GLIM malnutrition caused a 2.8-fold increase in the development of AF. Conclusions: Screening for malnutrition in the elderly is essential. According to GLIM criteria, malnutrition may play a role in the development of AF and increase one-year mortality in the elderly.Background: Atrial fibrillation (AF) is the most common rhythm disorder in the elderly. The AF can cause life-threatening thromboembolic complications. Therefore, there is a need to determine the risk factors of AF. In this study, we aimed to examine the association of markers of malnutrition with AF in individuals aged 75 years and older and to find the factors that may affect mortality. Methods: In this prospective study, 358 consecutive individuals aged 75 years and older presenting to the cardiology outpatient clinic were included. All participants were divided into AF and sinus rhythm (SR) groups. In addition, a questionnaire and scoring system were used to assess malnutrition status. Information was obtained from all patients through outpatient clinic visits or telephone interviews for one year. Death from any cause was considered as the endpoint. Results: AF was observed in 71 (19.8%) patients. Death was higher in patients with AF ( The AF is the most frequent arrhythmia in elderly individuals . In lateThe average life expectancy of the elderly has increased due to technological developments in health, early recognition of diseases, and innovations in treatment modalities. The increasing elderly population has led to the need for a detailed examination of nutrition in the elderly . NutritiElderly AF patients (≥75 years of age) have difficulty presenting to healthcare institutions due to multiple comorbid diseases and poor nutritional status. Consequently, they are not sufficiently represented in clinical trials . AlthougThis prospective, cross-sectional study included 388 consecutive participants aged ≥75 years who presented to the cardiology outpatient clinic from three centers between June 2022 and August 2022. Thirty of these participants were excluded from the study. The study was conducted with the remaining 358 patients . Data weThirty patients did not meet the criteria for the study. The exclusion criteria were as follows: moderate to severe rheumatic valve, prosthetic valve, acute or chronic infections, sepsis, malignancy, and life expectancy of less than three months.p waves and irregular R-R intervals are diagnostic for AF [Atrial fibrillation is the most common rhythm disorder in the elderly, characterized by irregular, chaotic beats in the atria, with an atrial rate ranging between 350–600 beats/min. On electrocardiography, the absence of evaluable c for AF . The AF c for AF . In patiEchocardiography was performed in all patients according to European and American echocardiography guidelines ,26. EchoCONUT is a nutritional index derived from albumin, total cholesterol, and lymphocyte count parameters. The serum albumin level indicates the protein reserve in the body. Total cholesterol represents the calorie expenditure of individuals. The total lymphocyte count is an indicator of the immune system. Malnutrition can lead to a decrease in lymphocytes due to a compromised immune system. Utilizing these three parameters to create a scoring system yields a total CONUT score. A high CONUT score indicates the severity of malnutrition . CONUT pThe prognostic nutritional index (PNI) assesses a person’s nutritional and immunological status. Research has evidenced that PNI can be a prognostic marker for malignancy, infectious diseases, and cardiovascular diseases. When the PNI score is lower, the risk of malnutrition is higher. The PNI score is calculated using the formula ‘10 × serum albumin (g/dL) + 0.005 × total lymphocyte count (mm3)’. Severe malnutrition was classified as PNI below 35, moderate malnutrition as PNI between 35–38, and PNI above 38 as normal .The Geriatric Nutrition Risk Index (GNRI) is a simple screening tool to estimate the risk of nutrition-related morbidity and mortality in older patients. This index was first reported by Bouillanne et al. Patients were divided into four groups according to GNRI: major risk group (GNRI: <82), intermediate risk group (GNRI: 82–<92), low-risk group (GNRI: 92–98), and no-risk group (GNRI: >98). The GNRI is also used to assess the prognosis of chronic diseases .This index evaluates the reliance of elderly patients on external aid to accomplish their daily physical activities and requirements. This index has ten questions, and the responses are based on the patient’s or their family’s opinions. It is divided into five categories: fully independent (≥100), partially dependent (91–99), moderately dependent (62–90), highly dependent (21–61), and fully dependent (0–20) ,31.Frailty was assessed with the FRAIL scale. It consists of a questionnaire with five questions  named after its initials. The questionnaire with yes and no answers is practical and easy to assess. Frailty score was categorized as 0 normal, 1–2 pre-fragile, and 3–4 fragile .This survey evaluates the cognitive abilities of older people and the effects of organic brain damage on them. The questionnaire covers verbal fluency, spatial cognition, arithmetic, and logical thinking. Parents or caregivers can provide information about the test. The scale consists of 10 questions. A high score indicates brain damage (maximum score: 10). Scoring is based on race and educational background. The scoring is as follows: 0–2, normal; 3–4, mild mental disorder; 5–7, moderate mental disorder; ≥8, severe mental disorder .Sarcopenia is a significant component of physical frailty. It is assessed with the SARC-F screening tool, which can be easily identified in daily practice. The SARC-F is a questionnaire consisting of five questions: Strength (S), Assisted Walking (A), Rising from a Chair (R), Climbing Stairs (C), and Falls (F). Each question can be scored from 0 to 2. The recommended cut-off value is ≥4 points. A correlation between SARC-F and clinical outcomes has been established in elderly and chronic diseases .Mini-mental state examination (MMSE) score can be utilized to ascertain cognitive impairment in acute or chronic illness. It assesses six mental abilities: orientation to time and place, attention/concentration, short-term memory, language skills, visual-spatial abilities, and the ability to understand and follow instructions. The MMSE was split into two parts: normal and anormal. A top score of 30 was attainable for the MMSE. A score of 25 or higher is classified as normal. If the score is below 24, the result is generally considered abnormal and indicates possible cognitive impairment .This consensus begins with a validated initial screening test to detect patients “at risk”. The Nutritional Risk Screening-2002 was used for the initial screening test. According to this test, a score of 3 or above indicated the individual was at risk of malnutrition. Individuals at risk of malnutrition were passed to the second stage. Assessing and grading the severity of malnutrition is the second step. This step is evaluated according to three physical criteria: low BMI, unintended weight loss, and a decrease in muscle mass, as well as two etiologic criteria—a decrease in food consumption or digestion and an inflammation/illness burden. According to GLIM criteria, at least one phenotypic criterion and one etiologic criterion must be present to diagnose malnutrition. Phenotypic criteria are used to grade the severity of malnutrition into stage 1 (moderate) and stage 2 (severe) .t-test was applied for those conforming to a normal distribution. The normality of data distribution was assessed by calculating skewness and kurtosis statistics. Patients were also categorized into three groups according to the CONUT and GLIM nutritional index. Pearson correlation analysis was performed between malnutrition scores. In addition, patients were followed up for one year. Comparisons were also made between those who died and those who survived. Using Kaplan–Meier analysis, survival tables were created. Finally, a multivariate logistic regression analysis was performed for factors affecting the presence of AF. Bland–Altman plots were used to evaluate measurement bias. p-value < 0.05 was considered statistically significant.All data were analyzed using SPSS 22.0  software. When individuals over 75 years of age were divided into AF and SR groups, the Mann–Whitney U test was used to compare continuous variables for those not conforming to normal distribution, and an independent samples A total of 358 participants were included in the study. Of these, 187 (52.2%) were male, and the mean age was 82.71 ± 5.83 years. Coronary artery disease was the most common disease in 55 (15.4%) participants. AF was observed in 71 (19.8%) patients. The mean CONUT score (1.54 ± 1.58), PNI (37.88 ± 5.19), GNRI (95.41 ± 16.49), Pfeiffer Questionnaire (3.37 ± 2.30), FRAIL scale (1.20 ± 1.01), Barthel index (84.83 ± 20.51), SARC-F (2.91 ± 1.88), MMSE (20.25 ± 6.08) were obtained in all participants. GLIM malnutrition was present in 139 (38.8%). Seventy-two (20.1%) patients had a high CONUT score. The correlation analysis between the participants’ scores is shown in p = 0.583) was similar between the two groups, while male gender was higher in the AF group. Known diseases were similar between the two groups.Demographic and clinical characteristics of the AF and SR groups are shown in p = 0.010) and LVEF (p = 0.036) were lower in the AF group. LVEDD (p = 0.001) was higher in the AF group.Biochemical and echocardiographic results of the groups are shown in p = 0.122) was similar between groups.Participants were divided into two groups, with and without malnutrition, according to GLIM criteria. They were also categorized based on high and low CONUT scores. AF was noticeably elevated in the high CONUT and GLIM malnutrition groups. Comparison with other parameters is expressed in p ≤ 0.001) and low LVEF (p = 0.001) had the highest statistical significance in the dead group. The association with other parameters is shown in During the 1-year follow-up period, 22 participants died from any cause. When the survivors and the dead groups were compared, the presence of AF  and low LVEF  were found to be independent risk factors for AF (The presence of GLIM malnutrition, LVEF, albumin, hypertension, diabetes mellitus, chronic obstructive pulmonary disease, and GFR were included in the logistic regression analysis. The presence of GLIM malnutrition (B = 1.036, s for AF .2DS2-VASc score is the most commonly used scoring system for initiating anticoagulant therapy. According to this scoring, it is recommended that anticoagulant therapy be initiated in all patients with AF over 75 years of age to prevent cerebrovascular thromboembolism [The AF, characterized as chaotic atrial contraction, can cause severe complications in the elderly. Many risk factors for AF have been identified. Antiarrhythmic and anticoagulant drugs are used in the treatment of AF. Anticoagulant therapy could be the most critical aspect of treatment. The CHAembolism . In partembolism . Cognitiembolism . In thisAdequate nutrition and diet are some of the most essential basic needs for disease prevention and healthy living in elderly patients. Loss of appetite causes malnutrition in elderly patients, leading to muscle loss and, consequently, weight loss . A lack The literature has revealed that malnutrition is essential in the prognostic process. Generally, studies have utilized a single marker and method to define malnutrition. Therefore, diagnosing malnutrition using a single method may not produce accurate results . The oneThe risk of malnutrition in patients with heart failure varies between 16% and 90% in studies. The broad spectrum of malnutrition rates in these reports is owing to distinctions in the medical characteristics of the patients and various definitions of malnutrition . Sze et The extent of malnutrition may differ across different studies. Malnutrition was present in 80% of elderly patients with hypertension, and 65.7% of elderly patients with AF had malnutrition, as measured by at least one malnutrition score ,49. In aOral nutritional supplements positively affect energy intake and body weight in elderly patients with malnutrition. A sufficient diet for those who have undergone cardiac surgery has been found to correlate with an earlier discharge and more rapid recovery . StudiesAdverse clinical outcomes may occur in AF patients due to chronic disease, partly caused by malnutrition and arrhythmia. Malnutrition is a determinant of disease severity and causes the disease to become chronic. Chronic diseases also can result in malnutrition. Hypoalbuminemia is caused by AF, which triggers inflammation, leading to enhanced protein degradation and reduced protein synthesis. Blood contains albumin as its most plentiful protein, and it is responsible for controlling osmotic pressure and partaking in the transportation of drugs in the bloodstream. Studies have shown that low albumin levels increase cardiovascular disease and mortality rates . ChronicThe vicious circle between chronic diseases and malnutrition can explain the association of malnutrition with AF in elderly patients. AF is a chronic disease and is accompanied by an inflammatory process. Protein synthesis decreases and predisposes to hypoalbuminemia with the activation of inflammatory processes . The chrThe high CONUT score in some patients is due to statin therapy’s low serum cholesterol levels. Therefore, the CONUT score overestimates malnutrition in patients receiving statin therapy. PNI is calculated by serum albumin level and lymphocyte count, reflecting immunologic nutritional status. In addition, the PNI score does not include mild malnutrition and, therefore, overestimates moderate and severe malnutrition. Since the PNI and GNRI scores do not include cholesterol compared to the CONUT score, they show less malnutrition in patients receiving statin therapy. Therefore, the GNRI score seems more appropriate to determine malnutrition in our study. Regardless of the malnutrition scores used in our study, malnutrition in elderly AF patients was associated with unfavorable clinical outcomes. However, in our study, GNRI and PNI were similar between the dead and survivor groups, whereas the CONUT score was higher in the dead group. This was because no participants were receiving statin treatment in the dead group.Our study has some limitations. First, this study included elderly people in a specific region. Different results may be obtained in different races. Secondly, the patients’ socio-economic conditions were not considered in this study. Therefore, it may not be generalizable to the whole population. Third, the participants’ nutritional indices were assessed during inclusion. Changes within one year were not taken into account.Malnutrition in the elderly may lead to an increased likelihood of AF. Calculation of malnutrition risk scores is essential in the clinical examination of patients. Improving malnutrition status is important in preventing the development of AF, which can cause serious problems, especially in the elderly. Our study showed that malnutrition predisposes to the development of AF. In addition, an increase in 1-year mortality was observed in those with AF and malnutrition defined according to GLIM criteria.


We characterized the physical and physiological profiles of high-level female Portuguese handball players and examined the relationships between their anthropometric characteristics, general motor performance and cardiopulmonary fitness. Twenty-four high-level female handball players with an average age of 23.6 ± 5.5 years, height of 173.6 ± 5.1 cm and body mass of 72.6 ± 9.1 kg volunteered to participate. A Pearson correlation test was used to assess the relationship between variables. Direct relationships were observed between the players’ height and arm span (r = 0.741), as well as between their squat jump and countermovement jump performances with regard to body mass . The 9 m jump shot has a large relationship with the 7 m standing throw (r = 0.786) and between left hand dynamometry and body mass index (r = 0.595). The 30 m sprint has a relationship with the 7 m standing throw (r = −0.526) and the 9 m jump throw (r = −0.551). Oxygen uptake has a relationship with the players’ height (r = −0.482) and time limit (r = 0.513), while the fitness index has a relation to the players’ height (r = −0.488) and arm span (r = −0.422). Our results should be considered when using physical testing to plan optimal physical training regimens in elite team handball. Handball is one of the most popular team sports, played in two 30 min halves with a 15 min break ,3. The pConsequently, physical performance should be well developed to match the game requirements, with muscular strength and specific skills requiring considerable development to become an elite player . These aThe physiological response of handball players has also been examined, showing that, although low-intensity activities  in, for example, female elite team handball constituted about 75% of mean effective playing time, the players demonstrated a mean relative workload of around 80% of maximal oxygen uptake during the periods of effective match-play . ConsideAchieving and maintaining higher physical and physiological conditions also require strategies to prevent and monitor health status. As a part of general health, well-being and quality of life, oral health status is an indisputable determinant to take into consideration in sports, particularly at the high standards required in elite performance ,13. TherTwenty-four high level female handball players, who were healthy, had no previous injuries, competed at national and international level, and were currently playing for the Portuguese national team, voluntarily participated in the current study. All players were assessed in two different occasions over a one-week period (with a minimum of 24–48 h between tests). In the first session, anthropometric and motor performance general characterization was conducted. In the second session, players completed a square wave transition running test from rest to the velocity at maximal oxygen uptake until voluntary exhaustion. Verbal encouragement was given during the exercise protocol. Players were informed about the experimental procedures and possible risks involved and signed a consent form to participate. The experimental procedures were approved by the Ethics Committee (CEFADE282020), following the Declaration of Helsinki and the guidelines of the World Medical Association for research on humans. Regarding the anthropometric traits, stature was measured using a Seca 206 stadiometer , arm span was assessed with an anthropometer and a sliding caliper  and body mass, body fat and fat-free mass were obtained using a bioimpedance scale . The anthropometric technical error of measurement was 0.8 cm for stature, 0.9 cm for arm span, 0.1 cm for hand breadth and length, and 0.9 and 0.3 kg for body mass and fat-free mass, respectively. All measurements followed the International Working Group on Kinanthropometry protocols .(i)Lower-limbs explosive power was estimated by performing a squat and a countermovement jump on a Bertec 6090 force platform  operating at 2000 Hz and a horizontal jump with upper limbs swing ;(ii)Abdominal muscular strength and endurance was estimated by the maximum number of sit-ups in 60 s ;(iii)Shooting velocity was performed at 7 (standing) and 9 (jumping) m from a standard handball goal, without opposition, and measured with a Stalker II radar gun  positioned on a tripod at 2 m from the back of the goal;(iv)Static handgrip strength was calculated using a hand-held digital dynamometer , and performing force in a standing position was calculated with the dominant and non-dominant upper limbs fully extended ;(v)Upper-limb explosive power was estimated by throwing a 3 kg medicine ball from chest height while in a seated position against a wall ;(vi)Running velocity was estimated by performing a 30 m sprint using a photoelectric cell system Speed Trap II  with 2.3% CV .The players general motor performance was assessed using the following functional tests:Two trials were performed for sit-ups and sprints, and three trials were conducted for vertical jumps, with the best value used for posterior analysis . Three t−] analysis was collected from a fingertip at the third minute after running.After an individualized running warm-up (of ~5 min duration at low intensity), handball players performed a square wave transition running test on a treadmill with 0% grade   from res2∙kg−0.73∙min−1) was also calculated [The collected respiratory and pulmonary gas exchange data were examined to exclude occasional errant breaths caused by swallowing, coughing or signal interruptions. Only data between oxygen uptake mean ± 3 standard deviations were included for analysis, being subsequently smoothed using a moving and time average of 3 breaths and 10 s, respectively ,20. Indilculated . Oral health screening was undertaken by an experienced dentist. The examination was conducted via a visual oral inspection of molar relationship (in accordance with Angle class), malocclusions, number of decayed, filled and missing teeth and periodontal status  . The oraComputations were completed using GraphPad Prism 6, with mean and standard deviation values for descriptive analysis being reported for all variables. The Levene and Shapiro–Wilk tests were used to assess data homogeneity and normality. A Pearson correlation test was used to assess the relationship between the players anthropometrics, general motor performance and physiological variables and was interpreted as small (0.1–0.3), medium (0.3–0.5) and large (0.5–1.0). Significance level was set at 5%.−2. From the 24 players, 5  were left hand dominant.The high-level handball players’ average physical characteristics were as follows: 23.6 ± 5.5 years of age, a height of 173.6 ± 5.1 cm, a seated height of 137 cm, an arm span of 176.0 ± 7.9 cm, a body mass of 72.6 ± 9.1 kg, a fat free mass of 58.1 ± 6.5 kg, a body fat of 20.2 ± 0.1% and a body mass index of 24.1 ± 2.6 kg∙mp < 0.05).n = 1), open bite (n = 2), edge-to-edge bite (n = 3) and dental crowding (n = 2). Overall, 15 players were free from decayed teeth, with the worst findings observed in 3, 5 and 1 players affected by 1–2, 3–4 and >4 dental caries, respectively. Meanwhile, 8 subjects presented at least 1 tooth filling, and, excluding third molars, only 2 players had missing teeth . An absence of supragingival plaque was verified for 16 players, and very good and good overall oral health hygiene were attributed to ~79% of the sample. The use of mouthguards during training or actual games was not reported by any player.Data related to molar relationship, the presence of malocclusions, dental and periodontal health, and oral hygiene status are presented in The relations between the assessed variables are displayed on The aim of the current study was to characterize the physical and physiological profile of Portuguese high-level female handball players by relating their anthropometric characteristics, general motor performance and cardiopulmonary variables and by examining their general oral health status. Our results indicate that the female handball players had a good average body mass and good general motor performance ,8. In adIn female handball, players’ physical characteristics are critical for obtaining success and are differentiated by appropriate morphological and motor features . It was It has been showed that the game requires heavier players to not only block, push and pull the opponent but also to attack and shoot . The cur−1∙min−1, with mean values similar to those of the Brazilian female national team [The intermittent nature of the handball game enhances the need for a well-developed aerobic capacity that allows the players to maintain a high level of performance over the playing time . In factnal team  but lowenal team  and Norwnal team .Complementarily, it is described that subjects engaged in sports, especially at professional and high-level standards, are more likely to develop poor oral health conditions. These includes dental caries, periodontal disease and wisdom tooth eruption, which enhance the risk of body inflammation, infection and muscle injuries, impacting overall health and the expected performance outcomes ,35. RegaOur data allow the formation of training plans according to players’ individual and overall performance, which can particularly improve general motor performance and the physiologic characteristics that have a direct impact on the game outcome. In addition, data from the oral status are valuable as they help to prevent situations  that could keep the players away from training or competition. A limitation of the current study was the lack of a larger sample that might have introduced selection bias in the results and that would allow for comparisons in terms of player seniority and position. Future studies should consider the influence of match efficiency. Since oral health assessments were conducted using simple dental tools and without specialized equipment , our oral health-related data also need to be carefully interpreted because some oral diagnoses could be underestimated or not fully detected.The current study provides valuable insights into the physical fitness profile of female handball players, particularly focusing on high-level Portuguese handballers. Our results highlight the importance of anthropometric characteristics related to general motor performance and cardiopulmonary fitness, which are crucial factors for success in the game and the general development of female players during the training season. In addition, oral health seems to be poorly monitored in the currently studied group, evidencing the need for oral health assessment in elite sports. Strategies to prevent oral disease and promote oral health need to be developed and evaluated in collaboration with a sports dentistry team.


Surgeons who receive reliable feedback on their performance quickly master the skills necessary for surgery. Such performance-based feedback can be provided by a recently-developed artificial intelligence (AI) system that assesses a surgeon’s skills based on a surgical video while simultaneously highlighting aspects of the video most pertinent to the assessment. However, it remains an open question whether these highlights, or explanations, are equally reliable for all surgeons.Here, we systematically quantify the reliability of AI-based explanations on surgical videos from three hospitals across two continents by comparing them to explanations generated by humans experts. To improve the reliability of AI-based explanations, we propose the strategy of training with explanations –TWIX –which uses human explanations as supervision to explicitly teach an AI system to highlight important video frames.We show that while AI-based explanations often align with human explanations, they are not equally reliable for different sub-cohorts of surgeons , a phenomenon we refer to as an explanation bias. We also show that TWIX enhances the reliability of AI-based explanations, mitigates the explanation bias, and improves the performance of AI systems across hospitals. These findings extend to a training environment where medical students can be provided with feedback today.Our study informs the impending implementation of AI-augmented surgical training and surgeon credentialing programs, and contributes to the safe and fair democratization of surgery. Surgeons aim to master skills necessary for surgery. One such skill is suturing which involves connecting objects together through a series of stitches. Mastering these surgical skills can be improved by providing surgeons with feedback on the quality of their performance. However, such feedback is often absent from surgical practice. Although performance-based feedback can be provided, in theory, by recently-developed artificial intelligence (AI) systems that use a computational model to assess a surgeon’s skill, the reliability of this feedback remains unknown. Here, we compare AI-based feedback to that provided by human experts and demonstrate that they often overlap with one another. We also show that explicitly teaching an AI system to align with human feedback further improves the reliability of AI-based feedback on new videos of surgery. Our findings outline the potential of AI systems to support the training of surgeons by providing feedback that is reliable and focused on a particular skill, and guide programs that give surgeons qualifications by complementing skill assessments with explanations that increase the trustworthiness of such assessments. Kiyasseh et al. compare the quality of feedback provided to surgeons by artificial intelligence (AI) to that provided by human experts. Teaching an AI system to explicitly follow human explanations improves the reliability and reduces the bias of AI-based feedback. Such feedback can be automated, in theory, by artificial intelligence systems9. To that end, we recently developed a surgical artificial intelligence system (SAIS) that assesses the skill of a surgeon based on a video of intraoperative activity and simultaneously highlights video frames of pertinent activity. We demonstrated that SAIS reliably automates surgeon skill assessment10 and also examined the fairness of its assessments11. In pursuit of developing a trustworthy AI system capable of safely automating the provision of feedback to surgeons, we must ensure that SAIS’ video-frame highlights, which we refer to as AI-based explanations, align with the expectations of expert surgeons 13 and be equally reliable for all surgeons 14. However, it remains an open question whether AI-based explanations are reliable and fair. If left unchecked, misguided feedback can hinder the professional development of surgeons and unethically disadvantage one surgeon sub-cohort over another .Surgeons seldom receive feedback on how well they perform surgery15 is used, such as natural language processing16 and protein modelling18. An element with a higher attention score is assumed to be more important than that with a lower score. Similarly, with a transformer architecture which operates on videos, SAIS10 generates an attention score for each frame in a surgical video, with high-attention frames assumed to be more relevant to the surgeon skill assessment. When such an AI system’s explanations align with those provided by humans, it can direct surgeons to specific aspects of their operating technique that require improvement while simultaneously enhancing its trustworthiness19. As such, there is a pressing need to quantify the reliability of explanations generated by surgical AI systems.Although an explanation can take on many forms, it often manifests as the relative importance of data (attention scores) in disciplines where the attention-based transformer architecture22 or clinical variables23. However, these studies remain qualitative and thereby do not systematically investigate whether explanations are consistently reliable across data points. Studies that quantitatively evaluate AI-based explanations often exclude a comparison to human explanations25, a drawback that extends to the preliminary studies aimed at also assessing the fairness of such explanations27. Notably, previous work has not quantitatively compared AI-based explanations to human explanations in the context of surgical videos, nor has it proposed a strategy to enhance the reliability and fairness of such explanations.Previous studies have investigated the reliability of AI-based explanations that highlight, for example, important patches of a medical image10 –that we previously developed and which was shown to reliably assess the skill level of surgeons from videos. Through evaluation on data from three geographically-diverse hospitals, we show that SAIS’ attention-based explanations often align, albeit imperfectly, with human explanations. We also find that SAIS generates different quality explanations for different surgeon sub-cohorts , which we refer to as an explanation bias. To address this misalignment between AI-based and human explanations, we devise a general strategy of training with explanations –TWIX –which uses human explanations as supervision to explicitly teach an AI system to highlight important video frames. We find that TWIX enhances the reliability of AI-based explanations, mitigates the explanation bias, and improves the performance of skill assessment systems across hospitals. With SAIS likely to provide feedback to medical students in the near future, we show that our findings extend to a training environment. Our study suggests that SAIS, when used alongside TWIX, has the potential to provide surgeons with accurate feedback on how to improve their operating technique.In this study, we quantify the reliability and fairness of explanations generated by a surgical AI system –SAISIn a previous study, we trained and deployed an AI system (SAIS) on videos of a surgical procedure known as a robot-assisted radical prostatectomy (RARP). The purpose of this procedure is to treat cancer by removing a cancerous prostate gland from the body of a patient. In general, to complete a surgical procedure, a surgeon must often perform a sequence of steps. We focus on one particular step of the RARP procedure, referred to as vesico-urethral anastomosis (VUA), in which the bladder and the urethra are connected to one another through a series of stitches. To perform a single stitch, a surgeon must first grasp the needle with a robotic arm (needle handling), and push that needle through tissue (needle driving), before following through and withdrawing the needle from the tissue to prepare for the next stitch . In this study, we leveraged SAIS’ ability to assess the skill-level of needle handling and needle driving when provided with a video sample depicting these activities.To obtain video samples from videos of surgical procedures, we adopted the following strategy. Given a video of the VUA step ( ≈ 20 min), we first identified the start and end time of each stitch (up to 24 stitches) involved in completing this step. We subsequently identified the start and end time of the needle handling and needle driving activities performed as part of each stitch. A single video sample reflects one such needle handling (or needle driving) activity ( ≈ 20 − 30s). As such, each VUA step may result in approximately 24 video samples for each activity , we only focused on needle handling for the purpose of our analysis  were collected under Institutional Review Board (IRB) approval from the University of Southern California in which informed consent was obtained (HS-17-00113). We have also obtained informed consent to present surgical video frames in figures.28). EASE was formulated through a rigorous Delphi process involving five expert surgeons that identified a strict set of criteria for assessing multiple skills related to suturing . Our team of raters comprised medical students and surgical residents who either helped devise the original skill assessment taxonomy or had been intimately aware of its details.We assembled a team of trained human raters to annotate video samples with skill assessments based on a previously-developed skill assessment taxonomy  upon watching a video sample. This reduces the level of expertise that a rater must ordinarily have in order to annotate a video sample. Second, the raters involved in the annotation process were either a part of the development of the EASE taxonomy or intimately aware of its details. This implied that they were comfortable with the criteria outlined in EASE. Third, and understanding that raters can be imperfect, we subjected them to a training process whereby raters were provided with a training set of video samples and asked to annotate them independently of one another. This process continued until the agreement of their annotations, which was quantified via inter-rater reliability, exceeded 80%. We chose this threshold based on (a) the level of agreement first reported in the study developing the EASE taxonomy and (b) an appreciation that natural variability is likely to exist from one rater to the next in, for example, the amount of attention they place on certain content within a video sample.10 where the lowest of all scores is considered as the final annotation.After completing their training process, raters were asked to annotate the video samples in this study with binary skill assessments (low vs. high skill). In the event of disagreements in the annotations, we followed the same strategy adopted in the original study28. In practice, we asked raters to exclusively annotate video samples tagged as low skill from a previous study . To recap, for the activity of needle handling, a low skill assessment is characterized by three or more grasps of the needle by the surgical instrument. For the activity of needle driving, a low skill assessment is characterized by either four or more adjustments of the needle when being driven through tissue or its complete removal from tissue in the opposite direction to which it was inserted. As such, raters had to identify both visual and motion cues in the surgical field of view in order to annotate segments of time as relevant. We reflect important time segments with a value of 1 and all other time segments with a value of 0.We assembled a team of two trained human raters to annotate each video sample with segments of time  deemed relevant for a particular skill assessment. We define segments of time as relevant if they reflect the strict set of criteria (or their violation) outlined in the EASE skill assessment taxonomyConsider that for a given video sample 30 s in duration, a human rater might annotate the first five seconds (0−5 s) as most important for the skill assessment. Based on our annotations, we show that ≈ 30% of a single video sample is often identified as important  of the criteria outlined in EASE are violated. Second, from an educational standpoint, studies in the domain of educational psychology have demonstrated that corrective feedback following an error is instrumental to learning [1]. As such, our focus on a low skill activity (akin to an error) provides a ripe opportunity for the provision of feedback. We do appreciate, however, that feedback can also be useful when provided for video samples depicting high skill activity . We leave this as an extension of our work for the future.To evaluate the reliability of AI-based explanations, we compared them to human-based ground-truth explanations. After normalizing AI-based explanations (between 0 and 1), we introduced a threshold such that frames with explanations that exceed this threshold are considered important, and unimportant otherwise. For each threshold, we calculated the precision; the proportion of frames deemed important by the AI system which are also identified as important by the human, and the recall; the proportion of all frames identified as important by the human which the AI system also identified as important. By iterating over a range of thresholds, we can generate a precision-recall curve.25.The precision-recall curve reflects the trade-off between the precision of AI-based explanations: the proportion of frames identified as important by the AI system which are actually important, and the recall of such explanations: the proportion of all important frames identified as such by the AI system. For example, recall=1 implies that 100% of the frames identified as important by a human are also identified as such by the AI system. However, an imperfect system can only achieve this outcome by flagging all frames in a video sample as important, irrespective of their ground-truth importance. Naturally, this is an undesirable outcome as the resultant feedback would no longer be temporally-localized, and would thus be less informative. We use the area under the precision-recall curve (AUPRC) as a measure of the reliability of AI-based explanations, as reported in previous studiesAlgorithmic bias is often defined as a discrepancy in the performance of an AI system across sub-cohorts of stakeholders. In this study, we define explanation bias as a discrepancy in the reliability of AI-based explanations across sub-cohorts of surgeons. The intuition is that such a discrepancy implies that a particular sub-cohort of surgeons would systematically receive less reliable feedback, and is thus disadvantaged at a greater rate than other sub-cohorts. To mitigate this explanation bias, we look to improve the reliability of AI-based explanations generated for the disadvantaged sub-cohort of surgeons, referred to as worst-case AUPRC.When dealing with live surgical videos, we measured the explanation bias against surgeons operating on prostate glands with different volumes, prostate glands with different cancer severity levels (Gleason Score), and against surgeons with a different caseload . To decide on these groups, we consulted with a urologist (A.J.H) about their relevance and opted for groups whose clinical meta information was most complete in our database in effort to increase the number of samples available for analysis. While it may seem out of the ordinary to define a surgeon group based on the prostate volume of a patient being operated on, we note that the distribution of such volumes can also differ across hospitals  . Greater attention, or importance, is placed on frames deemed more important for the ultimate skill assessment. Repeating this process for all data modalities, SAIS arrives at modality-specific video representations. SAIS aggregates these representations to arrive at a single video representation that summarizes the content of the video sample. This video representation is then used to output a probability distribution over the two skill categories (low vs. high skill).We outline the basic components of SAIS here and refer readers to the original study for more detailsN transformer encoders as the video representation, v . As the attention mechanism still applies to this video representation, we are able to measure its dependence on all frames in the video sample. The higher the dependence on a particular frame, the more important it is for the assessment of surgeon skill. We hypothesized that the temporal relationship between frames at the final layer of the transformer encoder is most strongly associated with the surgeon skill assessment, and as such, we extracted the attention placed on these frames. This method of explanations is referred to as attention in the Results section.To summarize a video sample with a single representation, SAIS adopts an approach often observed with transformer networks used for the purpose of classification; it learns a classification token embedding and treats its corresponding representation after the 10. Specifically, we trained SAIS using 10-fold Monte Carlo cross validation on data exclusively from USC. To ensure that we evaluated SAIS in a rigorous manner, each fold was split into a training, validation, and test set where each set contained samples from surgical videos not present in any of the other sets. Having formulated skill assessment as a binary classification task, we balance the number of video samples from each class (low vs. high-skill) in every data split . While doing so during training ensures that the model’s performance is not biased towards the majority class, balancing the classes during evaluation  allows for a better appreciation of the evaluation metrics we report. For evaluation on data from other hospitals, we deployed all 10 SAIS models (from the 10 folds). As such, we always report metrics as an average across these 10 folds.To train and evaluate SAIS, we adopted the same strategy presented in the original study32, we took inspiration from studies demonstrating the effectiveness of using explanations as a target variable36. To that end, we propose a strategy entitled training with explanations –TWIX –which explicitly teaches an AI system to generate explanations that match those provided by human experts .When dealing with the RGB frames of a video sample, SAIS first extracts spatial features from each frame and then captures the temporal relationship between these frames (see previous section for attention mechanism). Upon doing so, SAIS outputs both a single 10. In short, this involves 10-fold Monte Carlo cross-validation where each training fold consists of an equal number of samples from each class . The number of samples are provided in Supplementary Table We retrain SAIS on data exclusively from USC as reported in the original studypω, can now directly output the importance, θ which, in turn, are now affected by the inclusion of the importance loss, When SAIS is deployed on unseen data, the classification module, Further information on research design is available in the 25  by comparing them to those generated by human experts. To do so, we first trained and deployed SAIS to perform skill assessment on data from the University of Southern California (USC). We evaluated its explanations for the needle handling and needle driving activities by using the precision-recall (PR) curve25 Fig. a and nee25 Fig. b activit37. To measure this consistency, we trained SAIS on data from USC and deployed it on data from St. Antonius Hospital (SAH), Gronau, Germany and Houston Methodist Hospital (HMH), TX, USA  that previously experienced an explanation bias. Such an improvement would translate to a mitigation in the bias. To do so, we quantified the reliability of AI-based explanations for the disadvantaged sub-cohort of surgeons (worst-case AUPRC) after having adopted TWIX when assessing the skill-level of needle handling Fig. c and neeWe found that TWIX effectively mitigates the explanation bias across hospitals. This is evident by the marked increased in the reliability of SAIS’ explanations when assessing the skill-level of needle handling Fig. c for theAlthough TWIX was designed to better align AI-based explanations with human explanations, we hypothesized that it might also improve the performance of AI skill assessment systems. The intuition is that by learning to focus on the relevant aspects of the video, SAIS is less likely to latch onto spurious features. To investigate this, we present the performance of SAIS, both with and without TWIX (w/o TWIX), when assessing the skill-level of needle handling and needle driving Table .Table 2TAs expected, we found that TWIX improves AI-based skill assessments across hospitals. This is evident by the higher AUC values with TWIX than without it. For example, when SAIS assessed the skill-level of needle driving Table , it achi10. This was motivated by the promising capabilities demonstrated by SAIS and its impending deployment for the provision of feedback. Here, we show how variants of SAIS affect the reliability of explanations  of SAIS as that presented in the original studyons Fig. a and theons Fig. b, and whons Fig. a and theons Fig. b. These Our study builds the foundation for the future implementation of AI-augmented surgical training programs. It is very likely that, in the short run, SAIS will be used to assess the skills of surgical trainees and provide them with feedback on their performance. As with practicing surgeons, it is equally important that such trainees are also not disadvantaged by AI systems. We therefore deployed SAIS on videos from a training environment to assess, and generate explanations for, the skill-level of the needle handling activity performed by participants in control of the same robot otherwise used in live surgical procedures (see Methods) Fig. , c-f.38). Consistent with previous findings, we found that TWIX mitigates the explanation bias, as evident by the improvement in the worst-case AUPRC , which can automatically be decoded by systems like SAIS. Second, constraining AI-based explanations to match human explanations overlooks their promise for the discovery of novel aberrant  surgeon behaviour, contributing to the scientific body of knowledge and informing future surgical protocols. Although such discovery is beyond the scope of the present work, it is likely to yield value, for example, when associating intraoperative surgical activity with postoperative patient outcomes.51, in which explanations play an essential role52, its embodiment remains elusive. In pursuit of that definition, recent frameworks such as the feedback triangle53 may hold promise, emphasizing the cognitive55, affective, and structural dimensions of feedback. Third, while we have demonstrated that SAIS generates explanations whose reliability differs for different surgeon sub-cohorts, it remains to be seen whether this discrepancy will result in notable harmful consequences. After all, a discrepancy may only translate to an explanation bias if it is unjustified and harmful to surgeons56.Several open questions remain unaddressed. First, it remains unknown whether SAIS’ explanations accelerate the acquisition of skills by surgical trainees. To investigate this, we plan to conduct a prospective trial amongst medical students in a controlled training environment. Second, despite attempts to define optimal feedback57 in reference to learning how to perform surgical procedures. In contrast, AI-augmented surgical training programs can democratize the acquisition of surgical skills on a global scale59 and improve the long-term postoperative outcomes of patients.Surgical training programs continue to adopt the 20th century Halstedian model of “see one, do one, teach one”Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Description of Additional Supplementary FilesSupplementary InformationPeer Review FileReporting Summary


Both microbial pathways can yield nitrous oxide (N2O) as an end-product, which is a powerful GHG (2O emissions (2 (eCO2) atmosphere, rising temperatures, and water scarcity (Nitrogen (N) fertilization is required to improve crop yield. However, the inefficiency of agricultural systems, where only 30%–50% of applied N is used by crops, provokes significant losses to the environment in the form of N gas emissions or nitrate  remodels plant physiology, with enhanced photosynthesis and reduced stomatal conductance as the primary effects, leading to improved water use efficiency and potentially boosting plant productivity  assimilated , and dimethylpyrazol (DMP)-based NIs (Brachiaria humidicola and Sorghum bicolor (Leymus racemosus, a wild wheat relative with high BNI activity (Increasing N fertilization to address crop N demand in a climate change scenario seems undesirable, as excess soil N could further aggravate the aforementioned water and air pollution . Therefoased NIs . Howeverased NIs . As a reased NIs . Since t bicolor , the sea bicolor . Wheat cactivity , has rai2 is still far from being understood, with variable results shown in the literature (2 led to increased N2O emissions due to enhanced nitrification and/or denitrification. High rates of soil nitrification are predicted in the future because nitrifiers use CO2 as carbon source for growth and NH4+ as energy source (2, alone or in combination with increased temperature (2O losses regardless CO2 level, although further studies are needed to confirm their inhibition efficiency under eCO2 in the field and considering different soil types and environmental conditions. Additionally, anticipating how eCO2 will affect the plant’s capacity to synthesize and release BNIs, as well as their efficiency in suppressing nitrification, is of great relevance to propose effective strategies to increase NUE by crop plants under future conditions.How N cycling, and nitrification in particular, will be affected in agrosystems by future climatic conditions, as eCOterature . In a mey source . Indeed,perature . Althougperature . Bozal-L44+oxidation by inhibiting soil nitrification reduces N leakage while promoting NH4+ stability, thus presumably favoring a more NH4+-based nutrition. This will surely promote greater yield potential through a more efficient assimilation of co-existent N forms (2. Nonetheless, high NH4+ content in soil may entail a stressful situation for crop performance (4+ as N source are required. Because plant NH4+ assimilation is dependent on proper C-skeleton supply, conditions favoring photoassimilate production, such as eCO2 or direct carbon provision, have been shown to alleviate the symptoms associated with ammonium stress (2 may be advantageous for improving the performance of BNI-producing plants grown in the presence of enhanced NH4+ (From an evolutionary point of view, the BNI capacity is considered a plant response to adapt to N-scarce environments . Convers N forms , which iformance ; hence, m stress . Therefo4+ assimilation and plasma membrane H+-ATPase activity, stimulates BNI release (4+-based nutrition would act as a positive feedback regulatory strategy for BNI production and/or release (4+-tolerance mechanisms needs to be explored, and results of great interest for the future. Remarkably, the presence of nitrifying bacteria, but not denitrifiers, promotes the secretion of BNI compounds in wheat (2 conditions (Plant BNI capacity is dependent on soil conditions, of which rhizospheric pH, aeration, quantity, and form of available N are the main drivers of BNI synthesis and exudation . The pre release . Hence,  release . Whetherin wheat  and ricein wheat . Thus, ain wheat . In turnnditions  could po2 promotes not only whole plant and root biomass (Sorghum, or brachialactone from Brachiaria (2 to include compounds with BNI activity (2 lead to the rescheduling of secondary metabolism, with enhanced C-enriched metabolite production (2 on the production of BNI-active metabolites. However, root exudation in plants is affected by many factors; water availability is a determinant of exudation response to eCO2 (2, water availability, or temperature is the next step to further promote this trait for sustainable agriculture.Elevated CO biomass , but als biomass . The BNI biomass . This isachiaria . Therefoactivity Figure 1activity . Moreoveoduction . Therefo to eCO2 . In agre52 would boost both N assimilation and production of C-rich secondary metabolites. Although there are still many unresolved issues regarding factors that affect plant BNI capacity, BNI crops are promising candidates for future sustainable agrosystem production. In this context, selection of climate-resilient crop varieties adapted to the use of NH4+ as an N source is essential.Many uncertainties still exist in optimizing N management under future climatic conditions. However, to make agriculture more sustainable, it is mandatory to meet crop N demand, while reducing N losses derived from N fertilization. Improving soil N availability through the exploitation of plant BNIs is an outstanding opportunity. In this study, we hypothesize that BNI production would be promoted in a climate change scenario, since eCOIV-M and MG-M conceived the manuscript and supervised the whole writing process. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Culicoides and common knowledge shows high occurrence in Lusitano stud farms. This study aimed to compare skin tests and in vitro allergy tests for the diagnosis of IBH in Lusitano horses. The results of our study showed that skin prick tests presented a higher discriminatory diagnostic potential in IBH diagnosis. This increases our knowledge about IBH in Lusitano horses and could represent a step forward in the future development of specific immunotherapy.Insect Bite Hypersensitivity (IBH) prevalence in Portugal and in Lusitano horses is not known, but environmental characteristics are favorable to the activity of Culicoides nubeculosus (Cul n) and Culicoides obsoletus (Cul o) salivary glands and Culicoides nubeculosus Whole Body Extract (Cul n WBE). Addicionally, a cluster of six T and six C horses were also tested with Cul n 3 and Cul n 4 produced in insect cells and barley, as well as E. coli produced Cul o 3 and Cul o WBE. Allergen concentrations were 10 µg/mL for IDT and 100 µg/mL for SPT, and wheal diameters assessed at 20 min, 6 and 48 h. IDTs were considered positive when wheal diameter was ≥50% of the histamine wheal and SPT’s ≥ 0.9 cm. In vitro tests, allergen-specific serum IgE and sulfidoleukotriene (sLT) release assay were also carried out. Results showed that Cul n WBE, Cul n 7, 8, 9, Cul o1P and Cul o 2P were the best performing allergens for SPTs (p ≤ 0.0001) for the 1st allergen panel and Cul o WBE, Cul n 3 Bar and Cul n 4 Bac (p ≤ 0.05) for the 2nd, presenting a higher discriminatory diagnostic potential than IDTs, at a concentration of 100 µg/mL, with readings assessed at 20 min. Regarding in vitro tests overall, the sLT release assay performed best.Thirty controls (C) and 30 IBH-affected (T) Lusitano horses were evaluated. T horses were included based on anamnesis and physical examination, supported by questionnaires. All horses were submitted to skin tests, Intrademal (IDT) and Skin Prick Tests (SPT), on the neck with 14 specific allergens, 13 recombinant proteins (r-proteins) from  Culicoides [Culicoides hypersensitivity, summer eczema or Queensland itch [Culicoides extract, and sometimes also with other insect extracts, than healthy control horses [Culicoides allergens in IBH has also been demonstrated with functional in vitro tests such as sulfidoleukotriene (sLT) release  [C. nubeculosus, C.sonorensis or C. obsoletus extracts [in vitro sLT and histamine release in peripheral blood leukocytes from horses after a 40 min stimulation with Culicoides allergens [in vitro to given allergens. However, as stated above, even though intradermal skin testing is still currently used to identify the allergens responsible for the disease, sensitization to a particular allergen does not necessarily mean that the individual is allergic or will develop clinical signs of allergy [in vitro measurement of specific IgEs presenting in general a high specificity, they often present a lower sensitivity than SPTs in the diagnosis of IgE-mediated reactions [Culicoides spp. [The best characterized allergic disease in horses  is a typlicoides . It is mand itch . Insect and itch  and clinand itch ,5. The pand itch ,6. The pand itch ,8. As itand itch ,9,10. IBand itch ,11,12. Tand itch  and is band itch ,13. Variand itch . A few sl horses . The invries AG)  or histaextracts ,16,17, allergens , indicat allergy  previouseactions ,21. Advades spp. ,23,24.in vivo skin tests, SPT and IDT, and in vitro allergy tests, namely allergen-specific serum IgEs and sulfidokeukotriene (sLT) release assay in the diagnosis of IBH in Lusitano horses, using recombinant Culicoides allergens  and Culicoides extracts. The present study contributes to increase our knowledge about IBH in Lusitano horses and could represent a step forward in the future development of specific immunotherapy.In the present study, we evaluated the use of Culicoides spp. activity, and in the last few years, there has been an increase in IBH cases. Lusitano horse breeding plays an important role in the Portuguese economy and riders are making increasing use of the Lusitanos around the world [To the authors’ best knowledge, there are no studies about the impact of IBH in Lusitano horses, either in mainland Portugal, or outside. Nevertheless, Portugal presents all the environmental conditions for he world . IBH canin vitro allergy tests (sLT release assay and serum-specific IgEs), in Lusitano horses from stud farms in Portugal with a clinical diagnosis of IBH, as compared to healthy controls living on the same farms.The present observational cross-sectional study aimed at characterizing and comparing the results of skin allergy tests, both IDTs and SPTs, and Test Group (T)—30 IBH-affected horses presenting symptoms at the time of the tests;Control Group (C)—30 healthy horses.In total, 60 horses were tested, divided in two groups for the 1st allergen panel:For the 2nd allergen panel, a cluster of these horses, 12 animals (6 T and 6 C horses) in total, were simultaneously (1st and 2nd panels) tested in the same conditions, as described above.(a)AgesThe mean age of the T and C groups was 7 and 9 years with a standard deviation of 5.32 and 6.06, respectively. The horses presented an age range that varied between 2 and 25 years and 2 and 27 years, in groups T and C, respectively. They were grouped into several age ranges, namely, 2–5 years, 6–9 years, 10–14 years, 15–20 years and over 20 years, with the highest rate of horses occurring, in the case of the T group, in the age range of 2–5 years (57%) and 6–9 years (37%) in group C. The lowest rate of horses tested, for both groups, was ≥20 years with 3 and 7% in groups T and C, respectively.(b)Sexn = 16) and mostly were thoroughbreds; a total of 14, and only two geldings (1 each in C and T groups). In Portugal, most of the Lusitano females are breeders and live in an outdoor regimen all year round, being more exposed to the Culicoides spp., thus, presenting more signs of IBH than the males that are kept mostly indoors and less exposed to the Culicoides spp.Regarding sex, 73% of the animals tested were females; a total of 44 mares, 27 and 17 from C and T groups, respectively. Only a third of the tested animals were males , from at least the previous equivalent season;No glucocorticoid or antihistamine therapy within the two weeks prior to the tests was allowed.Horse breeds other than Lusitano;Gestational mares;Horses that presented other skin diseases;Horses that presented systemic signs of other diseases.Two different types of skin allergy tests were performed in all animals: SPTs and IDTs. These were mainly performed during springtime , corresponding to the onset of the symptoms for the IBH-affected horses. The readings of the papules’ diameters were assessed at 20 min, 6 and 48 h.E. coli [Culicoides nubeculosus and Culicoides obsoletus spp. salivary glands , as shown in Culicoides nubeculosus (Cul n) whole body extract (WBE).In total, in a first stage, 14 specific allergens were tested on all horses, which included 13 recombinant proteins, all expressed in E. coli ,23 from Culicoides extracts [In a second stage of the study, six other allergens were tested, but only on 12 horses, 6 IBH-affected and 6 C-group: they included the allergens Cul n 3 and Cul n 4, expressed both in insect cells and barley   and the extracts  and all extracts ,9.® 10 mg/mL, Orion Corporation; Espoo, Finland) 5 min before testing.Horses were sedated with intravenous detomidine hydrochloride 0.01 mg/kg  was inoculated per allergen .Papule diameters were measured 20 min after inoculation and at 6 and 48 h. Saline, PBS or the buffer in which the recombinant allergens were soluble  were useSkin test results, for both IDTs and SPTs, were evaluated by directly reading the papules’ diameter. The mean of two orthogonal diameters (mm) was then calculated. For the IDTs, results were considered positive when the papule size was at least half the size of the histamine wheal (positive control), as reported previously . For thein vitro sLT release assay, blood samples were collected into a vacuette containing ACD-B as anticoagulant  and shipped to the laboratory at the Vetsuisse Faculty, University of Bern, Switzerland within 24 h. The cellular antigen stimulation test  was performed as described previously [Culicoides nubeculosus (Cul n WBE) (2 µg/mL) and Culicoides obsoletus (2 µg/mL). After 40 min, plates were centrifuged, and the supernatants transferred into 96-well microtiter tissue culture plate to be kept at −20 °C until assayed. Released sLTs were measured using the CAST ELISA (Bühlmann laboratories) following the manufacturers’ instructions. For all further evaluations, values of the net stimulation were used, i.e., the spontaneous sLT release was subtracted from values obtained with the anti-IgE or Culicoides WBEs. Data from horses with sLT values < 250 pg/mL after stimulation with the anti-IgE were considered as non-responders [Culicoides WBEs. Three out of the 25 IBH horses and 4 out of the 24 controls tested in CAST were non-responders.To perform the eviously . To briesponders  and theiCulicoides exposure season, so the serological responses were evaluated after the IBH-affected horses started showing clinical signs. Frozen serum samples were then sent to the laboratory of the Clinical Immunology Group, Vetsuisse Faculty of the University of Bern. Allergen-specific IgE was measured by ELISA as described previously [Culicoides recombinant allergens at a final concentration of 2 ug/mL in 0.2 M Carbonate–Bicarbonate buffer, pH 9.4  for 2 h at 37 °C. After washing in 0.9% NaCl, 0.05% Tween 20, non-specific binding sites were blocked with blocking buffer  for 1 h at 37 °C. Plates were then washed twice with wash buffer and the sera, previously diluted 1:5 in blocking buffer, added in duplicates to the ELISA plate and incubated overnight at 4 °C. After washing, a monoclonal antibody specific for equine IgE  was added to the plates and incubated for 2 h at room temperature (RT) on a shaker. After a further washing step, an alkaline-phosphatase-conjugated goat anti mouse IgG with minimal cross reactivity to horse serum proteins  was added and incubated for 1.5 h at RT on a shaker. After final washes, plates were developed with 1.5 mg/mL phosphatase substrate  in 10% diethanolamine , pH 9.8 and absorption measured at 405 nm after 2 h. After subtraction of the blank, OD values were corrected for differences between the plates coated with the same allergen. The correction factor for each plate was calculated based on the average of the OD values of the positive controls included on all plates [For the serological tests, jugular blood was collected from all horses into 9 mL vacutainer serum tubes, about 10 min before the onset of the skin tests, and serum was then separated by centrifugation. In agreement with previous studies ,27,29,30eviously , for thep-value (p ≤ 0.05). Cut-off values were selected at a given specificity and at the highest accuracy possible.Statistical analysis was carried out using the SPSS program (IBM SPSS Statistics 23) and different statistical tests were used to evaluate the skin allergy tests’ data (size of the papule), namely ANOVA Repeated Measures and Discriminant Analysis with a classification matrix ≥70% for SPTs and ≥60% for IDTs, testing three independent variables: Exposure– two groups (control—C and IBH-affected—T), Time—over three time points  and Type of test—Skin Prick Test (SPT) and Intradermal Test (IDT). To determine cut-off values, non-parametric tests were used, namely Kruskal-Wallis’s test to determine statistically significant differences between groups (T and C) at each time, and Friedman’s test to compare the evolution over time, considering p ≤ 0.05 for all comparisons performed.As to the sLT release assay and the IgE serology, medians were used as the results were not normally distributed. The Mann-Whitney U test was used to compare median values of the IBH and control groups. To evaluate the performance of the tests, non-parametric Receiver Operating Curve (ROC) analysis was performed and calculations of the area under the empirical curve (AUC) were performed using PROC LOGISTIC (NCSS version 11). ROC Curves’ analysis was used to select the best cut-off values in the sLT release assay. Cut-off values were selected at the highest accuracy possible. The level of significance was set at All horses showed a positive response to histamine (positive control), in both skin tests performed, as shown in p > 0.05). Regarding IDTs, we had a total of 16 IBH-affected horses and 18 from C group that showed positivity to PBS, with the papules’ mean diameter of 1.6 cm (T) and 1.56 cm (C), for p > 0.05. Despite having positive values in T and C groups for both SPTs and IDTs, no statistically significant differences were detected; hence, no interference in the results of the significant allergens diluted in PBS was considered.Regarding negative controls, in case of PBS, there were some positive reactions in both T and C groups. In case of SPTs, we had a total of nine horses that showed positivity from T group and two from C group. The papules’ mean diameters were 1.021 cm (T) and 0.886 cm (C) with a standard deviation of 0.8110 and 0.8636 for T and C groups, respectively, with a significance of 0.31  and T (n = 30) groups for these allergens are shown in Differences between C (p ≤ 0.05). However, the number of horses tested was quite small. Papules’ diameters mean and standard deviation (σ) for these allergens are shown in For the second allergen panel, the allergens that presented statistically significant differences and better discriminated between groups C and T for both IDTs and SPTs were Cul o WBE, Cul n 3Bar and Cul n 4Bac  were statistically larger when compared to the control group (C), as seen in p ≤ 0.05) was used, with readings assessed at 20 min.On the other hand, statistically significant differences between C and T groups were only observed when a concentration of 100 µg/mL , over time, for both Skin Prick Test (SPT) and Intradermal Test (IDT), for IBH-affected horses (test group), for Cul n WBE.For the rest of the significant allergens, the diameter of the papules performed identically over time for both skin tests, IDTs and SPTs, reflected in identical and, therefore, overlapping graphs.Regarding the 1st allergen panel, it was also possible to determine that all the IBH-affected horses showed positivity for one to ≥ four of the statistically significant allergens. Furthermore, 70% of these T group horses presented positivity to at least four allergens .Regarding the control group, 10 horses showed positivity to just one of the statistically significant allergens, and only two horses presented positivity to ≥four of the statistically significant allergens, but the majority, 40%, presented no positivity to any of the allergens .In addition, all IBH-affected horses, without exception, were positive for the Cul n WBE allergen, and only six horses from the C group were positive to Cul n WBE . The secRegarding the 2nd allergen panel, results are shown in In case of IDTs, a classification matrix of 60% was determined and statistically significant differences were found for Cul n WBE, Cul n 8, Cul n 9 and Cul o2P .p ≤ 0.05). Papules’ diameters mean and standard deviation (σ) for these allergens are shown in In comparison with SPTs regarding the second allergen panel, the allergens that presented statistically significant differences and better discriminated between groups C and T were Cul o WBE, Cul n 3Bar and Cul n 4Bac  were statistically larger when compared to the control group (C), as seen in The authors also observed that for the tested allergens, the mean papules’ diameters in the IDTs were superior to those observed in SPTs , but we Over time, there was an increase in papules’ diameter, with the maximum mean value occurring at 6 h, and a decrease at 48 h .Regarding IDTs, readings at 20 min, 6 and 48 h are shown in p < 0.05) and more clearly, with Cul o WBE , as shown in The median sLT release was significantly higher in IBH horses compared to controls with Cul n WBE  of 0.892 for Cul o WBE compared to Cul n WBE (AUC 0.699). Decreasing the cut-off for Cul o WBE from 340 pg/mL to 250 pg/mL allowed an increase of the sensitivity to 73% at the same specificity of 90%. The best accuracy of the test was obtained using a cut-off value of 70 pg/mL and resulted in a sensitivity of 86% and a specificity of 85%.The results from the IgE serology are summarized in p ≤ 0.05) for the following allergens: Cul n 3, Cul n 4, Cul n 10, Cul o 2, Cul o 1P, Cul o 2P and Cul o 3 .The area under curve for the Culicoides allergens could distinguish between allergic and non-allergic horses. Based on the results obtained in the skin tests of the present study, Cul n WBE, Cul n 7, Cul n 8, Cul n 9, Cul o 1P and Cul o 2P may potentially be the most relevant of the tested allergens for the study of IBH in Lusitano horses. These seem to be promising candidates for an SPT diagnostic test panel, involving allergens which were chosen based on the geographical area. To determine which combination of specific allergens would result in the best panel, discriminant analysis by the stepwise method was used with IBH status as the outcome, as also used in another recent study [E. coli were used in the first group of horses, while the same allergens expressed in insect cells and barley [E. coli are often found in inclusion bodies and need a refolding step. As they lack posttranslational modifications, because eukaryotic proteins expressed in E. coli often form inclusion bodies and cannot always be refolded correctly, it may decrease the allergen’s functionality [Culicoides whole body extract (WBE) often result in a positive reaction, even in clinically healthy horses. Natural allergenic extracts are heterogeneous and may contain non-allergenic components, in addition to allergens [Culicoides nubeculosus WBE (Cul n WBE) was rather good. The specificity with some of the recombinant allergens was better, reaching 87%, but the sensitivity was usually much lower, at best 60% with Cul n 9. On the other hand, the use of recombinant allergens  may have an advantage compared to natural allergen sources, as they provide specific allergen products for the diagnosis and treatment of allergic diseases [Culicoides allergens (r- Cul) may provide a more specific diagnosis of IBH in sensitized horses, decreasing the number of false positive reactions. Hence, the use of r-Cul allergens may allow better diagnosis and better allergen immunotherapy [Culicoides obsoletus allergens [Culicoides allergen-specific IgE determination and an sLT release assay in horses with a history of IBH, and specifically in Lusitanos. IDTs were, until now, considered the most sensitive and confirmatory skin tests for horses [Culicoides spp. [in vitro measurement of allergen-specific IgEs [Culicoides allergens than healthy control horses [Culicoides allergens were determined in the sera of the C and T Lusitano horses. The T horses had significantly higher serum allergen-specific IgEs than the C group for Cul n 3, Cul n 4, Cul n 10, Cul o 2, Cul o 1P, Cul o 2P and Cul o 3. However, there was some overlap between the groups, meaning that some of the C horses also had serum-specific IgE for Culicoides r-allergens. This might be explained by a high degree of exposure to Culicoides bites resulting in some degree of asymptomatic sensitization [E. coli-expressed allergens, which is more important for functional tests such as SPT or cellular allergy tests than for binding of free serum IgE. Interestingly, barley or insect cell-expressed Cul n 3 and Cul n 4 led to significantly higher SPT reactivity in the T compared to the C group , the highest AUC was obtained for the CAST with Culicoides obsoletus extract (Cul o WBE) , when compared with Cul n WBE. Recombinant Culicoides allergens were not used in the CAST because the E. coli-expressed allergen did not or only weakly induced sLT release . Another limitation of the CAST is that fresh blood samples are needed. In conclusion, based on the results of this study for the SPTs, Cul n WBE, Cul n 7, Cul n 8, Cul n 9, Cul o 1P and Cul o 2P were the best performing allergens. Regarding in vitro assays, sLt release assay Cul o WBE was the best performing allergen, and even with a lower sensitivity and specificity, the serum allergen-specific IgEs, Cul n 3, Cul n 4, Cul n 10, Cul o 1P, Cul o 2P, Cul o 3 and Cul o 2, were determined as the best performing allergens in Lusitano horses.Our experimental observational cross-sectional study allowed us to analyze if IgE-mediated reactions to certain nt study . This shd barley  were tesionality . In the ionality  that IDTllergens . These ellergens . The assllergens . Almost llergens . As the diseases . Recombidiseases . Howeverdiseases . In humadiseases . Some prdiseases ,17,26 suotherapy . Our resllergens  were disr horses ,24,26,36r horses , accordir horses ,36,37,38r horses ,40. Thisr horses . IBH is r horses ,42 and sr horses , if therr horses . In SPTsr horses ,45. It ir horses . Furtherr horses . The tecr horses  and the r horses . Hence, r horses ,45. Thisr horses ,46. Alsor horses . In our r horses ,46 and wr horses , where tdes spp. , the autfic IgEs ,49 remaific IgEs . Skin tefic IgEs ,51. The fic IgEs . Accordific IgEs , the diafic IgEs . Neverthtization . A high tization . In humatization . Cul n 3 C group . This illlergens . The autllergens . Importaallergen . Some arallergen ,51,55. Itzerland . The CASCulicoides allergens involved in IBH in Lusitano horses using the allergen-specific IgE ELISA and the sLt release assay suggests that it will be worthwhile to test further Culicoides obsoletus recombinant allergens in SPT, probably increasing the performance of this test for the diagnosis of IBH. However, in agreement with other authors [in vivo and/or in vitro, is important to confirm the clinical suspicion of allergy, a thorough anamnesis and clinical examination remains essential for the diagnosis of allergy. Finally, the evaluation of new pharmacological or immunotherapeutic options for IBH will benefit from valid skin testing with a specific allergen panel, which may also allow for the comparison of results among study populations.The results of this study support the use of SPTs as a major contributor to the diagnosis of IBH and may represent a step forward as an IBH diagnostic tool and eventually in the establishment of patient-tailored, component-resolved specific immunotherapy. Moreover, the identification of relevant  authors , even th


The tolerance model rests on the thesis of a physiologically regulated, albeit unsustainable, systemic attempt to adapt to the catabolic challenge posed by acute prepubescent malnutrition even in its severe forms. The model centers on the immunological component of the attempt, positing reorientation toward a non-inflammatory form of competence in place of the classic paradigm of immunological attrition and exhaustion. The foundation of the model was laid in 1990, and sixteen years later it was articulated formally on the basis of a body of evidence centered on T cell cytokines and interventions with cytokine and hormonal mediators. The benefit originally suggested was a reduced risk of autoimmune pathologies consequent to the catabolic release of self-antigens, hence the designation highlighting immune tolerance. Herein, the emergence of the tolerance model is traced from its roots in the recognition that acute malnutrition elicits an endocrine-based systemic adaptive attempt. Thereafter, the growth of the evidence base supporting the model is outlined, and its potential to shed new light on existing information is tested by application to the findings of a published clinical study of acutely malnourished children. Finally, some knowledge gaps pertinent to the model are identified and its potential for growth consonant with evolving perceptions of immunobiology is illustrated. Pediatric malnutrition is considered by the World Health Organization to include both overweight and underweight, the latter presenting in two main forms, viz. acute (wasting) discerned on the basis of weight-for-height and chronic (stunting) discerned on the basis of height-for-age . The focSince the inception of nutritional immunology as a distinct research front, unregulated attrition has been an implicit governing postulate with respect to the inflammatory immune depression characteristic of acute pediatric malnutrition . A more The main purpose herein is to position the tolerance model within the purview of nutritional immunology by providing a realistic assessment of its evidence base and potential. This objective is addressed, first by tracing the origins of the model , second A secondary purpose of this article is to demonstrate the need, pointed out elsewhere ,7, for iThe core theme of the tolerance model was first proposed thirty years ago ,33 and rEndocrine hormonal characteristics were among the early points of interest in relation to the physiology of acute pediatric malnutrition. By the mid-1970s, the proposition was widely acknowledged that at least some malnutrition-associated endocrine characteristics represent adaptive attempts e.g., ,42,43), ,43, 42,4The cited early reports could not establish causation specific to the glucocorticoids, but they facilitated the formation of a connection, conceptually, between the endocrine and immunological components of the pathophysiology of prepubescent malnutrition in its acute forms. This point of understanding is now frequently invoked within the discipline  and is By the latter half of the decade of the 1970s, the concept connecting hormonal mediators and malnutrition-associated immune defences had achieved sufficient momentum to appear in secondary sources ,57, wherThe earliest intervention studies relevant to the tolerance model comprise a conglomerate of investigations that preceded the series outlined  with resOnly two of the cited investigations ,64 addreThe body of early intervention studies , includiIt is important to interject, at this juncture, that the concept of hormonally-regulated immunological change in response to malnutrition was articulated independently by three additional research groups within the ensuing decade ,22,23,24From its earliest days, the research effort regarding malnutrition-associated immune depression centered on the T cell system ,56,57,71+ and CD8+ T cell sub-populations attracted early attention because of the type of information available from clinical studies [+ and CD8+ sub-populations of T cells. Attention centered on a surface marker designated CD45RA because this protein identifies a quiescent, naïve-type subset with consequent stringent activation requirements. Thus, a series of studies using weanling mouse models revealed a particular abundance of CD45RA+ elements within CD4+ and CD8+ sub-populations both in secondary lymphoid organs wherein immune responses arise [+ sub-population was confined to the effector/memory (CD45RA−) subset [Balance in numbers between T cells and B cells and between CD4 studies ,57. Thes studies , the lym studies ,73,74 anes arise ,76,77 anes arise ,76,77. Ies arise ,76,77, a) subset . Intervention studies have provided the most persuasive evidence that immune defence plasticity is sustained into the advanced stages of acute malnutrition, even its severe prepubescent forms. Investigations of this type, outlined in Five investigations extended the database, revealing plasticity among innate defences. Triiodothyronine intervention prevented depression in natural killer cytotoxic activity in two mouse models of acute weanling malnutrition  and, in During this interval of time, a research front centered on leptin further extended the list of effective intervention mediators. Injections of leptin during a two-day starvation period enabled adult mice to generate a vigorous cell-mediated response, which was almost undetectable in the absence of the hormonal support . Soon thCollectively, the eight cited reports afforded a new breadth of support to the proposition that immune defences, both innate and adaptive, retain responsiveness to physiological mediators in advanced stages of acute malnutrition—perhaps even during prepubescence ,82. PartBy the beginning of the new century, an evidence base was established pointing to an emphasis on T cell quiescence in association with acute pediatric deficits of nitrogen and energy . In turnA small body of experimental and clinical evidence produced in the decade of the 1990s ,85,86,87Although easily overinterpreted, blood cytokine concentrations represent spillovers from extravascular sites and can be viewed as reflecting, while not representing, concentrations at extravascular sites of action . ArguablAs the body of information grew, the proposition was expanded to include cytokine-producing elements other than T cells and was refined to place emphasis on the potently anti-inflammatory and tolerogenic cytokine, IL-10 . The eviBlood cytokine levels are particularly informative when either complementary or opposing cytokines are considered together. This reasoning gave rise to an investigation of the blood concentrations of IL-10 and TGF-β in weanling mice subjected to acute forms of nitrogen and energy deficit . These cPrompted by the perception that the blood cytokine profile reflects a systemic immunological character, the “tolerance model” was articulated in 2006  to accomDuring this period, the possibility that benefits could accompany malnutrition-associated immune depression was put forward by two additional research teams ,30. The It is important to recognize the additional momentum contributed (to the idea that inflammatory immune depression might confer benefits) by these two independent propositions. It is also important to recognize, however, that the propositions ,30 diffeSeveral lines of evidence can be cited in the development of the platform for the tolerance model following its articulation in 2006. Broadly, these threads can be categorized as either primarily immunological in nature , or primarily metabolic in nature. These ongoing research fronts serve both to uphold the model and to highlight potential growth points. A timeline is shown in The reports regarding constitutive blood cytokine concentrations that prompted the articulation of the tolerance model proposition  focused In the meantime, young adult mice subjected to a wasting dietary nitrogen deficit were reported to exhibit low blood type 1 cytokine immunoactivities  but highDuring the same interval of years, additional studies ,37 confiThe foregoing notwithstanding, blood concentrations of two inflammatory cytokines in particular, TNF-α and IL-6, have been reported over a span of many years to be either elevated or unaffected in cohorts of acutely malnourished children e.g., ,103,104),104103,1It is also important to note that knowledge of the biological roles of IL-6 and TNF-α continues to grow in sophistication. Although generally considered invariantly inflammatory, these cytokines may be best understood as pleiotropic. For example, the IL-6–hepcidin axis promotes hypoferremia, a non-inflammatory defence against extracellular pathogens , and thi+ and CD8+ blood T cell populations from malnourished children exhibited type 2 cytokine polarization [+ blood mononuclear cells expressing intracellular IL-12 [An important research front, centered on blood mononuclear cells of acutely malnourished children, continued to grow through the efforts of a group based in Mexico City ,113,114.rization ,113. In rization ,114, andar IL-12 . Other rar IL-12  and INF-ar IL-12  in bloodDuring the same period of years, two studies of young adult rodents subjected to an acute dietary nitrogen deficit yielded broadly similar findings ,13. The The growing database pertaining to cytokine production and blood concentration profiles prompted an investigation to test and refine the tolerance model according to the prediction that acute prepubescent deficits of nitrogen and energy will elicit a shift toward a non-inflammatory identity within characteristically inflammatory lymphoid sites while sustaining the disposition of non-inflammatory sites . Using tTwo laboratory technologies, each applied to weanling rodents, are noteworthy at this juncture. In the first place, the application of bioassays rather than the understandably popular ELISA appears to have conferred an important benefit in terms of consistent clarity in the estimation of constitutive blood cytokine concentrations ,35,36,37The second key assay technology, the in vivo cytokine capture assay , provideEnlargement of the aforementioned database is needed both to increase the diversity of experimental systems and to expand the array of tested cytokines. In this context, however, it is relevant to note that the cited information was derived from the application of the bioassay and cytokine capture techniques to two longstanding and validated animal models. The particular merit of the models stems from their demonstrated relevance to severe forms of acute pediatric malnutrition and, in addition, from the database built up over three decades of investigation regarding their immunological characteristics. Although the critical physical and metabolic  characteristics of the two animal models have not been summarized in a single, consolidated source, they can be found recorded within the body of research based on the models, and some of these reports are cited herein ,97,120 bStudies reporting responsiveness to endocrine hormones, cytokines and other mediators or mediator-containing fluids  constituLeptin, having featured in studies leading to the formalization of the model , continu+ F4/80−/low) despite profound splenic lymphoid atrophy. Moreover, regardless of deepening catabolic disease, the exogenous cytokine supported a vigorous spleen-based cell-mediated adaptive immune response that was, otherwise, profoundly depressed. This report extended a previous piece of work using the same weanling model [Two additional interventions each probed responsiveness to a previously untested cytokine. In the first investigation , granulong model  in whichThe body of evidence continues to grow, therefore, pointing to ongoing competence and physiological responsiveness on the part of immune defences, both innate and adaptive, despite profound losses in terms of cellular numbers. Three of the six most recent intervention studies ,120,122,From its inception ,33,62 thThe foregoing considerations signaled, even decades ago, a response to acute dietary nitrogen deficiency that includes the controlled apportionment of nitrogen according to a new set of metabolic priorities. This perspective centered on metabolic attempts to adapt to catabolic malnutrition continues to grow and now includes, also, the metabolism associated with acute dietary caloric deficits. As an example, a recent report, albeit based on a young adult mouse model, points to a regulated diversion of energy away from inflammatory responses and toward maintenance of core temperature in response to a catabolic energy deficit . Other rIn another research front, bone marrow adipose tissue is reported to accumulate in anorexic humans ,132, in Of some interest in the present context, evidence that the remodeled marrow compartment serves as a repository for memory T cells  albeit  indicatiThe tolerance model is in need of testing to gauge its fitness to improve the depth of understanding with respect to immune defences in acute forms of malnutrition. Application of the model to the existing evidence base should be helpful in this regard, and the example that follows illustrates use of the model to re-evaluate the contribution of a published investigation of prepubescent humans. The example provides a basis for optimism that the model can be used to stimulate insights and questions, and ultimately advancements in understanding, that did not—and could not—arise through the classic lens of immunological attrition. Further such analyses appear worthwhile. A report regarding a cohort of Ugandan infants and children suffering severe acute malnutrition  is notewApplying the tolerance model to existing information should prove productive, but it must be kept in mind that the model remains a proposition. An assessment follows regarding the speculation that the systemic catabolism of acute malnutrition, in fact, creates a threat of inflammatory autoimmune pathologies . In addiAccording to the tolerance model, the primary benefit of fostering a tolerogenic form of immune competence is to minimize the risk of autoimmune inflammation pursuant to the catabolic release of self-antigens normally hidden from immunological view ,36,37,39Twenty years ago, epidemiological information was cited to the effect that autoimmune disease appears to be more prevalent in settings described as “affluent and more developed societies” than in settings described as “less developed” . AlthougThe foregoing speculations are enticing in relation to the tolerance model, but they are based entirely on association. Moreover, others  point ou+ T cells expressing Foxp3 [+ T cells together with an increased proportion of Foxp3+ T cells in the small intestinal lamina propria [Self-tolerance requires an array of cellular elements , and theng Foxp3 , a transng Foxp3 . Similar propria . Meanwhi propria , weanlin propria ) was notAs suggested more than 25 years ago , and reiA brief consideration of the “disease tolerance” concept may be instructive in the present context. Disease tolerance is a non-immunological defence strategy, increasingly acknowledged in recent years, that limits damage to an infected host without exerting a direct negative influence on the infecting pathogen ,107,148.For many decades, prepubescent malnutrition, at least in its severe and acute forms, has been widely considered to elicit diverse clinically significant deficiencies in immune defences e.g., ,57,149).,149.57,1Depressed inflammatory and febrile competence is a long-recognized clinical feature of acute pediatric and experimental weanling malnutrition ,150,151.A more insightful and nuanced grasp is needed regarding the inflammatory response in acute forms of pediatric malnutrition. In this connection, endotoxemia is a common feature of acute prepubescent malnutrition , and endRecently, mice subjected to acute malnutrition from weaning through adolescence were reported to present an amplified inflammatory response within the bone marrow in terms of cytokine profile and the influx of inflammatory monocytes . In turnAs a subdiscipline of immunobiology, the study of malnutrition-associated immune defence requires a paradigm that can grow with the larger field. In view of the prominent theme of immune regulation within the tolerance model, it is instructive to consider the model in juxtaposition with emerging insights regarding regulatory immunobiology. +Foxp3+ T cells [Peripheral anti-inflammatory suppressor cells are recognized as critical to immune regulation and self-tolerance ,155,156. T cells , and inf T cells ,9,34, Se T cells  that blo T cells ,155), isThe central tenet of the tolerance model—a regulated and adaptive modification of immune defences in the face of acute forms of prepubescent malnutrition—was first proposed more than thirty years ago ,33, and The most conspicuous and distinguishing implication of the tolerance model is that the immunological characteristics of acute prepubescent malnutrition confer physiological benefits and, in addition, reflect a larger adaptive metabolic response. Hints along each of these lines can be gleaned from recent animal-based studies that suggest benefits both in terms of energy-efficient preservation of diverse immune defence elements , Section and in tConnecting the immunological characteristics of malnutrition with a demonstrated adaptive benefit is a matter of some urgency to the development of the tolerance model and must be expected to require animal-based investigations demonstrably relevant to acute pediatric malnutrition. The need for improved attention to design and modeling in the experimental study of acute prepubescent malnutrition is documented ,7, and tThe potential of immunoenhancement to augment the management of acute pediatric malnutrition has been voiced for many years ,162,163,Leptin stands alone in having the support of an intervention study involving malnourished children , althougArticulation of the tolerance model  was moti