Cross-reactivity of allergens is the cause of various, sometimes unexpected, clinical reactions. There are no standard methods to investigate cross-reactivity. We present an experimental model of a two-sided inhibition test (IT) on ImmunoCAP membranes (CAP). We constructed the described model based on the known cross-allergy syndrome to red meat developing in people bitten by ticks . Some individuals who are bitten by ticks develop IgE antibodies specific to the carbohydrate determinant, galactose-α-1,3-galactose , present in the tick’s saliva. These antibodies can cross-react with α-Gal molecules expressed on mammalian meat proteins. The well-known property of anti-α-Gal IgE antibodies binding by various sources of this allergen was used by us in the proposed model of the two-sided inhibition test on ImmunoCAP membranes. We expected that anti-α-Gal IgE antibodies bind allergens from mammalian meat and blocking them abolishes this reactivity, and the two-sided inhibition test model we proposed on ImmunoCAP membranes allowed us to observe such a relationship. We conducted the experiment three times on biological material from people with different clinical manifestations of allergy to α-Gal, each time obtaining similar results. In conclusion, the model of bilateral inhibition on ImmunoCAP membranes proposed by us seems to be an attractive, simple tool for direct testing of allergic cross-reactivity. The cross-reactivity of allergens is a result of the similar structure of their antigenic determinants, both their linear and spatial structure . It may Alpha-gal  is a carbohydrate present on glycoproteins in non-primate mammals and certain invertebrates. Primates, including humans, do not express the α-Gal epitope, as the -1,3-galactosyltransferase gene is inactivated in these species ,3,4. A lAGS can manifest as an immediate drug allergy to pharmaceuticals containing α-Gal and/or a delayed hypersensitivity response to the ingestion of mammalian meat products. The first references to an association between alpha-gal allergy and red meat allergy appeared around 2006 and concerned unusual allergic reactions in people treated with cetuximab. Later, it was noticed that people who had been bitten by ticks previously manifested similar symptoms after ingestion of the mammalian meat. In all cases, the common factor was the presence of IgE antibodies to the carbohydrate determinant galactose-α-1,3-galactose, referred to as α-Gal ,17.Although the mechanism for the huge variability of the clinical symptoms of AGS and the delay in response is not entirely clear, it is known that IgE antibodies specific for α-Gal resulting from primary sensitization to tick saliva may bind this carbohydrate determinant expressed on mammalian meat proteins ,20,21,22The aim of the study was to evaluate the usefulness of the experimental two-sided inhibition test (IT) on ImmunoCAP membranes for the study of cross-reactivity of allergens. In order to achieve the assumed aim of the work, an exemplary model based on the known reactivity of α-Gal specific IgE antibodies with mammalian meat allergens was used.Two-side ImmunoCAP inhibition tests with two allergens  were performed to assess the cross-reactivity between α-Gal antigen and mammalian meat allergens.The study was carried out by case-to-case analysis. The serum of three patients with positive results anti-α-Gal IgE and anti-beef IgE, negative results anti-poultry IgE and different clinical histories associated with meat consumption and different underlying atopic status was used in the experimental inhibition assay.Patient 1—A 65-year-old man was diagnosed with recurrent anaphylactic reactions, which he initially associated with the consumption of various types of alcohol. Clinical history revealed that alcohol consumption was always accompanied by a meal consisting of a meat dish. In addition, episodes of anaphylaxis also occurred in association with exercise immediately before or immediately after eating a meat meal. The first episode of anaphylaxis occurred about 9 years ago. The man showed no allergic reactions before this event. It was established that about a year before the first episode of anaphylaxis, the man was bitten by a tick. The species of the tick could not be determined. The reason for the determination of anti-α-Gal IgE antibodies in this patient was clinical symptoms related to meat consumption and the presence of specific IgE antibodies to mammalian meat allergens detected in the ALEX2 multiplex test.Patient 2—A 60-year-old man was treated at an infectious disease clinic due to skin erythema associated with a tick bite. The bite occurred 2 months earlier. In addition to a diagnosis appropriate to the clinical situation, the man was tested for the presence of anti- α-Gal IgE antibodies as part of another clinical study conducted by our team, which aimed to determine the frequency of these antibodies in people bitten by ticks. The man denied any symptoms of allergies, including meat allergies. He ate the meat regularly and had no negative effects.Patient 3—A 40-year-old woman diagnosed with oral allergy syndrome (OAS) symptoms after consuming a variety of fruits, vegetables and nuts, including sweet cherries, nectarines, cherries, apples, hazelnuts and almonds. The patient had previously been diagnosed with bronchial asthma and inhalation allergy to hazel, birch, grass, weeds, Alternaria, mites, cats and dogs. In the course of the conducted diagnostics, the patient was found to have IgE specific for mammalian meat allergens. The patient denies the symptoms of an allergy to meat, which she consumes regularly. The woman was not sure if she had ever been bitten by a tick. The patient was tested for IgE antibodies to alpha-gal due to the positive IgE results of mammalian meat allergens in the ALEX2 test.E112i  tests, and the concentration of IgE specific for mammalian meat allergens , gelatin and chicken meat .The following laboratory tests were performed in the blood serum collected from each patient: the concentration of specific IgE antibodies to α-Gal , wide-profile multiplex tests with the ALEX2  and ISACIn the sera, two-side inhibition tests were performed on two types ImmunoCAP matrices: o215  and f27 (beef meat extract). To assess the effectiveness of the inhibition test and the cross-reactivity between α-Gal and meat allergens, the following tests were performed in the material collected after the inhibition tests: specific IgE concentration for α-Gal and beef (ImmunoCAP) and the multiplex ALEX2 test.Each patient’s serum was processed separately. Serum pooling was not performed before or after the inhibition test.E112i test, ALEX2 test), and experimental two-side inhibition tests on ImmunoCAP matrices were used in the experiment.A standard, certified for in vitro diagnostic (CE IVD), laboratory methods  is the in vitro test system for the quantitative measurement of specific IgE in human serum or plasma. In the ImmunoCAP system, the FEIA (fluoro-immuno-enzymatic) method is use. The allergens covalently coupled to ImmunoCAP, reacts with the specific IgE from the patient sample. After washing non-specific particles enzymes labeled anti-IgE  are added to form the complex. Following incubation, unbound conjugate (enzyme-anti-IgE) is washed away, and the bound complex is then incubated with a developing agent . After stopping the reaction (by sodium carbonate 4%), the fluorescence of the eluate is measured. The higher the response value, the more specific IgE is present in the sample. To evaluate the test results, the responses for the patient samples are transformed to concentration with the use of a calibration curve, with a calibrator range of 0–100 kUA/L. The Phadia 100 system was used in the experiment.E112itest  is an enzyme immunoassay solid phase based on biochip (microarray) technology. Allergen components applied in triplets to a glass plate (slide) bind their specific IgE antibodies from the patient’s blood serum. The specific IgE bound in this way is then detected with fluorescently labeled anti-human immunoglobulin E (anti-human IgE) antibodies. Fluorescence is measured with a biochip scanner . The obtained images are then analyzed using Phadia Microarray Image (MIA) software. The ISACE112i is a semi-quantitative test, and specific IgE antibody levels are reported in ISAC Standardized Units (ISU-E) and are divided into four categories according to the level: <0.3 ISU-E (undetectable); 0.3–0.9 ISU-E (low); 1–14.9 ISU-E (medium/high); >15 ISU-E (very high). The CapitalBIO LuxSCAN system with PhadiaMIA software was used in the experiment.The ISACThe ALEX2  is an immunological, microarray test based on nanotechnology. The ALEX2 enables the simultaneous measurement of the concentration of specific IgE for whole allergen extracts, allergen molecules and total immunoglobulin E. The ALEX2 is a quantitative specific IgE and semi-quantitative total IgE test. The measured immunoglobulin concentration is expressed in kUA/L for specific IgE and kU/L for total IgE. The measuring range of the ALEX2 test for specific IgE is 0.3–50 kUA/L and for total IgE 1–2500 kU/L. The sensitivity of the test for specific IgE is 0.1 kUA/L. The results for specific IgE were expressed in classes 0–4, where individual classes were assigned the concentration of specific IgE, respectively: class 0 , class 1 , class 2 , class 3  and class 4 . The manual Image Xplorer system with Raptor software was used in the experiment.Singleplex and multiplex results were analyzed separately.We designed a three-step experimental procedure to evaluate the cross-reactivity between α-Gal and different mammalian meat allergens .As a source of allergens, we used two types of ready-made ImmunoCAP membranes: coated with α-Gal (o215) and coated with beef extract (f27) .The source of specific IgE antibodies were sera of patients with positive specific IgE antibodies to α-Gal (necessary condition) and beef (necessary condition) and various allergens of mammalian meat  and negative specific IgE antibodies to poultry meat allergens (necessary condition).The scheme of the experiment is presented in The elements of the ImmunoCAP system  were used in the experiment. The ImmunoCAP method is a standard method for the determination of serum specific IgE or total IgE concentration. The ImmunoCAP membrane is a cellulose matrix that is the solid phase in the ImmunoCAP method. The membrane placed in the plastic reaction cup is the CAP of the ImmunoCAP system. The ImmunoCAP membrane is coated with allergens or anti-human IgE antibodies. In the standard ImmunoCAP procedure, it binds antibodies to the serum.Step 1This stage was aimed at preparing immunocap membranes for binding IgE antibodies from patients with membrane allergens (solid phase), as in the standard ImmunoCAP procedure.g; 15 min) to remove the wash buffer from the CAPs. The CAPs prepared in this way were placed in clean Eppendorf tubes.In the first stage of the experiment, the ImmunoCAP membranes were pre-prepared. For this purpose, ImmunoCAPs (CAPs) were placed in Eppendorf tubes and 50 µL of the washing solution was added to each CAP. The washing buffer used in this step of the experiment is part of the ImmunoCAP system (Art. No. 10-9422-01). The buffer was prepared in accordance with the manufacturer’s instructions. The CAPs sealed in Eppendorf tubes were then centrifuged . After completion of centrifugation, the ImmunoCAP membranes were removed, and the collected filtrate was used in the next stage of the experiment (Step 3).In this stage of the experiment, we assumed that the anti-α-Gal IgE and anti-beef IgE antibodies present in the sera and other anti-mammalian meat IgE would be inhibited by α-Gal and/or beef antigens coated on the ImmunoCAP membranes. For this purpose, 40 µL of anti-α-Gal IgE and anti-beef IgE positive sera and anti-mammalian meat  were applied to the pre-prepared ImmunoCAP membranes. ImmunoCAP membranes with applied sera were tightly sealed in tubes and subjected to two-stage incubation: first 30 min at 37 °C and then 24 h in a refrigerator (2–4 °C). After incubation, the tubes containing the cups of ImmunoCAP were centrifuged (5000× Two types of filtrate were obtained for each sample: (A) the filtrate after the inhibition test on the ImmunoCAP f27  and (B) the filtrate after the inhibition test on the ImmunoCAP o215 .Because the volume of serum required for the third stage of the experiment was 210 µL (for each patient), 6 CAPs of ImmunoCAP o215  and f27 (beef-coated membrane) were prepared for each patient. A 240 µL volume of the final serum of each patient was obtained.Step 3In this stage of the experiment, the effectiveness of the performed inhibition tests: f27-IT (Step 3 A) and o215-IT (Step 3 B) was verified. For this purpose, the concentration of IgE antibodies specific for beef and alpha-gal in the collected filtrates using the ImmunoCAP technique was determined, and the ALEX2 multiplex test was performed.In the presented experimental inhibition test, we focused on blocking antibodies to mammalian meat allergens and to alpha-Gal, which is the result of cross-reactivity. The test should have no significant effect on the concentration of IgE specific for other allergens. Because we used the multi-parameter ALEX2 test in the experiment, it allowed us to detect the presence of IgE antibodies specific for various allergens in the patients’ sera. In this way, we obtained an individual sensitization profile for each patient, which was used as a control for inhibition specificity. We compared the results of the ALEX2 test performed before and after the inhibition test, focusing on changes in IgE concentrations specific to allergens other than mammalian meat. The comparison was made individually for each patient. Two non-parametric tests were used for the statistical analysis of the test results: the Wilcoxon test and the Friedman ANOVA test.Only the results of inhibition tests for IgE antibodies to mammalian meat allergens and a-Gal were analyzed. The very small sample size limits the interpretation of the results of this analysis. No statistical analysis was performed for the controls due to the individual nature of these results.All results were divided into four groups: results before ImmunoCAP inhibition tests (IT), results after ImmunoCAP inhibition test with α-Gal , results after ImmunoCAP inhibition test with beef  and control results. All of them are presented in The total serum concentration of IgE in each of the described patients was very high and, in each case, exceeded the value of 100 kU/L, which is considered the upper range of reference values for adults. The level of IgE was particularly high in Patient 1 (2602 kU/L) and Patient 2 (714 kU/L) .E112i. Positive results as measured by the ImmunoCAP monospecific test were proportional to those measured by the ISAC test in both Patient 1 (302 kUA/L vs. 12 ISU-E) and Patient 2 (35.8 vs. 0.3 ISU-E). The concentration of IgE anti-α-Gal in the serum of Patient 3 measured by the ImmunoCAP test was very low (0.7 kUA/L), which was reflected in the negative result of the semi-quantitative ISACE112i test for this parameter (<0.3 ISU-E)  IgE antibodies were found in the serum of every patient by the monospecific ImmunoCAP test. This was the first inclusion criterion for the experiment. The concentration of these antibodies in Patient 1 was extremely high and amounted to 302 kUA/L (quantification of the concentration required dilution of the serum). In two cases (Patient 1 and Patient 2) the presence of IgE antibodies against α-Gal was confirmed by multi-component ISAC3 ISU-E) .The ratio of anti-α-Gal IgE to total serum IgE  between patients varied widely, being 11.61% in Patient 1, 5.01% in Patient 2 and only 0.52% in Patient 3 .The presence of anti-beef IgE was the second inclusion criterion for the study. IgE antibodies to beef allergens  were detected in the serum of all patients, but their concentration varied. Although these antibodies were detected in each of the three tested patients by both the ImmunoCAP monospecific test and the ALEX2 multiparameter quantitative test, the anti-beef IgE concentrations obtained in both tests were not mutually proportional. In Patient 1, the concentration of anti-beef IgE in the ImmunoCAP test was high (26 kUA/L), whereas in the ALEX2 test it was only 2.03 kUA/L. The inverse relationship occurred in Patient 2, where the concentration of IgE anti-beef antibodies in the ImmunCAP test was significantly lower than that measured with the ALEX2 test (0.38 kUA/L vs. 3.18 kUA/L). In turn, in Patient 3, both tests obtained similar results for this parameter (ImmunoCAP vs. ALEX2: 0.51 kUA/L vs. 0.53 kUA/L) .All patients were also tested for the presence of IgE antibodies to meat allergens of other mammals  with the single-parameter ImmunoCAP tests. The IgE antibodies to these types of meat were present only in the serum of Patient 1. None of the examined patients had IgE antibodies specific for porcine gelatin. No antibodies to chicken meat were detected in any of the sera tested by the ImmunoCAP test .The allergenic profile to mammalian meat allergens obtained in the ALEX2 multiparameter test basically corresponded to the allergenic profile to these allergens obtained in the ImmunoCAP single-parameter tests only in Patient 1. In this patient, the presence of IgE antibodies in beef, pork and rabbit meat was confirmed by the ALEX2 test results. The presence of IgE antibodies to the mutton  has not been confirmed. Additionally, in Patient 1, the presence of horse meat IgE antibodies was detected with the ALEX2 test . In PatiNo IgE antibodies specific for the main allergenic components of beef (Bos d 6) and pork (Sus d 1) were detected by ALEX2 in any of the patients .The IgE antibodies specific for poultry meat allergens  were not detected in the serum of any of the Patients . This waThe decrease in total IgE concentration after the o215  ImmunoCAP inhibition test (o215-IT) occurred in the serum of each patient and ranged from 14.93% to 97.2% .The decrease in anti-α-Gal IgE antibody concentration from baseline was observed in the serum of each patient  .The anti-α-Gal IgE to total IgE ratio for Patient 1 and Patient 2 was higher than before the o215 ImmunoCAP inhibition test . However, in Patient 3, it was lower than the initial one (0% vs. 0.52%) .After blocking on an α-Gal(o215-IT)-coated ImmunoCAP membrane, a reduction in the concentration of IgE antibodies to beef allergens was observed in the mono-parameter test (ImmunoCAP) in each patient. The lower base concentration of anti-beef IgE antibodies corresponded to its higher percentage decrease after the o215-IT test .In the results of the ALEX2 test, no positive IgE antibodies were observed for beef allergens in all the tested sera after the o215-ImmunoCAP inhibition test .For the other mammalian meat allergens , a significant or complete decrease in serum IgE levels was observed compared to the initially positive results of these antibodies in individual patients .Antibodies to poultry and bovine serum albumin (Bos d 6) and porcine serum albumin (Sus d1) remained negative after the o215-ImmunoCAP inhibition test .A decrease in total IgE concentration after the ImmunoCAP f27 (beef) inhibition assay (f27-IT) was observed in the serum of each patient. It was lower than after the o215-IT test and ranged from 4.47% to 42.99% .After inhibition on a beef-coated ImmunoCAP membrane (f27-IT), a reduction in the concentration of IgE anti-α-Gal antibodies in the sera of all patients was observed. The reduction was lower than when inhibited on the ImmunoCAP membrane coated with α-Gal (o215-IT). The relationship between f27-IT vs. o215-IT was for Patient 1: 46.69% vs. 85.56% and Patient 2: 27.09% vs. 88.04%. For Patient 3, the efficacy of both inhibition tests was 100% .The anti-α-Gal IgE to total IgE ratio for Patient 1 and Patient 2 was similar to before the o215 ImmunoCAP inhibition test . However, in Patient 3 it was lower than the initial one (0.07% vs. 0.52%) .After inhibition on a beef-coated ImmunoCAP membrane (f27-IT), an almost 100% reduction in IgE antibody concentration for bovine allergens was observed, which was measured by the mono-parameter test (ImmunoCAP). In Patient 2 and Patient 3, the concentration of the anti-beef IgE antibody in the blocked sera was below 0.1 kUA/L, which corresponded to a 100% decrease in their concentration. In the serum of Patient 1, whose baseline anti-beef IgE concentration was several dozen times higher than in Patient 2 or Patient 3 (26 kUA/L vs. 0.38 kUA/L vs. 0.51 kUA/L) after the inhibition test on the beef-coated membrane (f27-IT) anti-beef IgE antibodies were still present, but their concentration decreased by 96.19% .The multiparameter ALEX2 platform showed a 100% reduction in anti-beef IgE antibodies (anti-Bos d_meat IgE) after f-27-IT in all sera, regardless of the initial concentration of these antibodies .The concentrations of IgE antibodies specific to non-beef mammalian meat  after inhibition on the f27 coated ImmunoCAP membrane (f27-IT) decreased by less than 6% .Antibodies to poultry and bovine serum albumin (Bos d 6) and porcine serum albumin (Sus d1) remained negative after the f27-ImmunoCAP inhibition test .In the ALEX2 test performed on the material collected after the inhibition tests (o215-IT and f27-IT), no characteristic changes in individual sensitization profiles to various airborne and food allergens  were observed compared to the ALEX2 results obtained before the inhibition tests. The results are presented in Changes in the concentration of specific IgE antibodies not exceeding 10% were observed in each patient in both inhibition tests (o215-IT: −6.82% to 5.12% and F27-IT: −6.45% to 3.12%). These changes were multidirectional even for the same allergens. Both a decreases and increases in the concentration of specific IgE for individual allergens after inhibition tests were observed.Inhibition tests based on immunoblots and ELISA are successfully used to test cross-reactivity between allergens, especially of natural origin. Solid phase inhibition tests based on immunoblots and ELISAs are successfully used to test cross-reactivity between allergens, especially of natural origin. However, they have some limitations resulting from the methodology and low availability of standardized allergen extracts. These types of tests often use self-extracted allergens from natural sources. The composition and stability of these types of extracts are often incomparable. This limits the reproducibility of the results obtained with these methods. Differences in allergen extraction techniques as well as a number of other methodological differences make it difficult to compare the results of ELISA inhibition tests between different research centers. On the other hand, the use of liquid extracts of allergens in inhibition tests (liquid phase inhibition tests) leads to dilution of the test serum and changes in the immune reaction environment, which may affect the final results of the experiment and make their interpretation difficult ,10,11,12In our experiment, we used two types ImmunoCAP membranes (CAP): one coated with α-Gal (bovine thyroglobulin) and the other with beef protein extract. We performed the inhibition tests in sera collected from three patients with different atopic status. We assumed three conditions qualifying the sera for the experiment: the presence of specific anti-α-Gal IgE antibodies, the presence of specific anti-beef IgE antibodies and the absence of poultry-specific IgE antibodies. Allergy to α-Gal results in adverse reactions to mammalian meat, with tolerance to poultry  and fish . PoultryTo determine anti-α-Gal IgE antibodies, we used two currently available in vitro diagnostic (IVD) tests: the single-component ImmunoCAP method and the multi-component ISAC platform. We found that the results obtained with both methods are consistent only when the serum concentration of anti-α-Gal IgE is high. The higher the concentration of anti-α-Gal IgE antibodies measured by the single-component technique, the higher the concentration of these antibodies in the ISAC assay. Very low levels may not be detected in multi-component techniques. These differences result from the different sensitivity and specificity of the singleplex and multiplex tests used in the routine diagnosis of allergies. They may also be due to the quality and sourcing of the allergens used in the various tests .Antibodies against different mammalian meats were present in the sera of our patients, and the sensitization profile was different. The antibody profile for mammalian meat in the ImmunoCAP assay was not consistent with the antibody profile in the multiparameter ALEX assay. This may be due to methodological differences between these tests and possibly to different sources of allergen extracts used in the two techniques. It is worth noting, however, that antibodies to beef, constituting a selection criterion for us, turned out to be the only antibody to meat allergens present in every patient and detected by every test. Beef sensitization is mentioned by some authors as the main, and sometimes even the only, carnivorous symptom of α-Gal allergy ,25,26. OIn our study, we observed that bovine allergens abolished the reactivity of patients’ sera to α-Gal by an average of 58%. On the other hand, α-Gal reduced serum reactivity to beef by an average of 91% as assessed by the ImmunoCAP test. In the ALEX2 assay performed with sera blocked to ImmunCAP α-Gal membranes, the inhibition was 100%. Such results seem to confirm that the experimental model of inhibition test on ImmunCAP membranes used by us is effective in the study of cross-reactivity. The differences in the inhibition strength observed between the systems used are probably due to the amount of α-Gal molecules present on the ImmunoCAP membranes. Kollmann et al.  observedIt is noteworthy that the inhibition on the “ImmunoCAP/beef” membrane f27-IT) was not as effective as the inhibition on the “ImmunoCAP/α-Gal” membrane (o215-IT) for both α-Gal-IgE antibodies and mammalian meat allergens. Binding of anti-α-Gal IgE antibodies to bovine allergens was less efficient than binding to bovine thyroglobulin . However, in the case of reactions specific to allergens of mammalian meat, the reaction with beef allergens was mainly blocked. This result fully meets our expectations and is probably related to the natural distribution of α-Gal glycoprotein in mammalian tissues -IT was n,33,34,35It also seems necessary to note that in natural sources, the availability of an antigen for specific antibodies also depends on the relationship between the various molecules present in that material. Many independent factors affect the binding of an antigen to a specific antibody ,38. ThisIn our experimental inhibition tests, we used ready-made ImmunoCAP membranes, which are the standard in routine in vitro allergy diagnostics. ImmunoCAP allergen membranes have previously been successfully used in inhibition assays for cross-reactivity testing by our team ,11. OtheOur experience also has a few limitations that we are aware of. The use of natural sera as a source of specific antibodies entails consequences resulting, for example, from the fact that they are mixtures of polyclonal antibodies directed against different epitopes of the same antigen. Different patients were naturally immunized under different conditions, so results obtained with sera from different patients may not be consistent. It is also possible that for this reason the results of such an experiment may not be reproducible. In addition, we used research techniques routinely used in diagnostics, but not in accordance with their intended purpose. Undoubtedly, this could have influenced the obtained results. We also realize that carrying out the experiment on only three sera is not reliable. However, we were unable to obtain more α-Gal positive sera, as allergy to α-Gal in Poland is not a common phenomenon. So far, only two reports on this topic have been published—one population study  and one However, these are the results of preliminary studies, which certainly require additional testing using different pairs of cross-reacting allergens with known mutual reactivity. Despite these shortcomings, we believe that the inhibition test model we propose is effective in testing cross-reactivity. We believe that it may find wider application in the assessment of this phenomenon for various allergens. According to our opinion and suggestions of other authors , all inhIn conclusion, the example system of cross-reactive allergens  tested on the proposed experimental model of the two-sided inhibition test on ImmunoCAP membranes allowed to observe that both the beef allergen extract and α-Gal (bovine thyroglobulin), coated on the ImmunoCAP membrane bind anti-α-Gal IgE antibodies present in the serum, which reduces the reactivity of anti-α-Gal IgE sera positive after the inhibition test with mammalian meat allergens, especially beef. These preliminary studies suggest that the experimental model of the two-sided inhibition test on ImmunoCAP membranes proposed by us may be an attractive, simple tool for direct  testing of cross-reactivity between allergens. However, because these are preliminary reports from a small sample, they certainly require further research on various allergens with known cross-reactivity.Our study has some limitations. First of all, it was carried out only in three patients, which significantly hinders both the analysis of the results and conclusions. This strategy also makes any statistical analysis of the obtained results impossible. The α-Gal syndrome, however, is an extremely rare phenomenon, especially in the area of northern Poland, where the study was conducted. It turned out to be impossible for us to gather a larger study group. In preparing this project, we tested a group of 70 patients, 30 of whom had a confirmed tick bite history, 30 had positive IgE specific antibodies to beef and/or pork allergens and 10 were never bitten by ticks and had no IgE specific positive results. Of this group, only four patients were anti-α-Gal IgE positive, and only three of them were found to have both anti-α-Gal IgE and anti-beef IgE antibodies. We included these three patients in our experimental project, and we performed both inhibition tests (o215-IgE and f27-IgE) in the sera collected from them. Therefore, we are aware that inference based on the results obtained by us may be biased and certainly requires further research on a much larger group of patients. However, this is only a preliminary report on the results of the project, which we will continue with more samples and for more allergens. The concept of the experience seemed interesting and worth presenting at this initial stage.Despite these limitations, it seems to us that the presented experimental models can be a useful tool for studying the cross-reactivity of various allergens.


Doses were sufficient to cause up to 80% MEK inhibition. Across all species approximately 10 μg/ml zapnometinib was appropriate to inhibit 50% of peripheral blood mononuclear cells (PBMC) MEK activity. In mice, a 50%–80% reduction of MEK activity was sufficient to reduce influenza virus titer in the lungs by more than 90%. In general, while >50% MEK inhibition was reached in vivo at most doses, 80% inhibition in PBMCs required significantly higher doses and appeared to be the practical maximal level obtained in vivo. However, the period of reduced phosphorylated extracellular-signal regulated kinase (pERK), a measure of MEK inhibition, was maintained even after elimination of zapnometinib from plasma, suggesting a sustained effect on MEK consistent with regulatory effects or a slow off-rate. These data suggest a target plasma Cmax of at least 10 μg/ml zapnometinib in further clinical studies.The mitogen-activated protein kinase (MEK) inhibitor zapnometinib is in development to treat acute viral infections like COVID-19 and influenza. While the antiviral efficacy of zapnometinib is well documented, further data on target engagement/pharmacodynamics (PD) and pharmacokinetics (PK) are needed. Here, we report zapnometinib PK and PD parameters in mice, hamsters, dogs, and healthy human volunteers. Mice received 25 mg/kg/day zapnometinib . Syrian hamsters received 30 mg/kg (15 mg/kg twice daily) or 60 mg/kg/day once daily. Beagle dogs were administered 300 mg/kg/day, and healthy human volunteers were administered 100, 300, 600 and 900 mg zapnometinib (once daily p. o.). Regardless of species or formulation, zapnometinib maximum plasma concentration (C The coronavirus disease 2019 (COVID-19) pandemic and the influenza pandemic in 2009 have renewed interest in therapeutics for respiratory viruses . As demoA new approach for antiviral therapy is the inhibition of host cell factors that viruses require for replication. This could overcome the current limitations of antivirals and may also circumvent the problems of resistance development . SignaliH2 to a TH1 immune response, reduction of regulatory T cell (Treg) response and a prolonged clonal expansion of T cells  and in other cell lines  and pharmacodynamic (PD) parameters in mice, hamsters, dogs, and healthy human volunteers. Our goal was to establish the degree of MEK inhibition over time following various peak plasma exposures and to associate this with apparent antiviral activity in influenza virus infected mice. These data support zapnometinib dose selection in future clinical trials for antiviral efficacy.®, CRL-2936™) for the determination of viral titers in foci assays were purchased from ATCC and cultured in Iscove’s Modified Dulbecco’s Medium  supplemented with 10% (v/v) fetal bovine serum , 100 U/mL penicillin and 100 μg/ml streptomycin . Cells were maintained in a 37°C and 5% CO2 atmosphere. Human peripheral blood mononuclear cells (PBMCs) for the half maximal inhibitory concentration (IC50) determination were isolated from healthy volunteers registered with the Biobank of the Department of Immunology at the University of Tuebingen. Ethical approval was obtained upon review by the Ethics Board of the Medical Faculty of the Eberhard Karls University Tuebingen and the University Hospital Tuebingen (887/2020BO2). For the virus inhibition experiment influenza A virus (IAV) strain A/Regensburg/D6/09 (H1N1pdm09) was used. The virus was provided by the Robert-Koch Institute of Berlin.Madin-Darby canine kidney cells . The animals were fed with standard food. Drinking water was available ad libitum. The mice were housed in type 2 cages with four animals per cage under BSL-II conditions. During this study, assessments included mortality checks and body weight. Mice were sacrificed 24 h post infection. The mouse study was reviewed and approved by the Regional Council Tuebingen, registered under number IM01-13. In total, 10 eight-week-old male Syrian hamster  with a body weight of 86–102 g at administration were used for the pharmacokinetic and pharmacodynamic study . The hamster were sacrificed 24 h post the first treatment. The animals were fed with standard diet. Drinking water was available ad libitum. The hamster animal experiments were carried out at Synovo GmbH . The hamster study was reviewed and approved by the Regional Council Tuebingen, registered under number SYN 03/21–0001. In total, 10- to 24-months old beagle dogs  with a body weight of 6.6–9.2 kg at the start of treatment were used for the pharmacokinetic and pharmacodynamic study. Each dog received each formulation with a dosing holiday of at least 2 days between occasions to allow for washout. The animals were fed with standard food. Drinking water was available ad libitum. The animals were group housed in stainless steel dog cages equipped with an automatic watering system. The dog study was performed at Citoxlab North America . During this study, assessments included mortality checks, clinical observations , behavioral changes, and body weight. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and registered under the study number 2018–1,172. The animals were returned to the Citoxlab spare colony after the last blood sample collection.In total 15 eight-week-old female C57BL/6 mice  with a body weight of 21–24 g at administration were used for the antiviral and pharmacokinetic study . For treatment of mice, 10.5 mg zapnometinib were dissolved in 5% (v/v) dimethyl sulfoxide (DMSO)  and further diluted with 15% (v/v) Cremophor EL , and 80% (v/v) phosphate-buffered saline (PBS) . Each mouse received a dose of 25 mg/kg zapnometinib in an application volume of 200 µL of the formulation by oral gavage. The first treatment was given on day 0 (1 h prior to influenza virus infection) and the second treatment 7 h post infection. The liquid dose formulation for the hamster study was prepared as two-times concentrated stock solution of zapnometinib in 5% (v/v) DMSO, 30% (v/v) polyethylene glycol (PEG) 400, 20% (w/v) Glucose, 10% (v/v) cyclodextrin as Captisol® (stock was 40% (w/v) in water (H2O)). The 2x stock solution was then further diluted in strawberry flavored syrup and administered orally using a voluntary procedure. Each hamster received either a dose of 60 mg/kg once daily (OD) or a dose of 15 mg/kg zapnometinib twice daily (BID). The application volume of the zapnometinib formulation was 2 ml/kg for the hamster on day 0, and the second administration occurred 12 h after the first dose. For treatment of dogs, the liquid dose formulation (formulation 5) was prepared to a concentration of 10 mg/ml. Zapnometinib was dissolved in 5% (v/v) DMSO. Then 30% (v/v) of PEG 400 was added and stirred until homogenous. This was followed by the addition of an appropriate volume of 40% (w/v) Captisol® to obtain a final concentration of 7.4% (w/v) and stirred until homogenous. The pH was adjusted with sodium hydroxide and hydrochloric acid to a final pH of 7.4. The formulation was stored refrigerated (set to maintain at 2–8°C) pending use for dosing. Each dog received a dose of 30 mg/kg zapnometinib. The capsule formulation (formulation 1) contained a selected hot melt extrusion (HME) formulation (150 mg zapnometinib/capsule). The tablet formulation either contained micronized zapnometinib, coated for moisture/light protection, without enteric functionality (formulation 2), micronized zapnometinib, coated for enteric functionality (formulation 3) or non-micronized zapnometinib, coated for moisture/light protection without enteric protection functionality (formulation 4). Each tablet contained 50 mg zapnometinib. Each dog received 2 capsules for a total dose of 300 mg zapnometinib on day 1 or 6 tablets for a total dose of 300 mg zapnometinib on days 4, 7, and 11 as per schedule presented in Zapnometinib  (M = 409.56 g/mol) was synthesized at ChemCon GmbH . The doses used in this study were chosen based on previously unpublished preclinical data. In mice, a dose of 75 mg/kg zapnometinib was well tolerated. The toxicity of zapnometinib was evaluated in single- and repeat-dose p. o. studies of up to 1-month duration in rats and dogs. The genetic toxicity of zapnometinib was investigated in compliance with ICH S2; a standard battery of genotoxicity studies was performed, comprising an Ames test, an 2 (inclusive) were enrolled in this study. Exclusion criteria were clinically significant illnesses, positive urine test for selected drugs of abuse, positive alcohol breath test at screening and upon check-in at the clinical site, positive hepatitis panel (including hepatitis B surface antigen [HBsAg] and anti-hepatitis C virus [HCV] antibodies), and positive screens for human immunodeficiency virus (HIV) antibody. This Phase 1 study was a randomized, double-blind, placebo-controlled study  consisting of two parts. Part 1 was a SAD study with 40 healthy volunteers who were randomized to active or placebo treatment in a 4:1 ratio (4 cohorts with nZapnometinib)( = 2 in each cohort). Subjects receiving active treatment were treated with either 100 mg, 300 mg, 600 mg, or 900 mg zapnometinib. Part 2 was a MAD study with 30 healthy volunteers in three cohorts with the same randomization as in the SAD part (3 cohorts with nZapnometinib)( = 2 in each cohort). Each subject in the MAD cohorts received doses of zapnometinib of either 100 mg, 300 mg, or 600 mg once daily for seven days, or placebo. The safety assessment in this study included monitoring of adverse events (AEs), clinical laboratory tests, electrocardiograms (ECG), vital signs, and physical examination. The study protocol and its amendments were reviewed by the Commissie voor Medische Ethiek ZNA, Antwerp, Belgium. This study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Note for Guidance on Good Clinical Practice (GCP) (CPMP/ICH/135/95) and with applicable local requirements.Healthy men and women between 18 and 55 years of age (inclusive), who weighed at least 50 kg with a body mass index (BMI) between 18.0 and 31.0 kg/m5 foci forming units (ffu)  influenza A virus (strain: A/Regensburg/D6/2009 H1N1pdm09) in 50 µl PBS by inoculating 25 µL into each nostril.Mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (10 mg/kg) solution and then infected intranasally with 1.5 × 102-EDTA as anticoagulant at the following timepoints: pre-dose, 30 min, 1, 2, 4, 6, 9, 12, 15, and 24 h after treatment. The samples were kept on wet ice pending centrifugation. All samples were centrifuged at 1,500 x g at 4°C for 10 min. Plasma was collected and stored at −20°C or on wet ice until further analysis. The remaining blood pellet was used for isolation of peripheral blood mononuclear cells (PBMCs). For the study in human healthy volunteers 8 ml venous blood samples were collected in cellular preparation tubes (CPTTM tubes) containing lithium heparin as anticoagulant  at pre-dose, 30 min, 1, 2, 4, 8, 12, and 24 h post dose on day 1, and at pre-dose on day 2–4 (MAD part only). The samples were centrifuged at 1,650 x g at room temperature (RT) for 20 min. Plasma was collected and stored at −20°C until further analysis. The remaining blood pellet was used for isolation of PBMCs.For the study in mice, 30 µl blood samples were collected in microvettes containing lithium heparin as anticoagulant  at the following timepoints: pre-dose, 30 min, 1, 2, 4, 6, 8, 8.5, 9, 10, 22, and 24 h after treatment. The samples were kept on wet ice pending centrifugation. In the hamster study, blood samples of 50 µl were collected using heparinized capillaries at pre-dose, 30 min, 1, 2, 4, 8, 12, and 24 h after treatment. The samples were kept on wet ice pending centrifugation. In the dog study, 1 ml blood samples were collected into tubes containing KTM software, version 1.6.2. Plasma concentrations of zapnometinib of the human samples were determined at SGS Life Sciences  using a validated HPLC MS/MS method with a lower limit of quantification of 10 ng/ml for the analyte zapnometinib. Statistical analysis was performed by SGS  using SAS® version 9.4 .Pharmacokinetic analysis of the mouse plasma samples was performed using standard procedures at Prolytic GmbH . Hamster plasma samples were analyzed at Synovo GmbH  using HPLC MS/MS (Agilent 1260 HPLC with Sciex API4500) with linear response for plasma concentrations between 15 nM and 33 µM (6.1 ng/ml to 13.5 μg/ml). Pharmacokinetic analyses of the dog plasma samples were performed at Pharmascience Inc.  using an HPLC MS/MS method for the determination of zapnometinib plasma concentrations. The validated calibration range for this assay is from 0.6 μg/ml to 500 mg/ml for which all validation requirements were successfully evaluated. Regressions, calibration standard, quality control sample, animal sample result tables, acquisition methods and chromatograms were generated by Applied Biosystems AnalystTM tubes. After a 20-min centrifugation at 1,650 x g at room temperature, the plasma was collected for PK analysis as described in the section above (Blood sampling and preparation of plasma samples). The PBMC layer was collected in a 15 ml tube , and washed twice with PBS by centrifugation at 300 x g for 10 min at RT. The PBMC pellets were store at −80°C until further PD assessment.PBMCs were isolated from the same blood samples  used for pharmacokinetic analysis. After plasma separation as described in the section above (Blood sampling and preparation of plasma samples), the remaining red blood cells were lysed by adding 1 volume of red blood cell (RBC) lysis buffer  . The samples were incubated for 15 min at RT and centrifuged at 500 x g for 5 min (RT). The supernatant was removed completely, and the pellet was washed twice with PBS. The PBMC cell pellets were store at −80°C until further analysis. PBMCs of 8 ml human blood were isolated using CPT™ (Simple Western™) system which uses capillary electrophoresis to separate, identify and quantify proteins of interest. A chemiluminescence signal for each identified protein is detected and the area under the peak is calculated, which is then used for quantification. The 0.1x Sample Buffer, 400 mM dithiothreitol (DTT) solution, 5x Fluorescence Master Mix, and the Biotinylated Ladder  were prepared according to manufacturer’s instructions. For all species, PBMCs were lysed in 1x radioimmunoprecipitation assay (RIPA) buffer (containing 0.24% (w/v) tris base , 0.88% (w/v) NaCl , 0.2% (v/v) 500 mM EDTA (Sigma-Aldrich), 1% (v/v) Triton X-100 (Sigma-Aldrich), 0.5% (w/v) sodium deoxycholate (Sigma-Aldrich), 0.1% (w/v) sodium dodecyl sulfate (SDS) (Carl Roth), 10% (v/v) glycerol (Carl Roth), 0.05% phenylmethyl sulfonyl fluoride (PMSF) (Carl Roth), 0.01% Benzonase® Nuclease (Sigma-Aldrich), protease inhibitor cocktail , and phosphatase inhibitor cocktail (Roche) in H2O), further diluted with 0.1x Sample Buffer  to adjust the protein concentration to 1 μg/μl. Then the 5x Fluorescence Master Mix in DTT (ProteinSimple®) was added, and the samples were denatured for 5 min at 95°C, loaded onto the assay plate and analyzed using specific antibodies. The primary antibodies for phospho ERK1/2 (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb, Cat# 4370) and ERK1/2 (p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb, Cat# 4695) were purchased from Cell Signaling Technology  and used at a 1:50 (pERK1/2) or 1:100 (ERK1/2) dilution in Antibody Diluent (ProteinSimple®). The anti-rabbit secondary antibody (Cat# DM-001) was also purchased from ProteinSimple® and was ready to use. Data were analyzed with Compass software for simple western (ProteinSimple®). Using the ratio of pERK to ERK of each sample compared to control (pre-dose), the level of ERK phosphorylation was calculated and then converted into the percentage level of MEK inhibition.The pharmacodynamic assessment was performed using the Wes® foci assay on MDCK II cells as previously described  solution and permeabilized with 0.3% Triton X-100 in PBS. Cells were then immunostained with mouse anti-IAV nucleoprotein monoclonal antibody  for 60 min at RT, followed by washing and a 30-min incubation with goat anti-mouse IgG-HRP antibody . After washing, True BlueTM peroxidase substrate  was added for 10 min at RT. Finally, the number of foci were counted to determine the virus titer as foci forming units per ml (ffu/ml).Mice were sacrificed 24 h after zapnometinib treatment. Lungs were removed, weighed, and transferred into a Lysing Matrix D tube . 500 µL ice-cold PBS was added, the lungs were homogenized using a FastPrep FP 120 (Savant) and centrifuged for 10 min at 18,000 x g at 4°C. The supernatant was used to perform an AVICELescribed . Briefly2. Subsequently, PBMCs were isolated using 1x RBC lysis buffer and lysates were prepared in 1x RIPA buffer for further analysis with Wes™ technology. Using the ratio of pERK to ERK of each sample compared to control (DMSO), the level of ERK phosphorylation was calculated. For dose response curves and calculation of IC50 values, data were fitted using GraphPad Prism software version 9 .Human whole blood was treated with different concentrations (ranging from 0–100 μg/ml) of zapnometinib for 1 h at 37°C and 5% COt test with Welch’s correction. p values <0.05 were considered statistically significant.Unless otherwise noted, data were collected in MS Excel, and statistical analyses were performed using GraphPad Prism software version 9 (GraphPad Software Inc). Statistical details for each experiment are described in the corresponding figure legends. Differences in viral titer were analyzed by unpaired First, we investigated whether binding of zapnometinib to MEK inhibits phosphorylation of MEK and thus also prevents further phosphorylation of ERK. Since ERK is the only substrate downstream of MEK, usually reduction of ERK phosphorylation is measured to determine MEK inhibition. PBMCs from healthy donors were isolated, treated with zapnometinib, stimulated with Phorbol-12-Mystrat-13-Acetat (PMA) and analyzed for the reduction of MEK and ERK phosphorylation. We could show that zapnometinib directly prevents the phosphorylation of MEK and consequently causes inhibition of the phosphorylation of the MEK1/2-dependent kinase ERK1/2 . Becausein vivo. Therefore, we infected mice with a lethal dose of H1N1pdm09 (1.5 × 105 ffu) and treated them orally with 25 mg/kg zapnometinib twice daily, for a total dose of 50 mg/kg/day. The first dose was administered 1 h prior to infection and the second dose 7 h post infection (8 h after the previous dose). Blood samples were collected at different time points (as mentioned in the method section) for pharmacokinetic and pharmacodynamic assessments. Lungs of the sacrificed mice were taken 24 h post infection for the determination of the viral titer.One major objective of this study was to correlate pharmacokinetic and pharmacodynamic parameters with the antiviral efficacy of zapnometinib t = 8 h). Afterwards the level of MEK inhibition increased by about 10% following increased plasma levels and then steadily climbed to a second maximum of 80 ± 5% MEK inhibition at 22 h after the first treatment (14 h after the second treatment)  with a maximum concentration of 69 ± 24 μg/ml (Cmax). Prior to the second treatment (t = 8 h) the plasma concentration declined to 36 ± 8 μg/ml followed by an increase to 101 ± 16 μg/ml two hours after the second administration . At the end of the observation period (t = 24 h) the plasma concentration of zapnometinib had decreased to 15 ± 9 μg/ml.First, we investigated the level of MEK inhibition in PBMCs after treatment compared to pre-dose levels. Zapnometinib treatment resulted in MEK inhibition levels between 56 ± 20% to 80 ± 5% as measured by the reduction of ERK phosphorylation. A first maximum of MEK inhibition of 72 ± 10% was reached 2 h after the first treatment and thereafter declined between 59 ± 19% to 56 ± 30% prior to the second treatment (t = 24 h) 66 ± 19% MEK inhibition was apparent at a plasma concentration of 15 ± 9 μg/ml zapnometinib.Association of the %MEK inhibition  with the plasma concentration of zapnometinib  is presented in 10 ffu/ml  and 60 mg/kg (OD) compared to pre-dose level were examined (n = 5) at a Cmax of 61 ± 4 μg/ml. With the dose of 15 mg/kg, 40 ± 9% (n = 3) MEK inhibition was achieved with a Cmax of 15 ± 1 μg/ml (n = 4) was still observed 24 h after treatment with a mean plasma concentration of 0.2 ± 0.6 μg/ml zapnometinib (n = 4) measured at 24 h with a mean zapnometinib plasma concentration of 1 ± 0.2 μg/ml  and an AUC0–24 h 962 ± 52 μg/ml*h  at 4 h after treatment and AUC0–24 h (352 ± 53 μg/ml∙h)  or of 30 mg/kg (liquid formulation) led to different pharmacokinetic profiles. The liquid formulation resulted in the highest C μg/ml*h . Similar μg/ml*h . The capμg/ml∙h) .max and at 24 h post treatment for the five different formulations was investigated (max leading to an MEK inhibition of 79 ± 7% (n = 10) (n = 7) and 70 ± 11%  were reached with 93 ± 16 μg/ml (formulation 2) and with 98 ± 8 μg/ml zapnometinib (formulation 4) at Cmax. The weakest MEK inhibition was obtained with formulation 1 with 44 ± 16 μg/ml zapnometinib at Cmax leading to 42 ± 19% MEK inhibition (n = 10). Formulation 5 achieved the highest plasma concentration of 152 ± 25 μg/ml zapnometinib, but only a moderate MEK inhibition of 56 ± 15% (n = 10). At 24 h after a single administration of zapnometinib, MEK inhibition was still observed for all formulations (n = 10) the strongest MEK inhibition with a plasma concentration of 4 ± 8 μg/ml zapnometinib. Formulation 1 reached 44 ± 20% MEK inhibition with 6 ± 6 μg/ml zapnometinib. A mean MEK inhibition of 63 ± 12% could be obtained with 2 ± 2 μg/ml zapnometinib (formulation 4). Similar levels of 50 ± 19% (formulation 2) and 51 ± 23% (formulation 5) could be reached with mean plasma concentration of 3 ± 1 μg/ml (formulation 2) and 3 ± 1 μg/ml (formulation 5). When pharmacokinetic (red line) and pharmacodynamic parameters (blue line) were correlated, it was obvious that comparable with mice and hamsters, MEK inhibition in dogs was still present when zapnometinib was diminished from plasma at 24 h after treatment  . Comparaulations . Formulareatment . The conpre-dose .max, and was maintained despite elimination of the substance from plasma. The general similarity in the results suggested extension to human studies.These data show that in dogs, as in rodents, MEK inhibition was proportional to CTo test whether the PK and PD findings from different animal species translate to humans, we conducted a phase 1 clinical trial in SAD format with 10 healthy volunteers each treated with 100, 300, 600 or 900 mg zapnometinib.max was reached 2 h post dose for 100 mg zapnometinib and 4 h post dose for 300, 600 and 900 mg. A maximum plasma concentration of 9 ± 2 μg/ml was achieved with a treatment dose of 100 mg zapnometinib, 13 ± 4 μg/ml with 300 mg, 35 ± 7 μg/ml with 600 mg, and 46 ± 16 μg/ml with 900 mg zapnometinib. After Cmax, the zapnometinib plasma concentrations declined in a multi-phasic manner and in parallel for all doses , 41 ± 26% with C24 h = 4 ± 0.8 μg/ml (300 mg), 37 ± 20% with C24 h = 8 ± 2 μg/ml (600 mg) and 26 ± 26% (900 mg) with C24 h = 16 ± 6 μg/ml were found , then isolated PBMCs and measured apparent MEK activity via ERK phosphorylation. In line with the ICn by 50% .In summary, the results show that up to 80% MEK inhibition in PBMCs was observed after zapnometinib treatment regardless of species. 24 h after dosing, when almost all zapnometinib was eliminated from the plasma, MEK inhibition of up to 50% was maintained.Zapnometinib is a potent and selective MEK1/2 inhibitor that is currently in phase 2 clinical trials as a potential agent against COVID-19 (NCT04776044). Due to its mode of action, it could also be used against other viral infections, like influenza . Since zThe aim of this study was to investigate which dose of zapnometinib will lead to pharmacodynamically active concentrations in mice, hamsters, dogs, and humans. In addition, the study provided data on the duration of apparent MEK inhibition, and the effects of dose and formulation. These observations lead to questions on the degree of inhibition required for antiviral effects, the turnover of pERK, the mechanism for sustained MEK inhibition, the relevant tissues, and compartments to measure and the optimal dose regime for antiviral and anti-inflammatory effects.To measure drug interaction with the target, we used PBMCs as a model for lung tissue given that these can be easily obtained from healthy volunteers and patients. Since COVID-19 infection and also influenza virus infection occurs mainly in the lungs, lung cells would be more relevant. However, it is not possible to take lung samples from a living organism. Since blood cells are easy to collect and the signaling pathway is also activated in these cells by virus infection and is involved in many immune response processes, we use PBMCs. In follow-up studies, we will relate lung to PBMC values to understand how this parameter translate between peripheral blood and lung tissue. We found that zapnometinib leads to up to 80% MEK inhibition in PBMCs at doses that were well tolerated for the respective species. MEK inhibition in PBMCs was also detectable 24 h after treatment, when zapnometinib was mostly eliminated from plasma.max, we hypothesize that it indicates, inter alia, a slow off-rate. Alternatively, accumulation of substance to PBMCs or changes in regulation of the cascade may lead to this effect and we are examining the possible mechanisms of sustained levels of pERK after drug elimination.In mice, a 60%–80% MEK inhibition in PBMCs was sufficient to reduce influenza virus titer in lungs by >90%. Treatment of influenza virus infected mice with 25 mg/kg zapnometinib BID leads to a favorable pharmacokinetic profile and to an inhibition of MEK activity in PBMCs between 57%–80%. However, given the practical limits of sampling from infected animals over time, a detailed time course is not feasible and reduction of ERK phosphorylation, and thus MEK inhibition may also have been transiently higher than 80%. Although no substance was detectable in plasma after 24 h, MEK inhibition (measured as reduction of ERK phosphorylation) in PBMCs remained at 66%. The cause of the sustained effect on pERK levels remains to be clarified, however, based on other factors and the relationship with Cmax of 61 μg/ml. Treatment with 15 mg/kg (BID) resulted in a Cmax of 15 μg/ml and MEK inhibition of less than 50%. After 24 h, MEK inhibition levels were still relatively high at 55% (60 mg/kg) and 49% , despite the low zapnometinib concentration in plasma. Based on these data, higher doses of zapnometinib are recommended in a Syrian hamster SARS-CoV-2 infection model to achieve a constant MEK inhibition of more than 50%.Since the Syrian hamster represents a very suitable model to study COVID-19 , this anmax levels ranging from 44–152 μg/ml. The inhibition of MEK and the resulting reduction in ERK phosphorylation was 40%–80% at the time of Cmax. Interestingly, we did not find the strongest MEK inhibition in PBMCs at the highest Cmax value. Therefore, we hypothesize that the maximum achievable MEK inhibition of 80% has already been reached and that higher inhibition levels cannot be achieved even with a higher plasma concentration of zapnometinib. To better understand this observation, we are examining the kinetics of MEK inhibition onset vs. time to determine whether there is a lag between Tmax and maximal MEK inhibition. Again, after 24 h MEK inhibition levels were still between 44%–72%, although zapnometinib concentration was low in plasma (2–8 μg/ml). The difference between formulation 3 and 5 in terms of MEK inhibition may be due to the faster plasma levels achieved. Formulation 5 reached much faster higher plasma levels than formulation 3, but zapnometinib has high plasma binding. Therefore, we assume that not all of the compound reached the cell. And since formulation 5 reached Cmax faster, we suspect that it is too fast for zapnometinib to inhibit MEK in all cells, this could explain higher levels seen in the green bars . A further PK/PD study will be conducted with doses higher than 900 mg to address this question. In humans, we could not measure the concentration of zapnometinib in the lung, the main target organ of the drug in respiratory infection. We are currently planning another preclinical study in animal models to get a better insight into the correlation of plasma vs. lung concentration ratio.In summary, we could show the interrelationships of PK and PD for the MEK inhibitor zapnometinib in three relevant animal species and in healthy human volunteers. Independent of the species, we did not observe MEK inhibition of more than 80% but sustained activity to 24 h after a single dose when most substance was eliminated from the plasma. In the mouse model, we were able to show a correlation of PK/PD data with antiviral efficacy, where 60%–80% MEK inhibition was sufficient to reduce viral titers in the lung by more than 90%. These results provide a better understanding of the zapnometinib target engagement and will influence the design of further clinical trials.


Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the medium, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylation state of proteins when heterologously expressed, since their biochemical and physiological properties can be dependent on post-translational modification.In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate,  RRs can be chemically phosphorylated in vivo and in vitro with small molecule phospho-donors such as acetyl phosphate (AcP), carbamoyl phosphate (CP), or phosphoramidate2. Biochemical studies for two-component systems are routinely performed with purified RRs derived from recombinant protein production in Escherichia coli BL21 (DE3)4. These experiments are often supplemented with phospho-donors to ensure RRs are in their phosphorylated state6.Two-component systems (TCS) regulate bacterial responses to the environment. In canonical TCSs, histidine kinases (HK) phosphorylate cognate response regulators (RR), which when phosphorylated perform a function7. Bacteria, including E. coli, can use metabolically generated AcP to phosphorylate natively expressed RRs8. Post-translational modification by small molecules is speculated to have physiological roles in TCS regulation8. Although post-translational modification during recombinant protein expression is a known phenomenon9, the possibility for post-translational phosphorylation of response regulators to occur during recombinant protein production has never been systematically assessed. In this work, we report a systematic characterization of the phosphorylation state of RRs produced in E. coli BL21 (DE3) with phos-tag gels6.Of the available phospho-donors, AcP is a metabolic intermediate downstream of glycolysis with an important role in energy generation during periods of starvationPseudomonas stutzeri RCH210 (GCF_000327065.1), and tested their phosphorylation state by phos-tag gel12 and western blot. Phosphorylated protein loaded into a phos-tag gel will migrate more slowly than its unphosphorylated counterpart  homologs of cognate HKs that could engage in cross-talk with the non-native RRs or (ii) metabolic intermediates that can act as phospho-donors. In P. stutzeri RCH2, CopR II is activated in the presence of elevated levels of copper by their cognate HK, CopS13. CusS, the homolog for CopS in E. coli, is also activated in the presence of elevated copper14. To test if E. coli’s native CusS was responsible for the observed phosphorylation, we cultivated the CopR II expression strain in the presence and absence of copper (SF7). We found no significant change in the ratio of phosphorylated to unphosphorylated CopR II, indicating that CusS may not play a role in the phosphorylation of the heterologously expressed protein. To test the alternative hypothesis, we cultivated the CopR II expression strain in the presence and absence of magnesium  expression strain mutants, ∆cusS, ∆ackA, or ∆pta with the CopR II expression vector and characterized the resultant purified proteins with 12% and phos-tag gels . We note that heat treatment may lead to false apparent homogenous populations of unphosphorylated RRs in a phos-tag gel. Although it is common practice to heat treat protein prior to electrophoretic separation, we recommend that RRs should not be heat treated before phos-tag characterization.25 we know that despite the short half-life of acyl-phosphate bonds in small molecules26, the half-life within RRs varies significantly1. In our systematic study, we observed 12 partially or fully phosphorylated RRs. For CopR II, partial phosphorylation was observed even after long-term storage at cryogenic temperatures. Even after treatment with conditions known to destabilize the phospho-Asp bond, we observed that a percentage of the CopR II population remained phosphorylated. In agreement with the previous studies, our study suggests that the lability of the acyl-phosphate bond within RRs is dependent on the structure and conformation of the RR and is not necessarily unstable.From previous reports on the stability of phospho-Asp bonds in RRsOur study does not reveal the underlying mechanism for the observed phosphorylation of heterologously expressed RRs. First, we do not know how RR structure and homology determine phosphorylation state. Our observation that paralogs of RRs show inconsistent phosphorylation patterns suggests that RR phosphorylation is dependent on intrinsic properties of the RR that are distinct even between paralogs, such as phospho-Asp stability or susceptibility to phosphorylation by small molecule phosph-donors due to surface exposure of the active Aspartate residue. We suggest future studies to determine if orthologous RRs also exhibit distinct phosphorylation trends and structural comparisons of paralogous RRs to evaluate if differences in surface exposure of the active aspartate residue between paralogs is responsible for the differences in phosphorylation. Next, we could not address the mechanism by which RRs are getting phosphorylated. Although our results suggest some interplay between the metabolic intermediate AcP and the recombinant proteins, we could not attribute the observed phosphorylation pattern to its presence. We also cannot rule out the role of homologous cognate HKs in the observed phosphorylation for all of the RRs we tested. However our results with CopR II suggest that we can expect homologous cognate HKs to act as phosphatases when expression strains are grown in non-activating conditions. We propose an alternative hypothesis in which another metabolic intermediate that can serve as a phospho-donor, such as carbamoyl-phosphate, could be responsible for the non-canonical phosphorylation of RRs that are expressed and purified with recombinant protein production systems.P. stutzeri RCH2 as a representative set of RRs that were produced and purified in E. coli. Our observations of host-derived phosphorylation demonstrates how valuable it is to test the phosphorylation state of recombinant proteins. However, future studies must examine in a case to case basis the impact of the protein’s phosphorylation state in subsequent biochemical assay results.Overall our study provides the phosphorylation landscape for the majority of response regulators for Pseudomonas stutzeri RCH2 gDNA with regions homologous to the pET28 plasmid27. Parts were assembled via Gibson assembly28 and transformed into E. coli DH10b for plasmid propagation. The presence of the insert was confirmed by Sanger sequencing. Plasmids were purified and transformed into expression host E. coli BL21 (DE3) or deletion strains if specified. All plasmids generated are listed in ST4.All RRs were cloned into pET28 vectors encoding N-terminal 6X-His tags under T7 inducible promoters. The RRs were PCR amplified from cusS was made in E. coli BL21 (DE3) according to the Red recombinase method29. The gene integration template was prepared using primers previously designed for recombination with cusS (b0570)30. Colonies were screened via colony PCR for successful recombination of E. coli BL21 DE3 ∆cusS::frt Kan. E. coli BL21 DE3 ∆ackA:: frt Kan, ∆pta::frt Kan were provided by Alan J. Wolfe lab. All recombination strains were transformed with heat sensitive plasmid, pCP2030, at 30 °C. Single colonies were screened for successful kanamycin flip-out via colony PCR. pCP2030 was cured from confirmed knockouts by incubation at 42 °C. All primers are listed in ST5.A single gene knockout strain of 31) for growth at 37 °C, 250 RPM, for 5–6 h and were then transferred to grow at 17 °C, 250 RPM, overnight. Cell pellets were harvested and lysed at 37 °C for 1 h in a lysis buffer , 2.5 units/mL Benzonase nuclease (Millipore Sigma), 1 mg/mL Lysozyme (Millipore Sigma). Lysed cells were then clarified by centrifugation at 3214×g and further filtered in 96-well filter plates by centrifugation at 1800×g. To enable high-throughput processing, protein purification steps were performed with IMAC resin pipette tips (PhyNexus) using a custom automated platform with the Biomek FX liquid handler (Beckman Coulter). The expressed RRs were individually bound to metal affinity resin embedded within the IMAC resin pipette tips and washed in a wash buffer . The RRs were then eluted in an elution buffer  glycerol, 180 mM imidazole). The purified RRs were then quantified with the QuickStart Bradford Protein Assay (Bio-rad), normalized, flash frozen, and stored at − 80 °C for a minimum of 1 day and up to a week before applying gel-based characterization.Expression strains were grown overnight in LB and subcultured in autoinduction medium (Zyp-505231) or Terrific broth (TB) supplemented with Kanamycin (15 µg/mL). Cultures grown in TB were induced with 100 µM IPTG at OD600 0.6–0.8 at 37 °C and then incubated at 18 °C for overnight expression. Cultures grown in autoinduction medium were grown for 5–6 h at 37 °C and then transferred to 17 °C for overnight expression. Cell pellets were harvested and lysed at 37 °C for 1 h in a lysis buffer . His-tagged RRs were purified from clarified lysate using HisPur Ni–NTA resin (Thermo Scientific) or HisPur Cobalt resin (Thermo Scientific). The resin was washed in a wash buffer  and eluted in an elution buffer  glycerol, with 250 mM if Ni–NTA resin was used or 180 mM imidazole if Cobalt resin was used). RRs were quantified with the QuickStart Bradford Protein Assay (Bio-rad), normalized, and stored at − 80 °C until for a minimum of one day and up to a week before applying gel-based characterization, unless stated otherwise. Biological replicates are defined here as distinct subcultures for protein production. For reactions supplemented with a phospho-donor, 1 µL of 1 mM Acetyl phosphate was added to 30 µg of protein and allowed to react at 37 °C for 1 h.Expression strains were grown overnight in LB and sub-cultured the next day in either autoinduction medium (Zyp-5052Purified protein was mixed in 1:1 ratio with 2× laemmli loading dye (Bio-rad) prepared according to the manufacturer's instructions. 10 µL of the protein loading mixture was loaded into the wells of either 12% mini-PROTEAN TGX precast protein gels (Bio-rad) or 12.5% SuperSep Phos-tag precast gels . Band separation was achieved by running the gels in Tris–Glycine SDS buffer at 150 V for 1–1.5 h at room temperature. For gels with no ladder CopR II is used as size reference within all gels; the size of all other proteins are measured relative to CopR II, for expected molecular weights see ST4.−1 of anti-6X-His tag monoclonal antibody [HIS.H8] with an HRP conjugate (ThermoFisher) suspended in 10 mL 1× TBST for 0.5 h, and washed 3 times to remove unbound antibody with 1× TBST. To activate the HRP conjugate, membranes were incubated with ECL substrate for western blotting (Bio-rad) according to the manufacturer’s instructions. Chemiluminescence was imaged with the Amersham Imager 600  according to the manufacturer’s instructions. Raw image files can be found in the supplemental information. Two technical replicates of each experiment were performed for each condition. Proteins expressed and purified at different conditions confirm the reproducibility of the observed phosphorylation ratios.SDS-PAGE gels were blotted with Trans-Blot Turbo mini 0.2 µm PVDF membranes (Bio-rad) using the Trans-Blot Turbo transfer system (Bio-rad) according to the manufacturer’s instructions. After transfer, the membranes were blocked with 50 mL 5% bovine serum albumin in 1× TBS with 0.1% Tween-20 (TBST) for 1 h. The membranes were then washed 5 times with 1× TBST. Following washing, the membranes were incubated 1 mg LBands were quantified with ImageStudio Lite software (LI-COR v5.2.5). Unphosphorylated and background bands were identified by comparison with images of 12% gels. Phosphorylated and unphosphorylated bands were quantified with the median background method. Background was calculated by estimation of the area to the right and left of the band with a border width of 3 pixels. For images with CzcR and CopR II, all bands in gels and blots were quantified using BioRad Image Lab (Bio-rad v6.0). The background-subtracted densitometry of the bands was determined using multiple band analysis tools within the software. All quantification was conducted on raw images. Raw image files can be found in the supplemental information. Images were adjusted  for graphical representation in main text and supplemental figures.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.Supplementary Information 13.Supplementary Information 14.Supplementary Information 15.Supplementary Information 16.Supplementary Information 17.Supplementary Information 18.Supplementary Information 19.Supplementary Information 20.


To find a preoperative computed tomography-based method to predict the incidence of sacral screw loosening and assist surgical planning. t-tests, X2 analysis, Pearson correlation analysis, and ROC curve analysis.  Surgically treated patients for degenerative lumbosacral disorders with rigid pedicle screw fixation of patients with L5-S1 vertebra in our center from January 2016 to January 2021 were retrospectively included in the current study. CT scan attenuation of the horizontal plane of the sacrum was measured with Hounsfield units (HU). Postoperative X-ray tests were used to diagnose screw loosening. The data was analyzed by independent sample P < 0.01). In ROC curve analysis, AUC was 0.674 (95% CI: 0.592-0.756). A cutoff of 200 HU provided 64.8% sensitivity and 62.4% specificity, and a cutoff of 150 HU provided 90.2% sensitivity.  A total of 162  patients were included in the final analysis. Significant differences were found between the screw loosening group and nonloosening group concerning the HU value of the sacrum at the horizontal plane ( Analyzing 162 patients with at least 12 months of follow-up, we propose cutoff CT attenuation values of 200 HU and 150 HU to take moderate and radical measures of screw augmentation to prevent screw loosening in the sacral bone. In lumbar spinal stenosis, lumbar spondylolisthesis, and other lumbosacral degenerative diseases, lumbosacral decompression and pedicle screw fixation are often a necessity , 2. HoweAt present, techniques commonly used to enhance the strength of the sacral pedicle screw fixation include double and tricortical fixation, application of expanding screws, S1-2 combined screw fixation, iliac screw fixation, and the cement-reinforced screw fixation –9. HowevAll procedures were approved by the ethical committee of our hospital, and written consent was achieved from patients before treatment. All the procedures were carried out according to the Helsinki declaration. Surgically treated patients for degenerative lumbosacral disorders with bicortical pedicle screw fixation and interbody fusion of the sacral vertebra in our center from January 2016 to January 2021 were retrospectively included in the current study. Other inclusion criteria include the age of 50 years or older, computed tomography scans carried out before and after surgery, followed up for more than 12 months, no previous surgical intervention in the sacral region, and no previous congenital malformations such as congenital scoliosis. Patients with malignant tumor of the spine, previous surgeries at the lumbosacral region, and active inflammation before and after surgery were excluded.Computed tomography scans were carried out in the department of radiology of our hospital. Patients underwent CT scans  before the surgery. Two independent examiners used PACS  to measure the HU value. The region of interest (ROI) with a maximum diameter within the cortex of the sacrum was determined on the horizontal plane, and the radiologic attenuation was recorded in HU automatically . CT scanTwo senior spine surgeons independently evaluated screw loosening and bone fusion by the follow-up X-ray and CT scans. More than 1 mm of clear zone around the screw was used as a reference to diagnose screw loosening . Nonfusit-tests (measurement data) and X2 Fisher's exact test analysis (count data). Pearson correlation analysis and ROC curve analysis were used to further evaluate applicability of HU on the prediction of screw loosening. IBMSPSS 24.00 software was used for all statistical analysis. Continuous variables were recorded as mean ± standard deviation. The difference was considered significant when P < 0.05.Demographic characteristics between the two groups were compared with independent sample P > 0.05). The difference was significant between the groups concerning patient age, formation of pseudoarthrosis during follow-up, and HU value of the sacrum at the horizontal plane (P < 0.001). The average HU value of sacrum was 225.8 ± 62.4 and 205.6 ± 55.9 in the non-screw loosening group while it was 188.9 ± 54.6 and 166.5 ± 52.5 in the screw loosening group  patients underwent sacral screw fixation in the treatment of lumbosacral spinal disorders during the study period. They were treated for diseases including spondylolisthesis, lumbar spinal stenosis, degenerative scoliosis, and spondylodiscitis. The average time of follow-up was 18.8 ± 11.4 months (range 12-53 months). The average HU value of the sacrum was 212.6 ± 54.3. There were no significant differences between the screw loosening group and non-screw loosening group concerning patient gender, BMI, habit of smoking, and whether or not the patient had diabetes or suffered from spondylolisthesis (ng group .P < 0.01) and HU values measured on the horizontal plane (P < 0.01). Although pseudoarthrosis is significantly correlated to the incidence of screw loosening, considering the objective of the current study which was to find a prediction measure for sacral screw loosening, here, we only carried out further analysis on the sacral HU value on different planes. ROC curve analysis was carried out to find the predictive value of computed tomography on screw loosening after sacral screw implantation, and the results revealed that area under the curve (AUC) was 0.674 (95% CI: 0.592-0.756), indicating a higher predictive value of CT attenuation measured on the horizontal plane  between the screw loosening group and control groups, indicating the correlation between low HU value of the sacral bone and the high incidence of screw loosening of the sacral bone, which was further proven by the Pearson correlation tests (r < 0.01). Area under the curve in ROC curve analysis was 0.674 (95% CI: 0.592-0.756), indicating relatively high diagnostic value of CT attenuation. Further analysis on the statistical data showed a potential cutoff value of 200 with the sensitivity of 64.8% and the specificity of 62.4%. Although a HU value of 200 suggests more than 60% chance of screw loosening, considering the potential fatal complications of cement augmentation and complexity of revision surgery in cases of screw loosening after cement augmentation, we propose a relatively mild approach such as expandable screws to increase the stability of screws when the CT attenuation value of the sacrum on the horizontal plane is lower than 200 HU. In the meanwhile, there was 90% sensitivity when the HU value is lower than 150; it may be plausible to use more radical methods such as bone cement augmentation to increase pullout strength and avoid screw loosening in those cases.Although there were some reports on the correlation of HU value and the incidence of screw loosening in the lumbar spine, few studies were carried out to find HU-based criteria to predict sacral screw loosening. In the current study, we retrospectively analyzed the relations between Hounsfield units and the incidence of screw loosening after pedicle screw fixation in the sacral bone. In the current study, we measured the HU value of the sacral bone. Results of our study showed significant difference (Considering the relatively small sample size and retrospective nature of the current study, more prospective studies with larger patient inclusion should be carried out to further test our conclusion. Besides the HU value of the sacral bone, the HU value of the lumbar spine could also provide valuable information on the bone mineral density and the possible of screw loosening after surgery, which should be further analyzed in future studies. Except from the HU value, there are other factors such as patient age, segments of internal fixation, overall health status, and the vigor of the patient. In the meanwhile, most of the patients with pedicle screw loosening did not show any symptoms and require further treatment. All those factors should be taken into consideration when deciding whether or not to apply screw augmentation and to use what type of screw augmentation technique in the patient.Analyzing 200 patients with at least 12 months of follow-up, we propose 200 HU and 150 HU as cutoff points in the horizontal plane to take moderate and radical measures for screw augmentation to prevent screw loosening in the sacral bone.


Childhood wasting and stunting affect large numbers of children globally. Both are important risk factors for illness and death yet, despite the fact that these conditions can share common risk factors and are often seen in the same child, they are commonly portrayed as relatively distinct manifestations of undernutrition. In 2014, the Wasting and Stunting project was launched by the Emergency Nutrition Network. Its aim was to better understand the complex relationship and associations between wasting and stunting and examine whether current separations that were apparent in approaches to policy, financing, and programs were justified or useful. Based on the project's work, this article aims to bring a wasting and stunting lens to how research is designed and financed in order for the nutrition community to better understand, prevent, and treat child undernutrition. Discussion of lessons learnt focuses on the synergy and temporal relationships between children's weight loss and linear growth faltering, the proximal and distal factors that drive diverse forms of undernutrition, and identifying and targeting people most at risk. Supporting progress in all these areas requires research collaborations across interest groups that highlight the value of research that moves beyond a focus on single forms of undernutrition, and ensures that there is equal attention given to wasting as to other forms of malnutrition, wherever it is present. Reviews conducted by the ENN in 2013 and 2014 found that wasting was commonly viewed as a short-term, emergency problem, and stunting as a chronic, developmental problem, and that this was reflected in policy, financing models, and program designs  were initiated by ENN in 2014 with the aim of better understanding the complex relationships and associations between wasting and stunting and of further examining policy, financing, and program approaches to the 2 conditions. From 2014 until 2021, the project has gone through 3 phases and accomplished multiple milestones. During phase I, these milestones included developing an understanding of the relationship between wasting and stunting via a narrative literature review , and anaDuring phases II and III, the focus was on analyses of a much larger pool of secondary data. This enabled the calculation of pooled-prevalence estimates of data from 83 countries of the burden of children who are WaSt , examinaLancet viewpoint article  is associated with considerable excess mortality and a risk of death that is similar to or higher than that associated with severe, single-indicator measures of growth failure  , 12. RecBOX 1:A note on classification of undernutritionThe terms wasted and stunted were introduced in the early 1970s by John Waterlow to differentiate, among underweight children, those who had a low weight in relation to their height (wasted) and those who had a low height in relation to their age (stunted).The terms wasted and stunted are used to classify children by comparing them to a growth reference or standard based on data from a healthy population. Since 2006, the WHO child growth standards have been used for this comparison . A wasteThe terms wasting and stunting refer to a process rather than a category. Stunting refers to a slowing or halting of linear growth , and wasting refers to a loss of body weight or ponderal growth in relation to height . Both these processes can occur during infancy and childhood and also in utero. The use of the term “wasting” to mean “wasted” and “stunting” to mean “stunted,” however, is quite widespread.Underweight is a term used to describe children with a low weight in relation to their age (weight-for-age) when compared to the WHO 2006 growth standards. This is a composite indicator for wasting and stunting and is used for growth monitoring programs. All children who are WaSt are also underweight .These diagnostic criteria are also appropriate for assessing risks of death in infants under 6 months of age, as well as in older children. A risk of death in infants is traditionally assessed using a weight-for-length z-score (WLZ) for infants less than 6 months, but the WLZ cannot be calculated when length is <45 cm, due to the exclusion of infants <45 cm in length in the 2006 WHO growth standards . In a reThere are, however, limits in the use of anthropometry for identifying risks of mortality and growth faltering, because it can conceal crucial physiological variability, connections in pathways, or underlying physiology. We urgently need innovative and early markers to predict, identify, and monitor children at short-term and long-term risks of weight and linear growth faltering. Given that child undernutrition is often established by the time a child is born, we require more research focus on maternal factors that can potentially affect in utero and postnatal child weight and linear growth faltering. Such factors include maternal age, weight, anthropometric status, and education , as wellWork by the MAMI  group has recently outlined a range of assessment and screening tools or indicators to identify small and nutritionally at-risk infants (<6 months of age) and their mothers . Most ofAge and sex are also important when it comes to identifying risk. As well as the work discussed above that highlights high levels of being wasted and being stunted at birth, recent systematic reviews published by the WaSt TIG highlight population-level data that show that wasting, stunting, and being WaSt are all more prevalent in boys than girls, and that wasting is higher in younger children, while stunting is higher in older children , 20. PosLancet series on maternal and child undernutrition has now increased focus, compared to previous series, on the associations between wasting and stunting  helps in our understanding of the total burden of undernutrition in children. In a piece written by members of the WaSt TIG for the 2016 Global Nutrition Report, for example, we were able to say that more than a third of children in low- and middle- income countries (LMIC) were affected by either wasting or stunting ; this waIn order to further understand the burden of undernutrition, we also need to measure and report on the incidence of wasting. Given the transitory nature of wasting in particular, where a child can experience several acute episodes during a year, the use of cross‐sectional data is likely to lead to considerable underestimation of the actual burden. In Mali, Huybregts et al.  found thLancet (2021) maternal and child undernutrition update series, Victora et al. , it hinders our understanding of the relationship between the 2 conditions or processes and, ultimately, of the most effective responses , 28. Thea et al.  presentea et al. .Stunting and wasting share many causal pathways  yet, as High prevalences of wasting (or being wasted) and stunting (or being stunted) are present in infants at birth. Recent estimates from large, longitudinal cohorts suggest that 13% of infants in LMIC are born stunted, rising to 20% in South Asia , and 12%Mertens et al.  have alsWork in Nepal has shown that seasonality has different associations with a wide range of factors that impact early nutrition, such as maternal diet and food insecurity, maternal BMI, and neonatal anthropometry . SeasonaFuture research needs to unpack more precisely what seasonality represents as a proxy or marker for multiple environmental exposures that impact maternal and child nutrition in different contexts. These include the infectious disease burden; food prices, scarcity, or diversity; direct effects of temperature on fetal growth; and maternal physical activity and energy balance. To do this, as discussed in recent work by Marshak et al. , there iWork supported by the WaSt TIG and others that has drawn on longitudinal data has been valuable in better understanding the relationship between wasting and stunting, especially whereby episodes of wasting contribute to later episodes of stunting . LongituWhilst some data exist, there is a need for more robust longitudinal panel data and tracking of growth over time, including sufficient data collection frequency, improved data quality, the capture of more appropriate exposures , and the calculation of growth velocity as an outcome. This would help to improve our understanding of the incidence of wasting, the association between wasting and stunting at the population level, and the pathways and risk factors associated with becoming stunted or wasted. Additionally, it could help to elucidate how the process of stunting and wasting is affected by temporal factors, such as season, and by geography. More longitudinal data are also needed to fill particular research gaps linked to the relationship between being WaSt and mortality. For example, we do not yet know whether it makes a difference to mortality risk being WaSt occurs in a child who has been stunted for months or years then experiences rapid weight loss, compared to a persistently wasted child who slowly becomes stunted through limited linear growth. Nor do we know whether the heightened mortality risk is limited to those experiencing a tight time frame of concurrence , or whether episodes of wasting and stunting falling consecutively but not necessarily simultaneously also confers an elevated mortality risk. Whilst new data could help to fill these gaps, existing longitudinal data sets, reanalyzed through a wasting and stunting lens, might be equally as valuable.Many recent leading nutrition publications, strategies, and plans call for more and improved research to support the delivery of impactful actions for the prevention and treatment of child undernutrition. “No Time to Waste,” UNICEF's approach for the prevention, early detection, and treatment of wasting in early childhood, for example, highlights the need to unpack the context-specific determinants and drivers of wasting in order to develop appropriate preventive interventions that include the elements needed to address wasting alongside stunting . The UN'In order to respond to these calls, we need to improve several aspects of the way we design and conduct nutritional research. This paper has discussed several key areas that future nutrition research should prioritize, informed by several years of work by the WaSt TIG:Systematically report on the combined extent of wasting and stunting in the 0‐ to 59‐month population and on the concurrence of stunting and wasting, including better measurement and reporting of the incidence of wasting.Increase focus on the process of stunting and wasting rather than the outcomes themselves, including the composite stresses that impact mothers, especially during pregnancy, and the impacts of addressing 1 outcome  on the other .Develop a better understanding of the risk factors that drive child weight loss and linear growth faltering in different contexts at different times. This is critical for understanding and interrupting the process of becoming stunted and wasted. This includes the need to better understand how these factors differ for infants born stunted and/or wasted compared with infants that develop wasting and/or stunting around 3 to 4 months of age. More research therefore needs to include the preconception, in utero, and infancy periods, and studies should focus on both the mother and child.The importance of season is paramount to better understanding the drivers of infant and child weight and linear growth faltering. It relates to variability in factors that underlie early nutritional stress, such as women's agricultural workload, risks of maternal or child infections, availability of harvest for consumption, and seasonality of wages. To better understand the main seasonal drivers of poor fetal and child growth in different contexts, and how we can best mitigate the negative effects they are having, we need to improve both the design and analysis of nutrition seasonality research. Age and sex have also been highlighted as factors that drive vulnerability to undernutrition. As such, there is a need to power studies sufficiently to enable stratification by age and sex categories in order for important differences in risk between groups to be highlighted more widely.To enable this, we need more robust longitudinal panel data and to track growth over time, starting with measurements on the mother and not just the child. The calculation of growth velocity as an outcome, as well as the estimation of the prevalence of undernutrition, would support a better understanding of those growth trajectories prior to wasting, stunting, and being WaSt that indicate the highest risk of mortality. Whilst the collection of new data would help to fill these knowledge gaps, existing longitudinal data sets, reanalyzed with a wasting and stunting lens, might be as equally valuable. There is also a need to include gestational age and body composition measurements in studies that are looking at changes in nutritional status over time.Use diagnostic criteria that are known to better identify those infants and children at high risk of death. This may have implications for treatment that need to be explored. Work by the WaSt TIG has found that mortality risks differ according to the combination of deficits  that a child experiences, and suggests that different levels of treatment intensity may be appropriate based on the deficit(s) present . This is important to consider given both the increase in caseloads likely if a combined case definition  were to be adopted by treatment programs and the need to control program costs and resource implications.Develop more innovative and early markers to predict, identify, and monitor children at short-term and long-term risks of weight and linear growth faltering. This includes markers that can identify risks for inadequate fetal growth in pregnancy, such as nutritional deficits, placental pathology, or maternal disease, or physical or psychosocial threats to women.Supporting progress in all these areas requires research collaborations across interest groups that move beyond a focus on single forms of undernutrition and ensures that there is equal attention given to wasting, wherever it is present, as to other forms of malnutrition. Given the overlap in determinants of stunting and wasting, there are also considerable learning opportunities provided by robust program impact evaluations that examine impacts upon both stunting and wasting across both development and crisis settings . Donors,