retina  after treatment. There was no significant difference for HRF number in the SCP, the vascular density , and FAZ area before and after treatments.  The mean interval between baseline and follow-up with OCTA was 93.08 ± 5.00  days. Compared with the baseline, the number of HRF significantly decreased in DCP (7.52 ± 3.06 vs. 3.76 ± 1.48,  The number of HRF in DCP and outer retina might serve as an inflammatory biomarker in patients with neovascular AMD. The reduced HRF possibly represents the alleviation of inflammation after anti-VEGF treatment in patients with AMD. Age-related macular degeneration (AMD) is the main cause of irreversible severe vision loss in elderly individuals, with a prevalence that exponentially increases with aging. It has been estimated that about 288 million people worldwide will suffer from AMD by 2040 . AMD is μm [With the advancement of noninvasive retinal imaging technologies, such as optical coherence tomography (OCT), the hyperreflective foci (HRF) can be imaged, which attracted the researchers' attention , 5. HRF μm , 7. HRF μm . Subsequμm –11. AlthNowadays, many studies that support the hypothesis that activated microglial cells induced by an inflammatory response in the retina are a pivotal origination of HRF , 13. MicIn this study, we investigated whether HRF could serve as a possible biomarker of retinal inflammation in patients with neovascular AMD and its possible association with retinal microcapillary density. The number of HRF, vascular density in different retinal layers, and the foveal avascular zone (FAZ) area was measured and quantified with OCT angiography (OCTA) in neovascular AMD patients before and after 3 consecutive anti-VEGF injections.In this retrospective study, we enrolled 25 consecutive treatment-naïve eyes from 25 neovascular AMD patients who were diagnosed with comprehensive ophthalmologic examinations in the Department of Ophthalmology, Shanghai General Hospital affiliated to Shanghai Jiao Tong University, Shanghai, China, between January 1, 2020, and July 30, 2020. The participants with three consecutive monthly intravitreal injections of anti-VEGF agents were enrolled. This study was approved by the Clinical Research Ethical Committee of Shanghai General Hospital affiliated to Shanghai Jiao Tong University (Permits No. 2020KY205) and adhered to the principles of the Declaration of Helsinki. All individual participants provided written informed consent.The exclusion criteria were as follows: (a) previous treatments for neovascular AMD ; (b) other vitreoretinal diseases, including dry AMD, vitreous hemorrhage, subretinal fibrosis, and retinal vascular disease; (c) high myopia ; and (d) history of ocular surgery, uveitis, and apparent media opacification.All participants underwent full ophthalmologic examinations, including best-corrected visual acuity (BCVA), intraocular pressure (IOP) measurement, and anterior segment evaluation using slit-lamp biomicroscopy, fundus photography, and OCTA imaging at the baseline (M0) and 1 week after treatment (M3).One experienced surgeon performed all intravitreal injections. The intravitreal injection was performed at the temporal limbus through the eyeball's pars plana under aseptic conditions. All patients received three once-monthly intravitreal injections of aflibercept (2 mg/0.05 mL) using a 30-gauge needle. A 1-week variation was allowed for every injection interval.2 sections centered on the fovea. Enface images of the superficial capillary plexus (SCP), deep capillary plexus (DCP), and FAZ area were automatically recorded and analyzed by the OCTA autosegmentation software. The SCP was segmented as 3 μm below the internal limiting membrane and 15 μm below the inner plexiform layer (IPL); the DCP was set between 15 μm and 70 μm beneath IPL, while the outer retina was located beneath the DCP. In the SCP and DCP, two central circles divided capillary plexus into the “fovea area” (a disc with a 0.6 mm diameter) and “parafoveal area”  as shown in Retinal microvasculature was imaged using the RTVue XR Avanti OCT system , and measurements were acquired using the manufacturer's AngioVue software. The scan covered an area of 3 × 3 mmμm to 50 μm range in order to exclude small counting noise signals as HRF noise signals and prevent large hyperreflective clumps, which are presented as typical hard exudates in fundus photography. Poor quality images with a signal strength index < 4/10 were excluded from further analysis. The quantification of HRF was carried out by two experienced physicians independently.The number of HRF was manually counted within a 3 mm diameter centered on the foveal area using a fovea-spanning horizontal B-scan. HRF were subdivided into 3 categories according to different retinal layers: the SCP, the DCP, and the outer retina, as described above. The maximal diameter of HRF was limited within the 20 t-test was used to compare the number of HRF, the vascular density of SCP and DCP, and the FAZ area between the baseline (M0) and after 3 consecutive monthly anti-VEGF injections (M3). P value <0.05 was considered as statistical significance.The data were analyzed by using SPSS 22.0 software. All values are presented as a number or mean ± standard deviation. The visual acuity was presented as the logarithm of the minimum angle of resolution (logMAR). A paired Twenty-five eyes from 25 patients  with neovascular AMD were consecutively enrolled in the study, as shown in P < 0.01) and outer retina , compared to baseline; the representative images of HRF were shown in P = 0.12).Analysis of the OCTA parameters revealed that the HRF was distributed across all retinal layers, including SCP, DCP, and outer retina, in neovascular AMD patients. As shown in Regarding retinal microvascular density in the SCP and DCP analyzed with OCTA, no significant difference was found for the microvascular density in the foveal, parafoveal, and whole macular sectors before and after treatment as shown in 2  and 0.31 ± 0.08 mm2 , respectively, with no statistically significant differences (P = 0.11).The distribution of FAZ areas in the retina is depicted in Microglial cells are considered to have a vital role in the inflammatory response in the pathogenesis of AMD , 17, 18.The introduction of high-resolution OCTA has facilitated the study of different capillary plexus in vivo. Previous studies have suggested that HRF may be used as a marker of inflammation in AMD patients , 20. It In our study, the HRF was localized in almost every retinal layer, especially the outer retina of patients with neovascular AMD, indicating the migration and aggregation of microglia cells in the outer retina. Our results were consistent with the previous observations , 22, whiPrevious experimental studies have demonstrated that microglia-induced retinal inflammation is a relatively early process during AMD development, which precedes neovascularization, microvascular dysfunction, and retinal neurodegeneration . In thisPrevious studies have also indicated that HRF could be an inflammatory hallmark, contributing to retinal dysfunction in many retinal diseases. For example, in neovascular diabetic retinopathy, HRF was significantly higher in diabetic patients compared with healthy controls . FurtherHsia et al.  comparedThe present study suggested that HRF could be used as an indicator or sensitive biomarker for retinal inflammation in neovascular AMD. After anti-VEGF treatment, the reduced HRF number might indicate the alleviation of inflammation in the retina and, possibly, improvement in the visual function in patients with neovascular AMD. Yet, long-term observation and a study with a larger sample size should be performed to compare the relationship among HRF, visual outcome, intraretinal cysts, and similar.


The World Health Organization (WHO) launched an initiative in 2017 to promote universal access to rehabilitation when it hosted “Rehabilitation 2030: A Call for Action” [The WHO convened a Second Global Rehabilitation 2030 Meeting in July 2019 that brought together stakeholders, including member states, international and professional organizations, nongovernment organizations, rehabilitation service users, and rehabilitation experts— including journal editors. The first 2 days included presentations on the current state of affairs of rehabilitation services in various countries and efforts made by several member states to integrate rehabilitation into their health care systems. Further, participants discussed strategies to make rehabilitation a political priority.Over the next 2 days, the WHO’s staff, together with the Alliance for Health Policy and Systems Research, initiated a planning process for a health policy and research agenda related to rehabilitation. The objectives of this companion meeting were to (1) agree on a health policy and systems research framework for rehabilitation; (2) identify initial research questions; and (3) delineate enablers and barriers to building health systems and policy research capacity in rehabilitation.Alarcos Cieza, PhD, the WHO’s Coordinator for Blindness and Deafness Prevention, and Disability and Rehabilitation, wrote in an article titled, “Rehabilitation the Health Strategy of the 21st Century, Really?” [Her message highlights the critical work undertaken by the WHO to enhance access to rehabilitation, particularly in low- and middle-income countries. Accessible and affordable rehabilitation services are critical for people with chronic health conditions to maintain or increase their independence, participate in their communities, improve their economic productivity, and enhance their quality of life.The aging population not only represents a major challenge to high-income nations but to low- and middle-income nations as well. The comorbid increase in the prevalence of chronic conditions and the aging of the world’s population contribute to an increasing number of people who experience declines in functioning. It is now recognized that optimizing functioning at all ages is a major global public health goal. Recent estimates of the global effect of rehabilitation on persons with health conditions is estimated to be much higher than the 1 billion people in the World Report on Disability published in 2011 . RehabilIt is now essential to include data on functioning in health information systems in addition to the typical morbidity and mortality outcomes. These data can help us make better-informed decisions for the increased demand of rehabilitation services to enhance function as well as assist in the planning of health care systems for future expansion into these types of services.It is evident to us that rehabilitation must be integrated fully into a nation’s health system and be strengthened specifically at the primary care level in order to increase access and achieve its full potential. As the WHO highlighted, we agree that health systems must be strengthened to assure that everyone who needs rehabilitation receives it. Equity should be a fundamental goal regardless of one’s social, economic, demographic, or geographic situation. WHO member states must also find solutions to the paucity of trained rehabilitation professionals and mechanisms to pay for the implementation of such services.As editors-in-chief of rehabilitation journals, we unanimously accepted the invitation to participate in WHO’s Rehabilitation 2030 meetings and we embrace the concept of function as WHO’s third health indicator  along wi--Archives of Physical Medicine and Rehabilitation, Journal of Cancer Survivorship, American Journal of Physical Medicine and Rehabilitation, Journal of Prosthetics and Orthotics, Journal of Cardiopulmonary Rehabilitation and Prevention, European Journal of Physical and Rehabilitation Medicine, The American Journal of Occupational Therapy, Canadian Journal of Occupational Therapy, BMC Health Services Research.In order to encourage its wide dissemination this article is freely accessible on the following journal websites: --


Coronavirus disease 2019 (COVID‐19) has widely spread all over the world and the numbers of patients and deaths are increasing. According to the epidemiology, virology, and clinical practice, there are varying degrees of changes in patients, involving the human body structure and function and the activity and participation. Based on the World Health Organization (WHO) International Classification of Functioning, Disability and Health (ICF) and its biopsychosocial model of functioning, we use the WHO Family of International Classifications (WHO‐FICs) framework to form an expert consensus on the COVID‐19 rehabilitation program, focusing on the diagnosis and evaluation of disease and functioning, and service delivery of rehabilitation, and to establish a standard rehabilitation framework, terminology system, and evaluation and intervention systems based the WHO‐FICs. The “Novel Coronavirus Pneumonia Treatment and Treatment Plan ”According to the World Health Organization (WHO), as of 10 Based on the WHO’s International Classification of Functioning, Disability and Health (ICF) and its biopsychosocial model of functioning and disability, and relevant assessments, we use the WHO Family International Classifications (WHO‐FICs)This consensus focuses on a series of objectives as follows: (1) the establishment of a rehabilitation framework, terminology, and evaluation and intervention approach in accordance with WHO‐FICs standards; (2) the integration of rehabilitation services into the continuum of health services for prevention, treatment, rehabilitation, and health promotion in response to the rehabilitation needs of COVID‐19 patients; (3) the provision of multidisciplinary, interdisciplinary, and lifespan rehabilitation services to patients in medical institutions, rehabilitation institutions, and communities; (4) improvement of the quality and safety of rehabilitation services; (5) collection of big data on rehabilitation; and (6) the provision of an evidence‐based foundation for the establishment of clinical practice recommendations for the rehabilitation of COVID‐19 cases.22.1The WHO‐FICs include three reference classifications: the International Classification of Diseases (ICD),[Consensus 1] This consensus adopts the framework and approach of WHO‐FICs to build a rehabilitation protocol of COVID‐19 disease diagnosis, description and evaluation, coding and intervention of functioning , the Brief Model Disability Survey, and the Generic functioning domains (VB40). The functional evaluation based on the ICF, such as ICF‐Core set, can be used for patients’ overall functional assessment, rehabilitation‐needs assessment, and rehabilitation‐outcome assessment. This consensus recommends the use of these three standardized assessment tools in rehabilitation evaluation. The qualifiers of ICF can be used to standardize the results of functional assessment in the field of rehabilitation to comparable achieve comparable international functional data.According to the ICF, this consensus also recommends all evaluations mapped to ICF structure involve in four aspects: body function and structure, activity and participation, environmental factors and personal factors.According to the framework and scope of rehabilitation developed by the International Society of Physical and Rehabilitation Medicine (ISPRM white paper), ICF and ICHI β‐2, we develop a personalized intervention plan based on specific unmet needs of patients with COVID‐19.[Consensus 3] According to the menisfestion of COVID‐19 and related functioning, experts mainly recommend rehabilitation interventions related to lung function, physical activity, and psychological functions, which are widely used in chronic obstructive pulmonary disease (COPD). There were many recommendations in the rehabilitation clinical practice guidelines, and many studies provided relevant clinical evidences. The consensus also considers the environments in which interventions are applied, such as medical institutions, rehabilitation institutions, and communities, as well as the rehabilitation stage of patients. The clinical manifestation report from patients and the autopsy report for COVID‐19 are also provided evidences for this consensus.Psychological intervention is an important part of comprehensive rehabilitation, which is of great significance for people with major diseases or functioning. This consensus also refers to the rehabilitation recomendations of psychological disorders caused by major disability, such as post‐traumatic stress disorder and spinal cord injury, as well as the WHO’s relevant guidelines and recommendations on natural disasters and COVID‐19, and develops psychological rehabilitation recommendations for patients with COVID‐19.2.2.1In light of the WHO rehabilitation guideine: Rehabilitation in health system, rehabilitation for COVID‐19 survivors should be provided at tertiary‐, secondary‐, and primary‐care levels and integrated into the continuum of prevention, treatment, rehabilitation, and health promotion.[Consensus 4] According to the recommendations from WHO guidelines Rehabilitation in health system, considering the functioning caused by COVID‐19, including mental health issues and environmental support factors, we should implement the people‐oriented rehabilitation throughout lifespan and concerns social determinants, adopt multidisciplinary and cross‐domain approaches, and with approaches of universal design to bulid barriers‐free environments and to establish a comprehensive rehabilitation service system.33.1The new coronavirus is a coronavirus of β genus, with a capsule, round or oval particles, often pleomorphic, with a diameter of 60‐140 nm.The novel coronavirus is the main source of infection. Asymptomatic infections can also be a source of infection. The main route of transmission is through respiratory droplets and close contact. It is possible to propagate through aerosols when exposed to high‐concentration aerosols for a long time in a relatively closed environment. In addition, the existing data cannot exclude the possibility of fecal oral transmission. People are generally susceptible to the virus. Based on the current epidemiological survey, the incubation period is 1‐14 days, mostly 3‐7 days, and the longest period may be 24 days.3.2The results of a lung biopsy and autopsy in a COVID‐19 patient showed that the patient had pleural effusion, pleural thickening, and extensive and severe adhesion with the lung. The lung tissue showed dark red and gray white patchy changes in toughness, while a large amount of gray white viscous fluid overflowed in the lung tissue, and fiber cords were seen. White foam mucus was seen in the endotracheal tube, and mucus adhered in the lumen of the right pulmonary branch.3.3The main manifestations of patients are fever, dry cough, and fatigue. A small number of patients have nasal obstruction, runny nose, sore throat, myalgia, diarrhea, and other symptoms. Severe patients often have dyspnea and/or hypoxemia 1 week after the onset of the disease. Severe patients can rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction, and multiple organ failure. It should be noted that the course of severe and critical patients may be moderate to low fever, or even no obvious fever. Mild patients only show low fever, slight asthenia, and no pneumonia.It is necessary to establish a multi‐disciplinary rehabilitation team for patients with COVID‐19. According to the functioning characteristics and the rehabilitation stage of patients, individualized rehabilitation intervention strategies and approached are recommended.3.4Mild: The clinical symptoms are mild, and no pneumonia is found in radiology.Moderate: Fever, respiratory tract and other symptoms are present, and pneumonia is found in radiology.2)/fraction of inspiration O2 (FiO2) is <300 mm Hg (1 mm Hg = 0.133 kPa); (4) radiology shows that the lesions progress more than 50% in 24‐48 hours.Severity: Those complying with any of the following should be considered as having the heavy type: (1) when shortness of breath occurs, the respiratory rate is ≥30/min; (2) under the resting state, the oxygen saturation is ≤93%; (3) arterial oxygen partial pressure  respiratory failure occurs and requires mechanical ventilation; (2) shock; and (3) other organ failure occurs and requires intervention in intensive care unit.3.53.5.1WHO recommended to use U07.1 code for virus idendtified COVID‐19, and to use U07.2 code for virus not identified, including clinically‐epidemiologically diagnosed COVID‐19, probable COVID‐19, and suspected COVID‐19 cases in ICD‐10. In ICD‐11, the code for the confirmed diagnosis of COVID‐19 is RA01.0 and the code for the clinical diagnosis (suspected or probable) of COVID‐19 is RA01.1 (https://www.who.int/classifications/icd/covid19/en/).3.5.2A coding protocol of COVID‐19 with ICD‐10 had been issued by National Health and Health Commission and National Health Insurance Bureau.The “U07.100” code is used for novel coronavirus as a statistical code for all confirmed COVID‐19 cases. It is not used as a main diagnostic code for the front page of the medical record.The “U07.100x001: Novel coronavirus pneumonia” code is only applicable to the “confirmed inpatients with new coronavirus pneumonia” and must be used as the main diagnostic encode.The code is defined as follows: (1) It can only be used for confirmed cases of COVID‐19. For suspected cases, one of the following examples of etiological evidence is required: positive real‐time fluorescence RT‐PCR detection of new coronavirus nucleic acid; and/or virus gene sequencing highly homologous with the new coronavirus. Note: The novel coronavirus infection diagnosis is not limited to respiratory tract specimens or blood samples. Urine, stool, and conjunctiva specimens can be used as diagnostic evidence. The specific diagnostic criteria are detailed in the Novel coronavirus pneumonia treatment and treatment plan . (2) It can only be used for a confirmed COVID‐19 case in hospital, excluding outpatient, observation, shelter, home quarantine cases, etc.The “U07.100x002: Novel coronavirus infection” code is only applicable to “confirmed inpatients (excluding new coronavirus pneumonia)” and is used as the main diagnostic code.Code definition: (1) The novel coronavirus nucleic acid test is positive, but did not induce pneumonia in “atypical” patients. For example, no novel coronavirus pneumonia‐related clinical manifestations, or symptoms of gastrointestinal symptoms , heart‐related symptoms , or comprehensive neurological symptoms (such as headache) are present. (2) The case had been diagnosed with the novel coronavirus pneumonia and is in hospital, excluding outpatient, observation, shelter, and home quarantine cases.The “U07.100x003: Suspected case of novel coronavirus pneumonia” code is only applicable to suspected cases of new coronavirus pneumonia in hospital.Code definition: (1) The case is a new coronavirus or nucleic acid or gene test negative, and conforms to any one of the following epidemiological histories or any two clinical manifestations, or no clear epidemiological history, accompanied by any of three clinical manifestations. (2) The case is a suspect case with novel coronavirus pneumonia for hospitalization, excluding outpatient service, observation, shelter, home quarantine cases, and so on.The “Z03.800x001: Observed case of novel coronavirus pneumonia” code is only applicable to patients who have been discharged from hospital, but had been inpatients because of the new coronavirus pneumonia symptoms or signs before. The code should only be used as other diagnostic encode.Code definition: (1) The patient must be considered as having novel coronavirus pneumonia, and accompanied by any one of the following epidemiological histories, or no significant epidemiological history, or any 1‐2 of the clinical manifestations. (2) The patient must have been excluded from having novel coronavirus pneumonia during the hospital stay, for example, two or more nucleic acid tests are negative. (3) The patient must be in hospital. They do not include outpatient service, observation, shelter, home quarantine cases, and so on.3.6[Consensus 5] COVID‐19 cases have secondary functioning and disability. Using the rapid extended ICF core set, we can describe, evaluate, and code the functioning of cases with COVID‐19. This rapid ICF core set developed from ICF core set for COPD. See Table 4[Consensus 6] According to the disease classification, functional status, and rehabilitation needs of patients with COVID‐19, the following rehabilitation assessment and evaluation are recommended.As novel coronavirus is highly infectious and highly pathogenic, any rehabilitation assessment and intervention must first ensure the safety of rehabilitation personnels and the reasonable use of protective equipment. Use of systematic and comprehensive functional assessment and evaluations is recommended. When equipment assessment is necessary, effective protective measures should be ensured. Elderly, severe, extreme severe and critical patients may be complicated with multiple basic diseases or direct invasion of multiple organs and systems. If necessary or after discharge, comprehensive assessment and evaluation are recommended to develop a rehabilitation plan to improve intervention and outcome.4.1We recommend the use of standardized functional assessment tools based on the ICF to control quality of rehabilitation medical treatment, standardize the reporting of outcome, conduct functioning‐related statistical analysis, and improve the comparability of evidence‐based data. There are many functional assessment tools in the fields of rehabilitation. The ICD‐11 recommends three assessment tools in the supplementary chapters: the WHODAS 2.0,The ICF core set is a widely used standardized tool. There is no ICF core set for COVID‐19. We recommend a rapid and extended ICF core set from ICF core set for COPD for evaluation and description of functioning and disability for COVID‐19 cases Table .4.2According to the COVID‐19‐cases‐related body functions and structures and the assessments and evaluation tools commonly used in the fields rehabilitation, we recommend the following assessments and evaluations.4.2.1(1) Evaluation of dyspnea: Modified Borg Scale for daily follow‐up.(2) Subjective fatigue assessment: Rating of Perceived Exertion.(3) Limb pain assessment: Visual Analog Scale and Oral Rating Scales.(4) Evaluation of anxiety and depression: Zung’s Anxiety/Depression Scales, Self‐rating Depression Scale and Self‐rating Anxiety Scale for regular follow‐up evaluation.4.2.2(1) Vital signs evaluation: Record the body temperature, respiration, pulse, blood pressure, blood oxygen saturation, urine volume, and other indicators regularly every day, and make relevant records before and after the intervention treatment.(2) Imaging evaluation: Chest X‐ray; if necessary, CT and color Doppler echocardiography may be used to evaluate the morphology of heart and lung and provide an objective basis for making a treatment plan.(3) Assessment of bone, joint and muscle: Bed rest or disease consumption would reduce intake, and other reasons may lead to the decreased function of skeletal muscle, soft tissue level, and joint stiffness. It can regularly monitor the limb muscle circumference, subcutaneous fat thickness, grip strength, active or passive range of motion. As a simple index, grip strength can be closely related to prognosis and can be widely used in patients.(4) Motor function evaluation: According to the stability of the patient’s condition, carry out balance function test in the early stage or when necessary, such as one‐leg closed‐eyes time, 10‐m walking test, 6‐minute walking test, daily walking speed test, and other comprehensive evaluation tests of cardiopulmonary function should be implicated when patients are in the recovery period, discharged, or undergoing follow‐up outside the hospital. A 6‐minute walking test or cardiopulmonary exercise test can also be implicated, if necessary. Strength, Assistance with walking, Rise from a chair, Climb stairs and Falls can provide more information, such as balance, coordination ability, and so on, but requires a higher functional level of patients.(5) Pulmonary function evaluation: Patients followed up outside the hospital  should be assessed for their lung‐function test, which is helpful to understand the recovery of ventilation function. In cases where patients may still have the ability to transmit the virus, the examination should be avoided. If necessary, an individual handheld pulmonary function instrument may be used. In addition, dynamic lung capacity can also be used for evaluation of the expiratory flow rate per unit time, which can better reflect airway resistance.Diaphragm function assessment: COVID‐19 may not directly invade the diaphragm. However, some patients may have insufficient diaphragm function and diaphragm fatigue due to factors such as increased ventilation demand, decreased efficiency, bed rest, consumption of disease, and intake reduction. Therefore, inspiratory peak flow rate, and ultrasound diaphragm activity and thickness can be used for diagnosis according to patient’s stability and treatment needs. Direct or indirect evaluation can provide more reference information for patients with decreased activity tolerance and difficulty in offline.4.3We recommend the WHODAS 2.0, activities of daily living (ADL) assessments, and the 36‐item Short Form Health Survey (SF‐36) to evaluate patients’ activities of daily life and participation.4.3.1The WHODAS 2.0 is a standardized activity and participation assessment tool recommended by the WHO.4.3.2According to the severity of the patient’s condition, ADLs should be evaluated regularly for mild, medium, severe, and discharged patients, including basic ADL evaluation and instrumental ADL evaluation for cases who return to the community after discharge. The improved Barthel Index or other instrumental ADL scales are recommended. For severe cases, we recommend observational evaluation.4.3.3As there are no assessment scales specific to COVID‐19, we recommend using the Medical Outcomes Study SF‐36 or the COPD Assessment Test in order to shorten the time of evaluation.4.4Considering that COVID‐19 cases may have different health conditions and will be at different rehabilitation stages, we recommend not only collecting data of vital signs, laboratory tests, and other information on disease, but also evaluating pulmonary function, subjective fatigue, dyspnea, pain, joint and muscle function, activity level, and quality of life tailored to patients’ health conditions. Those assessments should be carried out pre‐ and post‐rehabilitation. Only comprehensive and systematic rehabilitation data will provide evidence for the effect of rehabilitation in the intervention of infectious diseases, such as COVID‐19.5[Consensus 7] We developed a protocol of rehabilitation intervention based on the ICF tailored to COVID‐19 diagnosis, functional status, and unmet needs of rehabilitation.5.1Within the framework of the WHO‐FICs, rehabilitation service delivery for COVID‐19 cases can be implemented in hospitals, rehabilitation institutions, and communities . Rehabilitation measures may include: preventive, therapeutic, health‐promoting, and palliative care according to their intervention purposes.5.25.2.1The main clinical manifestations of COVID‐19 are respiratory dysfunction, with pulmonary consolidation and airway secretion obstruction.5.2.2At present, there is no clinical evidence for the optimal time of rehabilitation intervention for the disease. The benefits and risks of patients should be balanced and cross‐infection should be avoided. For patients with severe or critical illness, rehabilitation treatment should be carried out when vital signs are stable, and the changes of vital signs should be monitored throughout the process. Rehabilitation doctors and therapists should strengthen interdisciplinary communication, and devise and implement intervention treatment together with clinicians. During the operation, therapists can use the early warning scores or the Awakening and Breathing Coordination, Delirium Monitoring/Management, and Early Exercise/Mobility Bundle (ABCDE)5.3According to the patient’s condition, the therapist can use body‐position drainage, vibration and clapping, active cycle of breathing techniques, and other techniques or equipment to clear the airway, and should pay attention to the local humidification of the whole body and airway to reduce the viscosity of the sputum.5.4For patients with dyspnea, cough, and other symptoms, an upright sitting or standing position can increase diaphragm activity, improve the Ventilation/Perfusion in the lung, increase tidal volume, improve peak flow rate of cough, and reduce the sense of breathing difficulty. When the secretion of the respiratory tract increases, the therapist can adjust the posture according to the change of vital signs, and assist in expectoration. In the intensive care unit, an electric vertical roll bed is recommended to help patients adjust their position.For mild, medium, and heavy patients, and patients after discharge, reducing the time spent in bed is helpful to reduce the risk of various bed complications, promote the improvement of cardiopulmonary function, and shorten recovery time.5.5The threshold loading inspiratory muscle training device is the most commonly used method of respiratory resistance training at present. Generally, the initial load is 30% of the personal maximum inspiratory pressure. According to the training purpose (endurance training or strength training), a gradual increase in the training load or training times is recommended, along with training 1‐2 times a day, 15‐30 rounds each time (for endurance training), or 8‐12 rounds each time (for strength training).Respiratory control can significantly improve the experience of breathing, reduce respiratory‐related oxygen consumption, and relieve the tension of patients. Generally, deep and slow breathing is used to increase the compliance of the respiratory system, reduce the work of breathing, and relieve the dyspnea of patients. Diaphragmatic breathing or abdominal breathing also have similar effects. If possible, physical therapy can be used for training, such as an electrical stimulation diaphragmatic trainer, electronic biofeedback, and so forth.5.6Exercise training is the basis of various rehabilitation measures. It can not only effectively improve the functional level of the cardiopulmonary muscle unit, reverse the decline of disuse function, but also increase the compensatory ability of the noninvolved organs, and provide more functional reserves for the disease. At present, it mainly includes aerobic (endurance) training, resistance (strength) training, balance training, and coordination training. According to the evaluation results, the four elements  of exercise prescription and the three principles  should be followed in the formulation of the plan, and individualized and precise prescription should be formulated to avoid sports injury while pursuing benefits.5.6.1(1) Intensity: Patients in the acute stage mainly take low‐intensity exercise without fatigue on the second day. In general, patients in better condition can try medium‐intensity exercise. After discharge, patients can carry out medium‐ and high‐intensity exercise training according to further evaluation results to obtain more benefits.(2) Frequency: According to the patient’s tolerance, they should carry out active and passive training once or twice a day. If the patient’s condition is serious and the tolerance is poor, they should shorten the training duration and increase the frequency accordingly to ensure the training quantity.(3) Time: Patients with good state and those discharged from hospital can accumulate aerobic exercise time to 60 minutes every day, and those in good condition can increase the time moderately; for patients with heavy or critical disease, exercise should be accumulated to about 30 minutes every day, if possible.(4) Activity mode: A patient should carry out fast walking, slow walking, standing, sitting upright, and other sports modes according to the environmental conditions and their own condition.In aerobic training, it is best to monitor blood pressure, heart rate, and blood oxygen saturation.5.6.2(1) Intensity: Patients in the acute stage should take low‐ and medium‐ intensity load training with the goal of maintaining muscle mass or delaying muscle atrophy. If possible, they can start from 30% to 40% of 1 RM, and if not, they can use 1‐5 kg. According to the patient’s tolerance degree and the change of the condition, the load or bodyweight training should be gradually increased; after discharge, the patients may use medium‐ and high‐intensity load training with the goal of recovering or increasing muscle mass. The effective prescription for improving muscle strength was 65% 1 RM for three groups, three times a week. Load training for patients with novel coronavirus pneumonia should be reduced as appropriate.(2) Frequency: Low‐ and medium‐intensity load trainers can try 1‐2 times a day, and medium‐ and high‐intensity load trainers can try one time every other day.(3) Methods: Progressive resistance training is a commonly used program. Resistance load can come from self‐weight, external resistance . There are many application videos of elastic band resistance training on the internet, which can be used to carry out alternating and repeated training of large muscle groups of limbs.5.6.3Long‐term bed rest can lead to joint stiffness, contracture, and other changes. Patients should be guided to actively carry out active and passive motions of the spine and limb joints to maintain their normal range of motion, which can be arranged 1‐2 times per day and can be completed by joint and position. Severe patients may not be able to complete the active whole joint motion effectively. At this time, they may need other people or special equipment to carry out the passive motion of joints, including the limbs, head, neck, and waist, to reduce the risk of deep vein thrombosis.5.7(1) Ultrashort wave therapy: Ultrashort wave therapy can promote the exudation and absorption of the lung and improve ventilation function. It can be used for patients with lung exudation and the specific prescription is mainly short‐term with micro or without heat; however, fever is a contraindication. Small ultrashort waves can be used for bedside treatment to reduce the impact of high‐frequency electromagnetic fields on monitoring equipment, but the ultrashort wave machine, its electrodes, and wires should be disinfected and protected according to the protection level.(2) Ultraviolet therapy: Whole‐body ultraviolet irradiation can increase immunity function, which may be applicable to mild and medium patients, but severe and critical patients may have immune disorders, so it is not recommended.(3) Low‐frequency neuromuscular electrical stimulation therapy: Neuromuscular electrical stimulation can improve the respiratory muscle and peripheral muscle function, so it can be used in bed patients for respiratory muscles or limb muscles, to delay muscle disuse atrophy, and to increase neuromuscular function.5.8Deep‐breathing control is a special form of training. It can be skillfully mastered by patients in a short period of time through a series of carefully choreographed action routines  and with the help of words, pictures, videos, and other communication methods, which is very suitable for patients. During breathing training, it is necessary to pay attention to the coordination of diaphragm movement with trunk and limb movement so that diaphragm‐function training, breathing‐mode training, and body and joint training can be carried out at the same time.5.9For some COVID‐19 cases, there may be some negative stress responses, mainly emotional disorders, such as panic, anxiety, and somatization symptoms, which affect the mood, state, sleep and overall mental health level.For hospitalized patients with emotional disorders, such as anxiety or depression, we recommend: the implementation of psychological intervention as early as possible (including cognitive therapy and behavior therapy); the elimination of stressors; the improvement of patients’ anxiety or depression; establishing a positive and optimistic mood; and seeking support from families, medical staff, and psychologists. In addition to psychological and behavioral interventions, drug therapy and biofeedback therapy can also be used in the treatment of severe anxiety or depression.For COVID‐19 patients who receive rehabilitation in the community, we recommend: the establishment of a psychological support service system; the relief of panic about infectious diseases at the community level; the establishment of a psychological support and assistance system between community members, family members and patients; and the provision of special psychological services for those who suffer critical psychological events in the pandemic situation, such as the death of family members, to ensure that patients will not suffer from serious psychological obstacles that reduce their quality of life.In view of the psychological problems caused by COVID‐19, we recommend to provide mental health services for patients and their families, assist them to acquire and understand the correct information about the pandemic and the impact of COVID‐19, prevent them from panic and long‐term stress state, and help them to establish a positive lifestyle and behaviors.5.10taijiquan, wuqinxi, baduanjin, and other physical activities is helpful to regulate breath, dredge meridians, and improve stability of the core muscle group and balance ability. These activities are especially suitable for elderly or weak patients with low physical abilities and can be carried out by mild, medium, and discharged patients, using group mutual aid mode or at home through video exercises.Participating in 5.11We recommend the following criteria for interruption of intervention for COVID‐19 cases with slight and medium functioning if one of the following conditions occurs: (1) there is obvious fatigue or a Borg Dyspnea Score >3, which cannot be relieved after rest; or (2) chest tightness, suffocation, dizziness, headache, unclear vision, palpitation, sweating, inability to maintain balance, and so forth.For COVID‐19 cases with one of the following conditions, we recommend the immediate cessation of intervention: (1) Respiratory system: blood oxygen saturation ≤90% or more than 4% lower than the baseline value; respiratory frequency >40 times/min; dyspnea or shortness of breath, aggravation, fatigue, and intolerable fatigue. (2) Cardiovascular system: systolic pressure <90 mm Hg or >180 mm Hg; mean arterial pressure <65 mm Hg or >110 mm Hg, or more than 20% change from baseline; heart rate <40 times/min or >120 times/min; new arrhythmia and myocardial ischemia. (3) Nervous system: poor state of consciousness.6[Consensus 8] Considering the importance of rehabilitation management and governance in modern rehabilitation services, we have developed a protocol of rehabilitation management and governance using the WHO‐FICs framework for COVID‐19 cases considering the functioning characteristics. This model is multidisciplinary and across sectors, covering health continuum of prevention, treatment, rehabilitation, and health promotion. COVID‐19 cases can receive rehabilitation services in hospitals, rehabilitation institutions, and community primary care institutions. These patients need to receive life span rehabilitation to improve their overall functioning and their quality of life. Community rehabilitation service is the main extended form of institutional rehabilitation service. Rehabilitation management is an important mechanism to ensure that patients receive high‐quality rehabilitation services from medical institutions, rehabilitation institutions, and community based service providers. We should build a patient‐centered rehabilitation service system.It is necessary to establish a multidisciplinary rehabilitation team for patients with COVID‐19. According to the functioning characteristics and the rehabilitation stage of patients, individualized rehabilitation intervention strategies and approaches are recommended.We should establish a comprehensive rehabilitation service system in all professional fields, integrating all levels of health services, and providing comprehensive rehabilitation services for COVID‐19 patients from clinical treatment to community rehabilitation services. In the community, we should especially emphasize the empowerment of patients, enhance their self‐confidence, and improve their overall functions and their quality of life.6.1For patients in different rehabilitation stages, we should analyze their unmet needs of rehabilitation, main functioning disability and rehabilitation resources from medical institutions, rehabilitation institutions and communities levels, and establish different rehabilitation solutions to improve the quality and cost‐effectiveness of rehabilitation.6.2We recommend: the establishment of a rehabilitation service platform; cooperation with experts in epidemiology and clinical medicine; and the integration of rehabilitation into the modern health‐service system. For special patient groups, such as the elderly, people with disabilities, and children, we strongly recommend the consideration of their special needs and obstacles, as well as multiple functioning and its impact on COVID‐19 rehabilitation.In the community, we recommend the all members pay attrention to the negative influences and discrimination against COVID‐19 cases. The proposed measures include the provision of correct information, the prevention of panic and bias, and the consideration of the psychological, social, and environmental factors in community rehabilitation services.Nothing to disclose.