Lifestyle intervention may be effective in reducing type 2 diabetes mellitus incidence and cardiometabolic risk. A more personalised nutritional approach based on an individual or subgroup-based metabolic profile may optimise intervention outcome. Whole body insulin resistance (IR) reflects defective insulin action in tissues such as muscle, liver, adipose tissue, gut and brain, which may precede the development of cardiometabolic diseases. IR may develop in different organs but the severity may vary between organs. Individuals with more pronounced hepatic IR have a distinct plasma metabolome and lipidome profile as compared with individuals with more pronounced muscle IR. Additionally, genes related to extracellular modelling were upregulated in abdominal subcutaneous adipose tissue in individuals with more pronounced hepatic IR, whilst genes related to inflammation as well as systemic low-grade inflammation were upregulated in individuals with primarily muscle IR. There are indications that these distinct IR phenotypes may also respond differentially to dietary macronutrient composition. Besides metabolic phenotype, microbial phenotype may be of importance in personalising the response to diet. In particular fibres or fibre mixtures, leading to a high distal acetate and SCFA production may have more pronounced effects on metabolic health. Notably, individuals with prediabetes may have a reduced response to diet-induced microbiota modulation with respect to host insulin sensitivity and metabolic health outcomes. Overall, we need more research to relate metabolic subphenotypes to intervention outcomes to define more optimal diets for individuals with or predisposed to chronic metabolic diseases. Driven by easy access to energy-dense foods and a sedentary lifestyle, obesity has become a major global health and socio-economic problem in the 21st century.Worldwide, the prevalence of obesity, insulin resistance (IR) and type 2 diabetes mellitus has grown dramatically since the 1980s (http://wwwfaoorg/3/a-i4646epdf). Many low and middle-income countries are facing currently the double burden of disease where infection and undernutrition may occur simultaneously with a rapid increase in overweight and obesity, particularly in urban settings. It is not uncommon that overweight and undernutrition co-exist in populations, households or even individuals (http://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight). Malnutrition in obesity may relate to a greater availability of inexpensive nutrient deficient foods, potentially inducing micronutrient deficiencies and a positive energy balance. Furthermore, the increased adipose tissue mass may sequester vitamins, leading to reduced circulating concentrations and a reduced supply of these nutrients to other tissues. For example, vitamin D deficiency, i.e. reduced circulating concentrations of 25-hydroxyvitamin D3, has been associated with obesity, IR and type 2 diabetes mellitus. Overall, a poor or altered intake of macro- or micronutrients in obesity or altered metabolism and pharmacokinetics in the obese individual may lead to an inadequate nutritional status. Additionally, treatment of extreme obesity involving bariatric surgery may sometimes worsen these nutrient deficiencies.Malnutrition may both be a cause as well as a consequence of overweight and obesity and refers to an abnormal physiological condition caused by inadequate, unbalanced or excessive consumption of macronutrients or micronutrients  with long-term (≥2 years) follow-up, showed that on average more than half the weight loss is regained after 2 years and more than three-quarters is regained after 5 years. This poses the question whether there should be more focus on the adoption of a healthy lifestyle and not on weight loss per se. Notably, it has been shown that moderate 5 % weight loss already improves metabolic function in multiple organs simultaneously. Additionally, lifestyle intervention in prediabetic individuals may reduce diabetes and metabolic syndrome risk by more than 50 % in different settings worldwide despite moderate weight loss–10. These lifestyle programmes were based on the general guidelines for a healthy nutrition and physical activity but tailored towards the individual lifestyle. Nevertheless, within these tailored lifestyle interventions, 30 % of the participants do not respond or adhere to the intervention. The current paper focuses on whether a personalisation based on a metabolic or microbial phenotype may be effective to improve the success of nutritional intervention with respect to metabolic health.One of the biggest challenges in the management of obesity and related complications is weight maintenance after successful weight loss. Weight regain may be partly driven by biological factors related to adipose tissue biology. IR may develop in different organs but the severity may vary between organs. Indeed, in the different prediabetic phenotypes the state of impaired glucose tolerance may be characterised by more pronounced peripheral or muscle IR, whilst the state of impaired fasting glucose by more pronounced hepatic IR. Consistent with this, we previously showed that skeletal muscle lipid handling was disturbed in the impaired glucose tolerance state, characterised by an impaired postprandial insulin sensitivity, an increased postprandial TAG extraction and a reduced muscle lipid turnover, compared with individuals with impaired fasting glucose,14.Insulin plays a central role in nutrient partitioning within the body. Whole body IR reflects defective insulin action in tissues such as muscle, liver, adipose tissue, gut and brain, which may precede the development of cardiometabolic diseases and lipidome and adipose tissue transcriptome profiles in individuals with either more pronounced hepatic IR or more pronounced muscle IR in the European multicentre DIOGenes trial in overweight and obese individuals; and used the Cohort on Diabetes and Atherosclerosis Maastricht and the Maastricht study, a large population-based cohort in the Maastricht area as validation cohorts. Based on a 5 or 7-points oral glucose tolerance test with insulin and glucose concentrations we estimated hepatic IR and skeletal muscle insulin sensitivity indices  as previously described and validated against a hyperinsulinemic clamp by Abdul Ghani et al., and optimised for the MISI calculation by means of cubic splining by O'Donovan et al.. For descriptive purposes, we divided individuals in having no IR, having muscle IR (the lowest tertile of MISI), having hepatic IR (in the highest tertile of HIRI) or having both MISI and HIRI.The question arises whether the severity of IR at the tissue level may determine the response to dietary intervention. A first question that has to be addressed then is whether these phenotypes are really distinct. We addressed this question by characterising the plasma metabolome–17. This is in line with studies in obese men and women using hyperinsulinemic euglycemic clamp techniques. Individuals with HIRI or combined HIRI/MISI in general have higher plasma TAG and a larger waist–17.In general, overweight or obese women have a lower HIRI as compared to males,24–26. We investigated whether in individuals with overweight or obesity but without diabetes, hepatic IR and muscle insulin sensitivity were characterised by distinct metabolic profiles. Metabolome profiles in DIOGenes were quantified by NMR metabolomics and results of DIOGenes were validated in the Maastricht study. Hepatic, but not muscle IR, was associated with lower ketone body concentrations (acetoacetate and β-hydroxybutyrate) and lower concentrations of the ketogenic amino acids leucine and tyrosine, indicating reduced ketogenesis and β-oxidation in the liver with progressing IR. Both muscle and liver IR showed a blood metabolome profile of elevated (branched chain) amino acids, lactate and TAG and lower glycine concentrations. Our findings showed that muscle and liver IR are characterised by partly distinct and partly overlapping metabolome profiles.The characterisation of the metabolic profile received renewed attention by the availability of new metabolomics techniques. In previous studies higher levels of branched-chain amino acids, lactate, acylcarnitines, glycolytic intermediates, long-chain fatty acids as well as reduced concentrations of ketone bodies, lysophosphatidylcholines and tricarboxylic cycle intermediates are associated with the development of IR and (pre)diabetes. We found that muscle IR was associated with higher circulating lysophophatidyl choline (LPC) concentrations, in particular of the LPC 18:2 and 18:1 species. This positive association was independent of body composition and body fat distribution. Several previous studies have shown associations of LPC and general indices of insulin sensitivity. Also, in vitro studies in male myotubes showed that extracellular LPC (16:0) and LPC (18:1) can act as lipid signalling molecules and may activate peroxisome proliferator activated receptor-δ-dependent gene expression and reduce lipid-induced inflammation and IR. Our data extend previous findings indicating that LPC may be specifically related to muscle insulin sensitivity.Subsequently, we analysed the lipidome profiles (by liquid chromatography–MS-lipidomics) in tissue-specific IR phenotypes in the DIOGenes study, also indicated as female advantage. Our findings of a worsening of blood lipid profile in women with the progression of hepatic IR are intriguing and might imply that women with hepatic IR ‘catch-up’ with men with regard to CVD risk and that the relationship between (hepatic) IR and cardiometabolic risk may be sex-dependent. In line, Kim and Reaven showed that the female advantage may not be explained by differences in insulin action per se but from an attenuation of the relationship between IR and cardiovascular risk, in particular in younger individuals.In women, but not in men, hepatic IR was positively associated with the sum of plasma TAG and diacylglycerol. Importantly, these observed differences between men and women were unlikely to result from the known differences in body composition since adjustments for body composition and body fat distribution did not affect the results. Furthermore, the women in our study had less hepatic IR, lower plasma TAG and higher HDL concentrations as compared to men. In general, healthy premenopausal women have a higher capacity for fat storage without inducing hazardous cardiometabolic health risks or in sex-related differences in insulin-mediated VLDL-TAG metabolism or de novo lipogenesis,31.The mechanisms behind this intriguing sexual dimorphism in hepatic lipid metabolism and IR remain to be determined. Notably, in our study most women were most likely in a premenopausal status. One potential mechanism may lie in the impact of oestrogens on hepatic lipid metabolism. Previous studies investigating the subcutaneous adipose tissue (ScAT) transcriptome have found an upregulation of ScAT inflammatory pathways and a downregulation of lipid metabolism pathways and/or mitochondrial respiratory pathways in individuals with whole body IR v. BMI-matched controls or in obese women with IR as compared to lean insulin sensitive women. To investigate whether differential ScAT transcriptome profiles were related to more pronounced muscle or hepatic IR, we analysed baseline RNA sequencing profiles in 368 individuals in overweight and obese individuals of the DIOGenes study and we subsequently validated mechanisms related to the systemic inflammatory profile in two independent cohorts Cohort on Diabetes and Atherosclerosis Maastricht and the Maastricht study.The IR state is accompanied by an increased adipose tissue mass and adipose tissue dysfunction, characterised by an impaired lipid buffering capacity, and infiltration of immune cells. Both systemic lipid overflow as well as low grade inflammation have been related to the development of IR of non-adipose tissues such as the liver and skeletal muscleWe showed that an altered extracellular matrix remodelling gene expression profile in abdominal ScAT was present in overweight and obese individuals with pronounced hepatic IR. Interestingly, an inflammatory gene expression profile was particularly present in individuals with primarily muscle IR. Based on the gene expression results, we proposed that an increased systemic inflammatory profile is a mechanism linking the increased expression of inflammatory genes in abdominal ScAT to muscle IR. In line with this, in the Cohort on Diabetes and Atherosclerosis Maastricht and the Maastricht study, an increased systemic low-grade inflammation profile, as reflected by the combined score of plasma markers of low-grade inflammation, was specifically related to muscle IR and not to liver IR. Although the relationship between adipose tissue inflammation and IR has previously been posed, the findings in the present study extend these previous unpublished results by indicating the tissue-specific nature of this relationship.Overall, the above results on metabolome, lipidome and transcriptome profiles indicate distinct tissue-specific IR phenotypes. The main characteristics of both phenotypes are summarised in ,14. There are indications that dietary fat quality may impact muscle lipid handling. The first mechanistic evidence came from myotube studies showing that SFA preferentially accumulate as diacylglycerol, whilst unsaturated fatty acids are readily converted to TAG. Also, a reduced fat oxidation was observed when diabetic myotubes were exposed to palmitic acid, whilst no differences were observed with oleic acid. Indeed, we previously showed that under in vivo conditions the insulin resistant muscle is characterised by a reduced muscle lipid turnover as well as an increased muscle TAG extraction in particular after a high SFA-mixed meal and not after a PUFA meal. Replacement of SFA by PUFA therefore would be protective against the development of peripheral IR. Manipulation of dietary fat quality by increasing the MUFA content or the n-3 or n-6 long-chain PUFA content of the diet in intervention studies has not shown consistent results on insulin sensitivity–39. Based on the finding of differential fatty acid handling, dietary fat quality and peripheral insulin sensitivity, it can be speculated that the effectiveness of dietary fat manipulation may depend on initial metabolic tissue-specific phenotype. Indeed, a post hoc analysis in the European project LIGENE, focused on the study of dietary fat quantity and quality in the metabolic syndrome showed that insulin resistant metabolic syndrome individuals were more susceptible to a health effect from substitution of a high saturated fat diet by either high MUFA and high (complex) carbohydrate (with added n-3 PUFA) diets. Unfortunately, in that study no distinction was made between liver and muscle IR.From the above it is evident the muscle and liver insulin resistant phenotypes are distinct which subsequently leads to the question whether these phenotypes respond differentially to dietary intervention. The above reported and previously shown disturbances in muscle lipid turnover in individuals with impaired glucose tolerance as compared to individuals with impaired fasting glucose were shown after ingestion of a high fat, high SFA mixed mealpost hoc analysis of the CORDIO-PREV-DIAB study. In the latter study, a low-fat high complex carbohydrate diet and the Mediterranean diet, rich in olive oil were compared with regard to outcomes related to glucose metabolism. A post hoc division in tissue-IR phenotype at baseline showed that individuals with muscle IR may benefit more from a Mediterranean diet showing a higher increase in disposition index (composite marker for insulin secretion adjusted for insulin secretion) as compared to the hepatic IR phenotype. Interestingly, individuals with hepatic IR have a more pronounced increase in disposition index on a low fat-high complex carbohydrate diet. Thus, although a diet according to existing dietary guidelines may be healthy for all, dietary prevention or treatment may require more personalised or sub-group based approaches to become most effective. Beside metabolic phenotype microbial phenotype may be of importance in personalising the response to diet. A landmark study within this field by Zeevi and coworkers showed that despite high interpersonal variability in postmeal glucose, personalised diets created with the help of a machine learning algorithm including dietary habits and physical activity and gut microbiota may successfully lower blood glucose responses. Personalisation based on microbial composition and phenotype has received increased attention, and in the following paragraphs this will be addressed targeting the microbial functionality with emphasis on the production of SCFA.Further evidence for an interaction between macronutrient composition and tissue-specific IR comes from a . Our gut microbiota ferment indigestible dietary components that are incompletely hydrolysed by our digestive enzymes. Carbohydrate fermentation yields mainly SCFA as well as lactate and succinate and produces gases such as methane, carbon dioxide and hydrogen. Most carbohydrate fermentation takes place in the proximal colon and is ending in the transverse colon, The major SCFA, acetate, propionate and butyrate have been associated with positive effects on body weight control and insulin sensitivity. Proteolytic fermentation, which occurs predominantly in the distal colon, yields a diverse range of metabolites: SCFA, gaseous products such as hydrogen, methane, carbon dioxide and hydrogen sulphide; the branched-chain fatty acids, isobutyrate, 2-methylbutyrate and isovalerate derived from fermentation of branched-chain amino acids and phenolic and indolic compounds derived from microbial fermentation of aromatic amino acids. Many of these compounds are toxic and have been considered as being detrimental for colonic and metabolic health (as reviewed in43).A growing body of evidence indicates an important role of gut microbiota and its functionality in the etiology of obesity, type 2 diabetes mellitus and CVDv. the distal intestines  in patients undergoing abdominal surgery. SCFA were highest in the inferior mesenteric vein and lowest in the radial artery (102 (se27) v. 22 (se8) μm). There was a 3-fold higher SCFA release from the distal intestine as compared to the proximal intestines. An implication of these findings is that slowly fermentable fibres that give lead to increased SCFA in the distal intestines may have a high potential to influence host metabolism.Studies on the effects of SCFA in metabolic health are often hampered by the lack of information on actual SCFA kinetics including the rate of intestinal SCFA production, and subsequent absorption and eventual appearance in the circulation. Insight into the fate of SCFA and/or intestinal absorption will help to develop more targeted nutritional strategies. In a previous study, we addressed this by assessing SCFA release in the proximal  intestines . Acetate administration in the proximal colon did not affect the metabolic profile. Subsequently, we showed that long-term supplementation with galacto-oligosaccharides, an acetogenic fibre, had no significant effect on peripheral and adipose tissue insulin sensitivity and no effects on faecal and circulating acetate and SCFA despite an increase in the abundance of bifidobacterium species. In view of our findings that acute distal colonic acetate administration, but not proximal colonic administration affected parameters of metabolic health, we hypothesised that the site of fermentation of galacto-oligosaccharides may have led to an insufficient increase in systemic acetate to induce pronounced metabolic effects. Thus, increasing dietary fibre availability and SCFA formation in the distal colon may be an important determinant of metabolic health. In particular, since a higher distal carbohydrate fermentation may decrease detrimental proteolytic fermentation. This ‘microbial substrate switch’ might provide a novel dietary strategy for preventing and/or treating metabolic diseases.Interestingly, we previously showed that acute distal colonic acetate administration increased circulating acetate concentrations and pronouncedly increased fasting fat oxidation (25 %), increased the concentration of satiety-stimulating hormone peptide YY and reduced concentration of the cytokine TNF-α in overweight malesin vitro model of the human colon (TIM-2), which was inoculated with faeces from lean and pre-diabetic overweight donors. At first, we selected the fibre combinations that led to the highest distal acetate production in vitro to subsequently execute an acute human intervention study with this mixture. In a first acute human study, we showed that a 1-d supplementation with a novel fibre combination of long-chain inulin and resistant starch led to pronounced effects on microbial fermentation as compared to supplementation with long-chain inulin only or placebo in lean but not in overweight prediabetic individuals the morning after the supplementation day. In lean individuals this was accompanied by an increased energy expenditure and carbohydrate oxidation and postprandial insulin sensitivity, whilst no metabolic effects were observed in the prediabetic individuals. Further mechanistic deepening on gut microbiota composition, SCFA in faeces and blood, as well as circulating hormones and metabolites will shed more light on these findings. These data show that an innovative fibre mixture to target and reach the distal colonic microbiota might be a combination of readily fermentable oligomers and more complex fermentable fibres with a high degree of polymerisation and side chains. Nevertheless, our data show a lack of response in the prediabetic phenotype, which is consistent with previous findings showing that a 4-week oral butyrate administration altered metabolism and insulin sensitivity in lean but not in obese insulin resistant individuals.Based on these findings we hypothesised that combining a fast fermentable  with a slow fermentable, acetogenic fibre will enhance distal acetate and SCFA production, thereby improving human substrate and energy metabolism. To test the hypothesis, we performed experiments in an –52. Of interest, we recently showed that long distal colonic transit, as measured with the radio-opaque methodology influences microbial diversification and fermentation in individuals with slow gastro-intestinal transit.In the future, it is important to take the initial microbial composition and metabolic phenotype into account when targeting the microbiota and its functionality. Due to a putative resistance of the microbiome–host metabolism axis to dietary intervention in prediabetic individuals, intervention with fibre mixtures may require longer periods of time to become effective. Additionally, in future studies other factors that may shape the microbiota composition have to be taken into account such as sex, age, the use of medication and the gut transit time,54. Overall, we need more research to relate metabolic subphenotypes to intervention outcomes to define more optimal diets for individuals with or predisposed to chronic metabolic diseases. In this, the different etiologies towards type 2 diabetes and cardiometabolic diseases have to be considered. Focusing on tissue-specific IR phenotypes as well as related microbial composition and functionality may be a targeted approach to define more personalised strategies for nutritional intervention. More prospective trials are required to provide the evidence for the implementation of these approaches.A diet based on existing guidelines for healthy nutrition may be a good diet for all. Nevertheless, these guidelines based on the reference person may not represent the optimal diet for all. Also, specific guidelines for the treatment (or prevention) of non-communicable diseases such as type 2 diabetes have been formulated


Acute coagulopathy is prevalent in adult and pediatric trauma patients and is associated with increased morbidity and mortality. While reasonable hypotheses have been created to explain the underlying perturbations of adult trauma coagulopathy , only a small number of studies have been performed to prove whether these mechanisms can likewise be detected in pediatric trauma patients. In addition, severe hypofibrinogenemia (<100 mg/dL) is a frequent finding in pediatric trauma patients (>20%). Although the probability of life-threatening coagulopathy is low with minor to moderate injury, it is present in almost all patients with an injury severity score >25, hypotension, hypothermia, and acidosis. As these multifactorial changes in hemostasis cannot be adequately and rapidly measured using standard laboratory testing, the use of viscoelastic measurements has been established in adult trauma management, but prospective studies in children are urgently needed. Apart from diagnostic challenges, several studies have focused on the impact of blood product ratios on the treatment of massively bleeding pediatric trauma patients. The majority of these studies were unable to show improved survival by using higher plasma to red blood cell ratios or higher platelet to red blood cells ratios, but there are no published randomized trials to definitively answer this question. A goal-directed transfusion protocol using viscoelastic tests together with early substitution with an antifibrinolytic and fibrinogen replacement is a promising alternative to traditional ratio-based interventions. Another crucial factor in treating trauma-induced coagulopathy is the early detection of hypofibrinogenemia, a common condition in massively transfused patients. Early treatment of hypofibrinogenemia is associated with improved morbidity and mortality in adults, but needs to be further studied in future pediatric trials. Pediatric trauma patients are not only threatened by coagulopathy-related bleeding but are also at higher risk for venous thromboembolism. Pediatric trauma patients with brain injury, central venous catheters, immobilization, or surgical procedures are at highest risk for developing a deep venous thrombosis. There are no specific pediatric guidelines established to prevent venous thromboembolism in children suffering from traumatic injury. Adult traumatic coagulopathy is a commonly studied, but not fully understood, condition that occurs early and is a major contributor to injury-related mortality , 2. Its While initial theories to describe trauma-related coagulopathy were based on the effects of hemodilution due to the administration of large amounts of coagulation factor-free solutions, the knowledge about the pathophysiologic changes have evolved substantially. The current understanding of trauma-induced coagulopathy is still evolving, but the main underlying causes are thought to be hemorrhagic shock-related hypoxia/hypoperfusion, and tissue injury. Hypoperfusion and hypoxia promote platelet and endothelial dysfunction that further trigger activation of protein C and impaired thrombin generation, as well as major changes in the fibrinolytic pathway . In addiIn addition to a brief description of TIC in children and discussion of the major differences between hemostatic changes in adults and children suffering from trauma, this review will focus on the management of coagulopathic bleeding in pediatric trauma patients. Finally, we will review the impact of trauma on the occurrence of venous thromboembolism, as this is an important but frequently overlooked counterpart of trauma-related bleeding complications.The incidence of TIC in severe pediatric trauma patients in the emergency room varies tremendously and is reported to be in the range of 10–77% . This reAlthough the TIC-related changes in hemostasis and fibrinolysis of severely injured adult patients can be likewise observed in children , age-depIn adult trauma patients, the presence of hyperfibrinolysis is highly correlated with mortality . FibrinoWhile the empiric administration of tranexamic acid to address hyperfibrinolysis has shown to reduce mortality in adult trauma patients, data in pediatric trauma are scarce . StudiesThere is a substantial difference in the underlying mechanisms of trauma in children as compared to adults: a relatively higher rate of blunt trauma and non-accidental trauma, including brain injuries, can be observed in children . This isIn summary, when TIC is present in pediatric patients the mortality rate appears to be markedly increased , 24. UnfIn addition to reviewing global plasmatic coagulation parameters  which provide only a simple assessment of the first phase of clot initiation, it seems worthwhile to review published evidence of other markers to detect TIC, such as platelet count, fibrinogen, signs of fibrinolysis and viscoelastic testing.Platelet count was significantly associated with mortality in a cohort of 102 pediatric trauma patients, and an abnormal platelet count was observed in 23% of all children . HoweverSevere hypofibrinogenemia (plasma fibrinogen levels below 100 mg/dL) was detected in 20% of the previously mentioned cohort of pediatric trauma patients . It remaWhen viscoelastic testing such as thrombelastography or thromboelastometry was used to detect TIC in pediatric trauma patients, signs of hyperfibrinolysis  , a prolon = 54) of all children , plasma and platelets in pediatrics. Unlike in the adult setting, several trials have failed to show any survival benefit by using a transfusion protocol with a predefined transfusion ratio –37. In aRecently, the successful resuscitation using whole blood at a dose of 20 ml/kg was reported in a cohort of 18 pediatric civilian trauma patients . Howeverp < 0.0001). Another important finding of that study was that almost all patients suffered from severe hypofibrinogenemia after arrival at the emergency room, which was successfully treated by administration of fibrinogen concentrate at a dose of 50 mg/kg, while a dose of 30 ml/kg of plasma failed to normalize plasma fibrinogen levels. The importance of detecting and treating hypofibrinogenemia early was also investigated in a prospective cohort study of 517 adult trauma patients . Although very young children tend to be protected from VTE by their quantitative and qualitative developmental differences in hemostasis , the risAlthough there is currently no consensus for routine administration of thromboembolism prophylaxis in the pediatric trauma setting, a group from Johns Hopkins created their own VTE prediction score based on risk factors identified in their risk assessment model . A retroAt present, the knowledge regarding hemostatic balance in severe pediatric trauma patients is mostly extrapolated from adults. More studies are urgently needed to understand the pathophysiologic changes in this patient population. Trauma-induced coagulopathy is not just one single entity, but a heterogenous group of hemostatic imbalances showing distinct phenotypes depending on timing, mechanism of injury, underlying conditions and other less understood factors. The conditions are even more heterogeneous in children, where the age of the children impacts the mechanism of injury as well the baseline hemostatic factors. As such, a rapid, frequent and reliable laboratory testing should be performed to detect the current and evolving clinical picture. Coagulation tests such as INR or PT may not accurately assess the hemostatic imbalance and may lead to over transfusion of plasma without improving the outcome. The implementation of a viscoelastic testing protocol has been beneficial in adult trauma treatment and should be validated in the pediatric setting. A targeted transfusion approach may offer a feasible alternative in pediatric bleeding management, with focus on the treatment of hyperfibrinolysis and hypofibrinogenemia. Although thromboembolism prophylaxis is not generally recommended in pediatric trauma patients < 12 years old, future studies should investigate whether viscoelastic testing can be used to predict thrombosis, thus giving a more global picture of hemostatic balance in this patient population.TH and MC conceptualized and wrote the manuscript. Both authors contributed to the article and approved the submitted version.TH has received lecturer's fees and travel support from Octapharma and Instrumentation Laboratory, and is a consultant for Octapharma. MC is a consultant for Cerus Corporation and Octapharma.


And a little child shall lead them”“Gress et al.; Desai et al.; Scott and Pratt].Nowhere is immunogenicity of therapeutic proteins of greater concern or impact than in children who potentially face a lifetime of treatment for chronic disease. This is brought into focus by three publications [Gress et al. make clear that the outcome for children with Inflammatory Bowel Disease (IBD) who lose efficacy due to Anti-Drug Antibodies (ADA) requires recurrent surgeries and eventually bowel resection and colostomy creation. While this outcome is hard for adults with IBD, it is devastating to children. To allow better Tumor Necrosis Factor (TNF)-inhibitor mediated control of IBD, this article suggests: 1) Keeping levels of TNF inhibitors over a critical level (which appears to preclude ADA in most patients) using therapeutic drug monitoring (TDM). 2) Accessibility to tests detecting ADA at the earliest time point allowing consideration of therapeutics that will prevent further escalation of ADA. 3) Development of immune tolerance induction protocols. The addition of co-administered immune suppressant agents  at the introduction of the biological therapy has been shown in numerous studies to reduce ADA development and potentially induce tolerance to the biological therapy. Whether patients co-administered these agents are then truly tolerant to the TNF antagonist has not been formally evaluated. Moreover, the duration of the dual immune suppressive therapy necessary for insuring tolerance to the biological component should be studied. Key to these approaches is the ability to readily test, at no or low cost, patient samples for levels of the TNF antagonist as well as ADA.Desai et al. present an elegant approach to addressing ADA that neutralize a life-saving enzyme replacement therapy (ERT), recombinant human alpha glucosidase (rhGAA). In a subpopulation of Infantile Pompe Disease (IPD) patients that lack endogenous GAA, immune tolerance induction (ITI) is essential to a favorable patient outcome. This protocol, in which a short course of methotrexate, rituximab, and IVIG are given concurrently with initial doses of ERT proved highly tolerogenic. In the absence of this protocol these IPD patients would lose motor milestones and die of cardiopulmonary failure. Patients treated with ITI protocol recovered their B cell populations over several months and mounted vaccine responses, demonstrating the transient nature of the safety concerns. A case study by Gupta et al. demonstrated the effectiveness of early ITI in a prenatally diagnosed patient with IPD who started treatment immediately after birth. This is the first report of successful ITI at such an early age. Critically, if patients were not treated concurrently with initial dosing of the ERT and did develop life threatening ADA, the prophylactic regimen failed to eliminate antibody producing plasma cells. Thus, adding a proteasome inhibitor (bortezomib) to the regimen, resulted in dramatic diminution and elimination of the ADA, facilitating immune tolerance to ERT and allowing patients to recover their motor milestones . In this issue, two reviews from Vaisman-Mentesh et. al. and Nabhan et al. provide an excellent introduction to the molecular and cellular mechanisms involved in the generation of ADAs. Additionally, Fitzpatrick et al. provide a fascinating overview of the role of recombinant Fc multimers in immune tolerance induction and suggest that monoclonal antibodies used for treatment of cancer and autoimmunity, may mediate tolerogenic effects. This could be mediated by immune complex formation or by antibodies coating target cells, triggering multiple mechanisms including antibody dependent cell-mediated cytotoxicity, complement activation and regulatory T cell expansion, that reset immune homeostasis in an antigen independent manner. Thus, an understanding of the underlying biology is essential for designing tools to assess and circumvent immunogenicity.Anticipating an ADA response to therapeutic proteins remains the holy grail. In the ideal world, reliable non-clinical methods would predict immune responses early in clinical development. There is an unmet need for two classes of tools that assess the likelihood that a drug will generate a significant immune response. The first is a suite of in silico and Jawa et. al. and Meunier et. al.. Jawa et al. list technical approaches to assess immunogenic potential and provide a very useful discussion of integrating immunogenicity data in regulatory submissions. These two articles also provide mechanistic context for immunogenicity and of the assays being discussed. The identification of T cell epitopes in biopharmaceuticals reveals multiple mechanisms leading to T cell activation, ADA response or regulation . Variability, diversity, and joining (VDJ) recombination of antibodies and somatic mutations resulting from affinity maturation processes contribute to making non germline sequences, which could be recognized as new T cell epitopes (neoepitopes). However, though not mutated, therapeutic protein counterparts of endogenous proteins such as recombinant hormones, growth factors and cytokines can elicit CD4 T cell responses and elicit ADAs. The basis for their immunogenicity may pertain to the insufficient expression of their endogenous counterpart in the body and thus failure to induce central tolerance. Functional T cells therefore escape from negative thymic selection by insufficient levels or affinity for self-antigens leading to failure of deletion or induction of anergy, although they are specific for self-sequences and not neoepitopes. These escaped T cells may be activated in the periphery by infusion of the therapeutic proteins . Both articles  also provide an important caveat to the use of T-cell proliferation assays to assess immunogenic potential. Although T cell activation is essential for high titer, class switched, affinity matured antibody response, they do not always promote ADA responses and might differentiate instead into regulatory IL-10 secreting T cells (Tr1), with IL-10 being able to dampen activation of effector T cells .Following up on the concept that immunogenicity goes beyond strict self/non self-discrimination, two comprehensive surveys of the current state of the art with respect to non-clinical immunogenicity assessments are provided by Karle provides an in-depth critical discussion of an important emerging technology. The MHC-associated peptide proteomics (MAPPs) assay is technically challenging but allows the direct identification of the naturally processed peptides derived from biopharmaceuticals displayed by HLA class II molecules on DCs. MHC class II molecules are immunopurified and the bound peptides are eluted by acidic treatment and sequenced by liquid chromatography-mass spectrometry. Karle describes how MAPPs is used to investigate immunogenicity risk of biopharmaceuticals but also to evaluate their capture by antigen presenting cells and the impact of post-translational modifications, folding, and aggregation on peptide presentation. The article provides a useful summary of all studies that have applied the MAPPs technology to therapeutic proteins, and by comparing multiple studies using the same therapeutic, shows that the technique is reliable and renders consistent results. Unfortunately, as with all non-clinical estimates of therapeutic protein immunogenicity, the clinical significance of the results remains the critical unknown.In addition to these broad surveys of the literature, Barra et al. report an artificial neural network (ANN) model, trained to predict T cell epitopes. The algorithm presented by Barra et al. joins multiple in silico methods that are freely available to estimate immunogenicity risk based either on HLA binding affinity or on MAPPs data.Data sets of peptides eluted from MHC molecules in MAPPs assays are rapidly growing and are being leveraged to improve in silico algorithms used in immunogenicity assessments. Peptide-MHC engagement is a necessary step in the initiation of an immune response to therapeutic proteins. Thus, algorithms that predict peptide-MHC binding affinities have become an initial rapid and inexpensive screen for potential immunogenicity. One reason why such algorithms overestimate immunogenicity risk is that not all potential high-affinity peptides are actually generated by the protease machinery of antigen presenting cells. By directly identifying therapeutic protein-derived peptides on MHC molecules, the results of MAPPs assays report on both peptide processing and presentation. Thus, in this Research Topic, Paul et. al. introduce the need of rigorous benchmarking to compare the different methods of identification of T cell epitopes and provide quantitative data to point out the benefits and insufficiencies of each method . While some algorithms have become increasingly accurate at specific tasks, e.g. estimating peptide-MHC affinity or which peptides will be presented by MHC molecules, the ability to predict clinical immunogenicity remains a key challenge. Predicting immunogenicity for biotherapies using patient and drug-related factors is attractive but no robust method has as yet been developed. With the growing ability to collect massive amounts of data, machine learning algorithms could identify predictive variables. Two studies applied machine learning models to predict ADA status  utilized clinical data collected in the multi-cohort of autoimmune diseases treated with biotherapies from the ABIRISK consortium. Duhazé et al. evaluated the predictive power of a custom-built machine learning model, the random survival forest model  step in the immune response leading to ADAs. However, the MHC genes are the most polymorphic in the human genome and occur at different frequencies in various human subpopulations. This creates a problem in putting together a suitable real or virtual cohort for immunogenicity assessments that is representative of the population of interest with respect to distribution of MHC variants. Atiqi et. al., Mehta and Manson, and Gress et al. describe the scope of the problem using the example of TNF-Inhibitors. These medications have revolutionized the management of rheumatoid arthritis and other diseases but only a small proportion of patients maintain long-term clinical response  the field is beset by challenges. ADA identification is technically difficult and not standardized, making interpretation of immunogenicity data and application in the clinic almost impossible (Mehta and Manson). However, Lallemand et al. reported on the potential of using a highly sensitive reporter gene assay to quantify both an anti-VEGF ADA and the therapeutic drug activity to monitor responder vs. non-responder patients. Overall, longer term information is needed to determine the utility of these approaches. This Research Topic, however, does also offer potential strategies to overcome some of these problems  and novel assays that may be more reliable .As discussed earlier, assessment of the potential for immunogenicity represents an unmet need not only for biomolecules in the early stages of development but also in the clinic. Most approved therapeutic proteins do exhibit various levels of immunogenicity and their clinical use would benefit from accurate, reproducible and clinically meaningful measurements of ADAs and TDM. response , 4. BoyeFu et al. address biotherapeutic immunogenicity in the context of the orchestrated function of highly differentiated T and B cells, including follicular helper CD4 T cells and germinal center B cells, for the optimal generation of antibody responses. They suggest that understanding the cellular and molecular mechanisms mediating the antibody responses against therapeutics could lead to novel strategies to reduce their immunogenicity. Kishimoto describes a promising approach to antigen targeted immune tolerance induction to prevent the formation of ADAs across a wide variety of biologics. Antigen-targeted tolerance is induced in several experimental animal models  by the incorporation of rapamycin in nanoparticles in the presence of therapeutic antigen at the time of administration (ImmTOR). The problematic immunogenicity issues pertaining to recombinant immunotoxins (chimeric proteins consisting of a targeting element such as a Fv antibody region bound to a toxin) are reviewed by Mazor and Pastan. These therapies have potential for use in a wide variety of diseases and have been tested and approved in cancer. This review outlines the strategies used to mitigate the immunogenicity of immunotoxins which has a major impact on the efficacy of these promising drugs. Finally, Waddington et. al. have presented original research that applies the rapidly expanding field of serum metabolomics to therapeutic protein immunogenicity. Their study shows that serum metabolites are a promising biomarker for early identification of ADA development in MS patients treated with IFNβ and could provide novel insights into mechanisms of immunogenicity.In keeping with our increased understanding of the complexity and diversity of immune responses four papers provide a glimpse into the application of new but rapidly emerging fields to therapeutic protein immunogenicity. Significant effort is spent during clinical development in commissioning robust, specific and sensitive ADA assays that are validated to support clinical studies for product approval. However, longer term evaluation is needed to facilitate translatability to routine clinical practice that can impact patient care. ADA status (ADA-positive vs. ADA-negative) is just the first tier for assessment. Clinical relevance of ADA on PK, PD biomarkers and safety  is more evident when assessed in the context of either quartile or tertile ADA titer groups as well as evaluation of neutralizing activity. Determining relevance of ADA on efficacy parameters is complex as the measured clinical outcomes are often distal from the site of drug action and more time is needed to observe the consequences of diminished drug efficacy due to ADA. This outcome is now seen with the class of TNF-alfa inhibitor drugs and enzyme replacement therapies for some lysosomal storage diseases. Determining a clinically relevant ADA titer can be challenging and therefore, TDM is an alternative approach as data indicate that keeping levels of some biological therapeutics above a threshold value diminishes the probability of ADA generation and improves patient outcome. Post marketing commitments and requirements are mechanisms for obtaining long term data to address practical questions. However, there are challenges with this approach. After approval or licensure, such testing is usually done in a CLIA or other regulated clinical diagnostic lab. The lack of standardized assays to assess drug concentrations and ADA further complicates interpretation of results and identification of the reason for the loss of response. Companies, in collaboration with health care providers and insurers, have a responsibility to address in clinical practice the key questions regarding ADA and therapeutic drug monitoring, provide actionable answers, and establish means to make such testing readily available to ensure patients receive efficacious drug dosing. Robust analyses of the economic impact of ADA may motivate payers to support these efforts.All authors contributed to the article and approved the submitted version.Research conducted in the laboratory of ZS is funded by FDA intramural grants.This article reflects the views of the author (AR) and should not be construed to represent FDA’s views or policies.SR is an employee of Sanofi and owns stock in the company.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Race and ethnicity are associated with disparities in pain management in children. While low English language proficiency is correlated with minority race/ethnicity in the United States, it is less frequently explored in the study of health disparities. We therefore investigated whether English language proficiency influenced pain management in the post-anesthesia care unit (PACU) in a cohort of children who underwent laparoscopic appendectomy at our pediatric hospital in San Francisco. Our primary exposure was English language proficiency, and our primary outcome was administration of any opioid medication in the PACU. Secondary outcomes included the amount of opioid administered in the PACU and whether any pain score was recorded during the patient’s recovery period. Statistical analysis included adjusting for demographic covariates including race in estimating the effect of language proficiency on these outcomes. In our cohort of 257 pediatric patients, 57 (22.2%) had low English proficiency (LEP). While LEP and English proficient (EP) patients received the same amount of opioid medication intraoperatively, in multivariable analysis, LEP patients had more than double the odds of receiving any opioid in the PACU . LEP patients received more oral morphine equivalents (OME) than EP patients , and they also had almost double the odds of having no pain score recorded during their PACU recovery period , although the precision of these estimates was limited by small sample size. Subgroup analysis showed that children over the age of 5 years, who were presumably more verbal and would therefore undergo verbal pain assessments, had over triple the odds of having no recorded pain score . In summary, English language proficiency may affect the management of children’s pain in the perioperative setting. The etiology of this language-related disparity is likely multifactorial and should be investigated further. Disparities in pediatric pain management associated with race/ethnicity are well-documented. In emergency departments nationwide, Black children receive opioids at significantly lower rates than White children when presenting with acute appendicitis . In the linguicism  language) and expressing that “language is a social practice that shapes subjectivity and establishes power relations among members of different racial and class groups” [In the United States, race and language are intricately connected, with scholars describing the practice of  groups” . English groups” ,7, is ho groups” . In addi groups” . In addi groups” .The present study is focused on further examining the role of language disparities in perioperative care. Specifically, we conducted a retrospective cohort study in order to understand whether English language proficiency influenced PACU pain management in a cohort of children who underwent laparoscopic appendectomy at an urban pediatric hospital in San Francisco, where a large proportion of residents do not speak English at home . We hypoWe used electronic medical record data from our academic pediatric hospital located in Northern California to conduct a retrospective cohort study among patients who underwent laparoscopic appendectomy between February 2015 and July 2019. Eligible patients were age 18 years or younger and were recovered in the PACU after surgery. All laparoscopic appendectomies were performed by pediatric general surgeons who used three port sites and administered local anesthetic subcutaneously at these sites during surgical closure. All anesthetics were administered by pediatric anesthesiologists who provided a balanced anesthetic with a volatile agent, a medium- to long-acting opioid , and boluses or infusion of an intravenous sedative/hypnotic (propofol). Other analgesics and sedative/hypnotics including short-acting opioids , ketamine, and dexmedetomidine were not routinely used. For each patient, follow-up extended from the four hours preceding surgery until they were discharged from the PACU.The primary exposure of interest in our analysis was English language proficiency, which we determined based on whether an interpreter was needed when the patient and their parent or guardian presented for their appendectomy or for any prior clinical encounter. Patients were categorized as having low-English-proficiency (LEP) if they indicated that an interpreter was needed and designated as English-proficient (EP) otherwise.Our primary outcome of interest was whether a patient received any opioid medication in the PACU. Opioid medications included fentanyl, morphine, hydromorphone, oxycodone, hydrocodone, methadone, and tramadol. We created an indicator variable that equaled one if the patient received any of these opioid medications in the PACU and zero otherwise. Secondary outcomes included the amount of opioid administered in the PACU, calculated in oral morphine equivalents (OME) per kilogram ,13. We aPatient demographics included in the present study were continuous age (0–18); categorical patient- or family-reported gender ; categorical patient- or family-reported ethnicity (Hispanic or Non-Hispanic); and categorical patient- or family-reported race. For race assignation, patients and their parent/guardian could decline to answer or self-identify one or more categories from the U.S. Census list of race categories  , “Other,We also compared LEP and EP patients in terms of American Society of Anesthesiologist ratings, preexisting medical conditions , use of opioids before admission, time in the operating room, presence of appendiceal perforation and/or peritonitis, opioids administered intraoperatively (in OME per kilogram), nonopioid adjunct medications administered intraoperatively (acetaminophen and/or ketorolac), and occurrence in the PACU of laryngospasm, postoperative nausea/vomiting, low respiratory rate (<10 breaths per minute), and low oxygen saturation (<90%). We did not include these variables in our subsequent statistical analyses because we hypothesized that they were related to the outcomes in the causal pathway solely as mediators, rather than as confounders.We used generalized logistic regression to estimate the association between language proficiency and PACU opioid administration. We hypothesized that demographic factors may influence how PACU nurses assessed and treated patients’ pain. This model was therefore adjusted for demographic characteristics as enumerated above. Ethnicity was not included in this model or subsequent models as only three patients self-identified as Hispanic.Next, we examined the association between language proficiency and the amount of opioid administered in the PACU in OME per kilogram. The observed distribution of OME per kilogram administered exhibited overdispersion due to a point mass at zero and right skew, which we accommodated by using negative binomial regression, adjusting for demographic characteristics as described above. We also conducted a supplementary analysis using this method to estimate the association between language proficiency and the amount of intraoperative opioid administered in OME per kilogram.Finally, we used generalized logistic regression to estimate the association between language proficiency and whether a pain score was incorporated in the PACU after adjusting for demographic characteristics. We then conducted a subgroup analysis by stratifying our cohort by age  to delineate differences in how nurses may interact with and elicit pain scores from young children versus older, school-aged, more verbal children.Statistical analyses were performed using R version 3.6.3  . This stDuring our study period, 257 pediatric patients underwent laparoscopic appendectomy and subsequently recovered in the PACU. Of these, 57 (22.2%) were identified as LEP. The most common language spoken by LEP patients was Spanish (80.7%). Characteristics of these patients are presented in For the remainder of the results, we report 95% confidence intervals but do not interpret results using null hypothesis significance testing. Rather, we attempt to determine if regression results imply an underlying relationship between exposure and outcome .In multivariable analysis, LEP patients had more than double the odds of being administered any opioid in PACU compared to EP patients  . LEP patLEP patients also had approximately double the odds of having no pain score recorded during their PACU recovery periods  . In subgOur retrospective cohort study identifies English language proficiency as a potential contributor to disparities in children’s perioperative pain management, in addition to the racial disparities that have been reported previously in the PACU ,18 and tOptimal treatment of pain requires balancing both objective and subjective assessments of pain and can prevent long-lasting complications in a post-surgical population. Overreliance on objective pain scales, without incorporation of clinicians’ and patients’ subjective assessments, can increase patient anxiety and diminish the therapeutic alliance among patients, family members, and their healthcare providers . HoweverIn our cohort, a large proportion of children underwent no objective measurement of pain during their PACU recovery period, with LEP children being more likely to have no recorded pain score and more likely to receive opioid medication in the PACU. Our data did not elucidate what types of subjective measurements clinicians may have used to assess pain before administering opioids, or whether LEP children were overtreated or oversedated. While there were almost no documented occurrences of nausea/vomiting, low respiratory rate, or low oxygen saturation in the PACU in either the LEP or EP groups, these outcomes are often transient and may not be adequately captured in our medical record. In our open-model PACU, a low oxygen saturation event may be shorter than a minute and be rapidly corrected by stimulation or administration of supplemental oxygen. Such transitory events would not automatically be entered into the patient’s medical record. Future studies should investigate the types of non-objective assessments being done in the PACU and whether these do indeed result in oversedation.The reasons behind language-related differences in care—including the likelihood not to use objective measures of pain—are likely multifactorial and related both to interpersonal and systemic factors. Interpersonal factors may include conscious or subconscious bias against those who do not speak English  ; patientThis retrospective study was based on language assessment data from inpatient and outpatient admissions personnel and may therefore be subject to selection or information bias. A bilingual child may have been denoted as LEP if their parent or guardian was LEP; thus, a nurse could potentially assess the patient’s pain in English in the PACU even if the parent or guardian may have needed interpretation for more complicated discussions. Further, our medical record does not include information on non-English languages spoken by the patient’s assigned nurse. These types of errors are common in language data collection , and in Children with generalized peritonitis and/or perforation experience more pain following laparoscopic appendectomy . While oOur data also illustrate the complexity in portraying constructs such as ethnicity. Only three of 54 Spanish-speaking patients self-identified as Hispanic, findings which are inconsistent with our city demographics . Self-idFinally, our study was conducted at a single academic pediatric hospital in San Francisco, and findings therefore may be unlikely to reflect the experiences of patients who receive care in other hospital systems due to meaningful differences in patient and staff demographics. Subsequent larger studies may provide more precise and generalizable findings regarding potential language-related biases in pediatric populations.In this retrospective cohort study of 257 pediatric patients, we found that English language proficiency may affect the management of children’s pain in the perioperative setting at our institution. Our study underscores the importance of communication in decisions surrounding pain management. Our findings motivate further investigation regarding the role that language plays in treatment decisions in pediatric patient populations, as well as efforts to identify strategies to optimize pain management for all of our patients regardless of their language.


Human infection challenge studies (HCS) involve intentionally infecting research participants with pathogens (or other micro-organisms). There have been recent calls for more HCS to be conducted in low-income and middle-income countries (LMICs), where many relevant diseases are endemic. HCS in general, and HCS in LMICs in particular, raise numerous ethical issues. This paper summarises the findings of a project that explored ethical and regulatory issues related to LMIC HCS via (i) a review of relevant literature and (ii) 45 qualitative interviews with scientists and ethicists. Among other areas of consensus, we found that there was widespread agreement that LMIC HCS can be ethically acceptable, provided that they have a sound scientific rationale, are accepted by local communities and meet usual research ethics requirements. Unresolved issues include those related to (i) acceptable approaches to trade-offs between the scientific aim to produce generalisable results and the protection of participants, (iii) the sharing of benefits with LMIC populations, (iii) the acceptable limits to risks and burdens for participants, (iv) the potential for third-party risk and whether the degree of acceptable third-party risk is different in endemic settings, (v) the conditions under which (if any) it would be appropriate to recruit children for disease-causing HCS, (v) appropriate levels of payment to participants and (vi) appropriate governance of (LMIC) HCS. This paper provides preliminary analyses of these ethical considerations in order to (i) inform scientists and policymakers involved in the planning, conduct and/or governance of LMIC HCS and (ii) highlight areas warranting future research. Insofar as this article focuses on HCS in (endemic) settings where diseases are present and/or widespread, much of the analysis provided is relevant to HCS (in HICs or LMICs) involving pandemic diseases including COVID19. Modern HCS are sometimes referred to as ‘controlled human infection studies’ because they involve HCS can accelerate the development of new vaccines (and therapeutics) because they can be substantially smaller, shorter and less expensive than other kinds of studies. Among other advantages, far fewer people need to be given experimental vaccines (that might not turn out to be safe or effective) in HCS than in field trials. The latter are much larger, and commonly involve thousands of participants; whereas most HCS involve less than 100 participants. HCS can also provide unique insights in to host-pathogen interactions.Although several prominent historical cases of unethical research involved the intentional infection of research participants,Despite the potential scientific advantages of (endemic and/or LMIC) HCS, such studies are nonetheless ethically sensitive. HCS raise ethical issues involving controversial aspects of research ethics more generally , but they also raise ethical issues that are more specific to HCS in particular .This paper summarises the findings of a project that examined the ethical and regulatory issues related to HCS in endemic and/or LMIC settings via (i) a review of relevant literature and (ii) qualitative interviews involving 45 scientists and ethicists with relevant expertise (table 1). Details of the search strategy and qualitative interview methodology can be found in the 10.1136/medethics-2019-106001.supp1Supplementary dataInsofar as this article focuses on HCS in (endemic) settings where diseases are present and/or widespread, much of the analysis provided is relevant to HCS (in HICs or LMICs) involving pandemic diseases including coronavirus disease 2019 (COVID19).In the limited ethics literature on HCS to date, there appears to be a consensus that intentional infection of research participants  can be permissible so long as certain ethical conditions are met.not performing HCS could lead to greater net harms including (i) longer delays to the development and implementation of beneficial new interventions for infectious diseases and/or (ii) the exposure of more participants to potentially greater risks in alternative study designs . Such considerations might especially favour HCS for diseases that cause persistently high annual burdens of disease  and/or emerging diseases . Given that HCS are often expected to lead to significant public health benefits more efficiently  than alternative research designs,inter alia, (i) HCS in endemic LMICs may be more scientifically valid and/or efficient in terms of producing results that are relevant to disease control in at-risk populations, (ii) in at least some cases participants in endemic LMIC HCS might benefit directly from research participation  If there is an ethical imperative to conduct HCS  that is grounded (in part) in the need to relieve significant burdens of infectious disease, then there is arguably an even stronger ethical imperative to conduct HCS in endemic settings and/or LMICs in particular because, There are thus strong ethical reasons that support conducting (more) endemic and/or LMIC HCS. However, even if there is an especially strong case for conducting such studies, certain (other) ethical issues related to their design and conduct warrant particularly careful attention. This is because (i) HCS may sometimes involve, or at least be perceived to involve, particularly high levels of risks (for participants and third parties) and other burdens for participants (and such studies must therefore be carefully designed and conducted to ensure that expected benefits outweigh risks and burdens), (ii) local and/or international community acceptance of HCS being conducted in endemic LMICs may be contingent on such studies being designed and conducted to especially high ethical (and scientific) standards and (iii) certain ethical considerations, although familiar in research ethics discourse, may have particular (underexplored) implications in the context of endemic LMIC HCS. The evaluation of these latter implications may both improve the design and conduct of LMIC HCS and/or inform ongoing debates in research ethics.A key link between the scientific rationale and the social value of a study is the generalisability of the findings (whether these consist of knowledge of disease pathogenesis or estimates of the efficacy of a novel intervention) to the population and context for which the eventual benefits of a research programme are intended.Certain choices in study design  might lead to results that are more or less generalisable to the populations most at risk of a particular disease. For example, LMIC HCS would arguably be more generalisable and thus have a stronger scientific and ethical rationale in cases where there are (more) relevant features  shared by potential (LMIC) study participants and the (LMIC) population(s) most at risk of the  infection in question.early phase research  in LMICs because such research is not intended to lead to the immediate development or approval of such interventions.The sharing of benefits with study participants and/or local populations is commonly cited as an ethical requirement for international research involving human participants, but controversy surrounds questions regarding the nature, content and weight of such a requirement.Research participation can involve a range of burdens; and some HCS designs entail relatively high levels of burdens for participants , especially in comparison with other studies involving healthy volunteers.inter alia (i) selection of study participants at lower risk of severe disease, (ii) use of challenge strains that produce less severe disease, (iii) early diagnosis and/or treatment, (iv) keeping participants as inpatients to enable particularly close monitoring for at least part of the study, (v) close monitoring of (any) outpatient participants and/or (vi) careful follow-up for long-term outcomes. Many of these means of reducing risks frequently entail increasing other kinds of burdens  and/or a reduction in generalisability . There may thus often be complex ethical trade-offs involved in study design.Recent HCS designs are generally considered to be safe. They have been approved by research ethics committees; there have been no reported deaths among participants; and significant lasting harms to participants are extremely rare .exceed what might commonly be thought to be acceptable limits in research with healthy volunteers who are not expected to benefit directly from study participation. For example, cholera HCS in North American volunteers led to some participants experiencing >13 L of diarrhoea (whereas Thai participants in similar studies had up to approximately 3.6 L—perhaps due in part to immunity from prior infection).With risk minimisation strategies in place, some HCS designs could plausibly be described as minimal risk , under a range of definitions of this term.per se presumably shouldn’t ethically preclude HCS.Aside from well-described risks, HCS participation sometimes involves significant uncertainty, particularly at the beginning of a research programme involving a novel HCS design  via which few (or no) people have yet been challenged. While HCS conducted under situations of uncertainty (at the outset of a research programme) might eventuate in unexpected harm, they can also yield scientifically important (but unexpected) results; and HCS research has revealed new and/or unexpected features of certain diseases.There is no universally agreed-upon upper limit to acceptable risk in ‘non-therapeutic’ research with healthy volunteers that do not have potential to benefit directly from research participation. Proposals include (i) no limit,A pragmatic reason for excluding very high-risk HCS is that the public reaction to severe harm that might otherwise result could lead to a moratorium on HCS—including those that are potentially beneficial but involve low risk as well as those that have very high expected benefits that outweigh significant but acceptable risks.Most commentators have argued that, as a general rule, HCS should involve infectious diseases that are treatable and/or self-limiting—that is, resolving in the absence of treatment, without causing serious harm.Requirements that there be no lasting harms are potentially important since some infections (that have been or could plausibly be studied via HCS) do involve a low probability of lasting harm. Many of these risks can be reduced through careful participant selection, diagnosis, treatment and follow-up. However, while some postinfectious syndromes have known risk factors, meaning that individuals known to be at higher risk can be excluded from HCS, such strategies often cannot prevent these outcomes entirely (table 2). The fact that lasting harms have not been documented among modern HCS participants for these pathogens may reflect careful selection practices and/or relatively low numbers of total participants  and/or publication bias . One HCS researcher interviewed was aware of at least one case of presumed postinfectious arthritis occurring after HCS with an enteric pathogen. More generally, interviewees agreed that such lasting harms were particularly concerning and, at a minimum, require (i) careful risk mitigation strategies, (ii) long-term follow-up of participants and (iii) systems to compensate any participants who experience harm. However, there was no clear consensus about what level of residual risk of such outcomes, if any, should be considered acceptable , one might think that, in some cases, this background risk reduces the marginal risk an individual would take on by participating in a challenge study.the absolute magnitude of risk increase is a key consideration, independent of background risk magnitude).Research ethics literature regarding background risk more generally,Since HCS frequently involve multiple study visits, blood draws and (inpatient or outpatient) monitoring by study staff, and since inpatient HCS in particular involve significant time away from normal activities, many HCS designs can be highly burdensome for participants. In some cases, such burdens might include adverse effects on mental health . Recent social science work with participants has suggested that HCS participation, particularly in inpatient studies, can lead to a wide range of burdens.Certain participant behaviours might lead to greater risks than those anticipated in the study protocol—for example, participants (i) choosing to withdraw from the study and/or (ii) absconding .The right of participants to withdraw from a study—at any time, for any reason, and without having to give a reason—is widely endorsed in research ethics theory and practice, although the practical implications of this right have been a matter of debate in particular contexts.Many types of research with infectious diseases can pose risks of infection to those not directly participating in the research .Some have argued that researchers have extensive ethical duties to third parties where such risks of being infected by research participants exist. For example, some have argued that informed consent should be sought from at-risk individual third parties and/or local communities prior to study commencement.In any case, the potential risks may vary in different contexts and with different modes of disease transmission. For vectorborne diseases (VBD) such as malaria, if there are no local vectors then there are minimal risks of third-party transmission (apart from blood donation by participants while infected). Many ‘endemic’ LMICs contain regions or cities with no vectors; and, with respect to third-party risk, these are arguably ideal locations for VBD HCS research.The potential for higher third-party risks in endemic, epidemic, and/or pandemic contexts can lead to controversial questions. How important, for example, is a small third-party risk and/or single episode of transmission  in the context of high local disease transmission ? Some individuals (and communities) may consider this additional risk negligible (and some interviewed experts from both HICs and LMICs held this view), while others may see each additional episode of transmission as highly  significant see . Given tCalls for more LMIC HCS arguably go against the idea that, insofar as they are considered ‘vulnerable’ due to poverty and/or other factors, LMIC populations should be excluded from research, particularly early phase studies. One concern regarding exploitation of vulnerable populations is that relatively privileged populations are often the primary beneficiaries of research conducted in more underprivileged populations.An increasingly recognised problem is that excluding vulnerable populations from research as a way of protecting them from further burdens can ultimately lead to these same populations being excluded from the benefits of research.It is sometimes thought that it would be more ethical to recruit those with higher levels of education as research participants because this may improve (participant understanding and thus) informed consent. This is one reason why some LMIC HCS have aimed to recruit university students in particular.One of the most controversial questions regarding HCS concerns when, if ever, it might be ethically acceptable to recruit children as participants in studies that could cause significant symptoms/disease among (child) participants. On the one hand, many pathogens of interest predominantly harm young children in endemic settings who would thus stand to benefit most from new vaccines (and thus the research in question). On the other hand, (i) HCS in carefully selected adult volunteers might in at least some cases be sufficiently generalisable to children at risk (and thus obviate the need to recruit children), (ii) children lack the capacity to provide informed consent to participation, (iii) challenge infection might sometimes be higher risk in children and (iv) public perceptions of the enrolment of children in HCS could undermine trust in HCS and/or research more generally.For these and other reasons, the only recent HCS performed with children have used (live-attenuated) vaccine strains of micro-organisms as the challenge agent.There was widespread consensus among those we interviewed that, even if disease-causing HCS in children could be conducted safely, there should be a presumption against enrolling children in such studies . Arguably, several requirements would  have to be met before (disease-causing) HCS involving children were initiated, including (i) a strong and very carefully described scientific rationale, (ii) international consultation  and (iii) clear demonstration of local community acceptance. A particularly strong theme that emerged in our stakeholder interviews was the importance of public and/or community acceptance and the risk of undermining trust in (HCS) research see .Since HCS participants endure costs and burdens while contributing to projects that primarily aim to benefit others , some level of payment might be considered ethically appropriate. Payment of research participants remains controversial, however, especially in studies involving  vulnerable populations and/or high levels of payment.Attitudes towards the payment of research participants vary across different cultures/countries, as do payment practices. Small reimbursements  are relatively uncontroversial, and there is also a recognition that studies involving large time commitments  may warrant greater payment.Payment intended to compensate for other burdens and/or incentivise research participation, however, is more controversial.Stakeholders we interviewed generally agreed that payment for participation in (LMIC) HCS was ethically acceptable, and that it should be proportional to the burdens of HCS participation . However, determination of the appropriate method(s) for titration of payment according to the burdens of participation was an area that was considered contentious see .Undue financial inducement might (or might be perceived to) (i) undermine informed consent by impairing decision-making Many of our interviewees were concerned about the potential for undue inducement to participate in HCS (in both HICs and LMICs) and acknowledged that poverty and cultural norms could alter local perceptions of payment for research participation. However, some also argued that this should not necessarily preclude the payment of participants in LMIC HCS. Indeed, as noted above, most stakeholders we interviewed agreed that the level of payment should be proportional to the burdens of participation. Several acknowledged the potential for undue inducement among economically vulnerable participants in HICs, emphasising that this potential issue was not unique to LMICs, but one that requires further work to determine fair levels of payment in different settings see .In addition to undue inducement, there may sometimes be other ethically salient effects of payment. First, payment may lead to the disproportionate recruitment of impoverished individuals and groups, which would in some cases arguably be unjust .HCS are sometimes perceived to be an unusual and/or particularly sensitive type of research, and thus some commentators have recommended the development of specific ethical frameworks/guidelines/principlesWith regard to specific ethical principles/guidelines/frameworks for HCS,new challenge models in particular ,Regarding potential special review procedures for HCS, different models have been proposed, including: (i) the appointment of a special committee  or subcommittee for review of all HCS ,Ultimately, policymakers in any given jurisdiction will need to adopt a policy regarding HCS review that is apt for the local context. Ethically, the important outcomes (regardless of the policy chosen) might include that burdens, including risks (to both participants and third parties), are appropriately minimised; that public trust in research is maintained and that scientifically valuable, acceptably low-risk studies are not unduly impeded by excessively costly or slow review procedures.As noted in other sections of this article, public controversies regarding (HCS) research have the potential to undermine support/acceptance of (other) research and/or public health endeavours—domestically and/or internationally. Thus, in some cases, international consultation and/or appeal to international agencies  may be appropriate. Whether or not a particular jurisdiction decides on specialised or standard review, international agreement on an ethical and regulatory framework for HCS may help to improve review and ensure that relevant issues are consistently addressed.There are ethical and scientific reasons in favour of conducting HCS in endemic and/or LMIC settings in order to address the persistently high global burden of infectious diseases, especially in disadvantaged populations. Careful attention to the ethically salient aspects of HCS design, relevant governance mechanisms and the acceptability of HCS among participants and communities will help to ensure the sustainability of this important area of research which will hopefully produce significant benefits in such populations. Insofar as this article focuses on HCS in (endemic) settings where diseases are present and/or widespread, much of the analysis provided is relevant to HCS (in HICs or LMICs) involving pandemic diseases including COVID19. Given the complexities of such studies, and the controversial and/or unresolved issues highlighted in this article, further work is needed by biological scientists, social scientists and bioethicists to support ongoing improvements in the ethical design, conduct and review of HCS.


The natural environments in which infants and children learn speech and language are noisy and multimodal. Adults rely on the multimodal nature of speech to compensate for noisy environments during speech communication. Multiple mechanisms underlie mature audiovisual benefit to speech perception, including reduced uncertainty as to when auditory speech will occur, use of correlations between the amplitude envelope of auditory and visual signals in fluent speech, and use of visual phonetic knowledge for lexical access. This paper reviews evidence regarding infants’ and children’s use of temporal and phonetic mechanisms in audiovisual speech perception benefit. The ability to use temporal cues for audiovisual speech perception benefit emerges in infancy. Although infants are sensitive to the correspondence between auditory and visual phonetic cues, the ability to use this correspondence for audiovisual benefit may not emerge until age four. A more cohesive account of the development of audiovisual speech perception may follow from a more thorough understanding of the development of sensitivity to and use of various temporal and phonetic cues. Studies of the development of speech perception in infancy and childhood have provided valuable information about the structure of phonological representations and the mechanisms of early speech and language learning ,2. HowevThe natural environments in which infants and children learn speech and language are specified by highly redundant and synchronous multimodal signals. These environments are also noisy ,4,5,6,7.Speech is a particularly rich multimodal signal. Visible articulations (and even head movements) correlate with multiple cues in the auditory speech stream, including onsets and offsets, amplitude modulations, and rhythm ,21,22,23Development of the ability to use visual speech cues to understand speech in noise extends into adolescence, with young children showing limited benefit, e.g., ,35. Thisare sensitive to the correspondence between auditory and visual speech cues, whereas child and adult studies measure whether children and adults use the correspondence to benefit from visual speech cues. As Shaw and Bortfeld [One interpretation of these findings is that development follows a u-shaped trajectory, wherein infants are sensitive to the correspondence between auditory and visual cues, but children are not. Another interpretation is that different studies and methods have different cognitive requirements. Infant matching studies only measure whether infants Bortfeld  eloquentSome measures compatible with testing infants have been used to assess audiovisual perceptual development across the lifespan. For example, the range of asynchronies over which we bind auditory and visual speech has been estimated at various ages and shown to decrease with age ,47,48. ARecently, Lalonde and colleagues ,52 adaptLalonde and Werner  also adaOverall, these results suggest that there is little change over development (from 6 months to about 30 years) in audiovisual benefit to speech detection, at least for simple speech signals such as single syllables . This eaAudiovisual speech perception involves a large network of subcortical and cortical structures. Midbrain structures such as the superior colliculus receive both auditory and visual input, e.g., . At the Neurophysiological and behavioral evidence indicates that this large neural network subserves multiple mechanisms of audiovisual speech perception benefit in adults ,63,64,65There are correlations between the temporal properties of visible articulations and acoustic speech, with visible mouth movements preceding acoustic speech by 100 to 300 ms . This meThere are many cross-modal temporal cues in speech, varying in complexity. The most basic is the onset of speech. Onset cues increase temporal expectancy, reducing uncertainty as to when auditory speech will occur. In general, temporal expectancy makes it easier to detect sensory information. For example, defining an observation interval with a light improves detection of tones in noise compared to when the observation interval is not defined . As prevBeyond basic cues to speech onset/offset, there are ongoing cross-modal correlations between the amplitude envelopes of fluent auditory and visual speech signals ,21,22. IBoth mouth movements and the amplitude envelope of acoustic speech are quasi-rhythmic and temporally modulated at a rate of 2 to 7 Hz, corresponding to the timing of syllables . This moBehavioral support for the use of ongoing cross-modal temporal correlations for audiovisual benefit comes from comparisons of degree of audiovisual benefit across sentences naturally varying in degree of cross-modal correlation. Grant and Seitz  demonstrRecent work has also demonstrated audiovisual benefit to sentence recognition from a visual analog of the acoustic speech amplitude envelope. When presented sentences in multi-talker babble, adults recognized speech with 3–5% greater accuracy with this visual analog  than when listening to auditory-only speech . No signFrom years of experience with speech and language, adults have learned which salient visual cues are associated with phonemes, syllables, and words in their native language. For example, adults learn that a lip closure represents a bilabial sound, such as /p/, /b/, and /m/, and not a velar sound, such as /k/ or /g/. Adults use this knowledge to differentiate between visually distinct speech sounds . In noisThis concept extends to the domain of lexical activation . The neiFurther support for the use of visual speech to activate lexical representations comes from the word superiority effect in audiovisual speech perception . Fort etThe temporal and phonetic mechanisms of audiovisual benefit provide a framework from which to interpret studies relevant to development of audiovisual speech benefit. From a neurophysiological perspective, the cortical structures used to access visual/multimodal phonetic/lexical representations are experience-based and show limited maturation during the first year of life. In contrast, the subcortical pathways that underlie sensitivity to auditory and visual synchrony are highly developed at 6 months of age ,92.In addition to distinguishing between temporal and phonetic cues, one has to consider the complexity and salience of the particular temporal or phonetic cues needed to perform a task. More basic auditory–visual temporal cues, such as onset cues, and more visually distinct or visually salient phonetic cues appear to contribute to audiovisual speech perception benefit earlier in development than more subtle and complex cues. Studies of general auditory–visual temporal perception in infancy indicate that sensitivity to synchronous onsets emerges first, with sensitivity to increasingly complex temporal cues  emerging sequentially and in a hierarchical fashion . SimilarThe importance of visual cue salience/distinctiveness extends to later development. One-year-old infants recognize mispronunciations in familiar audiovisual words if the mispronunciation involves a change in a visually salient feature (place) but not in a visually indistinguishable feature (voicing) . Three- A few developmental studies have been designed to differentiate between audiovisual speech perception benefits resulting from the use of temporal and phonetic cues, respectively. One approach is to degrade the acoustic signal in ways that minimize the use of phonetic cues and compare performance with and without those phonetic cues. A second approach is to limit the phonetic cues provided by the visual signal and compare benefit from full and cue-limited visual signals. A third approach is to vary the level of perceptual processing required by a task, from speech detection benefit  to higher-level tasks such as speech discrimination and speech recognition (which likely rely on phonetic cues). Fourth, by examining the patterns of errors in auditory and audiovisual word recognition, we can assess whether visual phonetic information was used for audiovisual benefit. Finally, different components of electrophysiological responses to audiovisual speech stimuli may vary in their dependence on temporal and phonetic cues.Baart and colleagues ,66 have Baart and colleagues  comparedNaïve observers are typically unaware that SWS is generated from a speech signal, but once informed of its speech-like nature, they can usually understand it . TherefoIn a follow-up to the infant experiment, Baart and colleagues  comparedIn previous sections we noted that (1) infants, children, and adults demonstrate equal audiovisual speech detection benefit for syllables ,52, and There is an assumption that cues that benefit speech detection also apply to higher-level tasks such as speech recognition, due to the hierarchical nature of speech perception. One must detect speech in order to discriminate its features and recognize phonemes and words. To test this assumption, Lalonde and Werner  conducteInfants can use more complex cross-modal temporal cues in fluent speech for audiovisual benefit. Hollich and colleagues  used a hAll groups were subsequently tested in auditory-only conditions to determine whether infants showed preference for the target word spoken by the same female target talker over a non-target word spoken by the same talker in the same manner. Infants who heard the synchronous, congruent passage demonstrated preference for the familiar target word, while those who saw a static image or asynchronous, incongruent speech did not. This suggests that infants used the congruent visual cues to segregate and attend to the target speech stream and to segment the target words from the passage. Importantly, infants who viewed the oscilloscope pattern demonstrated the same preference, suggesting that infants’ ability to benefit from the visual speech signal was related to the use of correlated amplitude envelope cues. Adults’ benefit from a visual analogue of the amplitude envelope of speech is also significant, but is greatly decreased in comparison to synchronous, congruent visual speech . It is iAs previously noted, adults seem to rely on different cues to benefit from visual speech depending on the level of processing required by an audiovisual speech perception task. They relied on onset/offset cues for audiovisual benefit to speech detection and relied on other salient differences and/or phonetic cues for discrimination . Among sLalonde and Holt  examinedAdults were three times more likely to substitute a consonant with a visually similar one in audiovisual conditions than in auditory-only conditions. Four-year-olds’ errors followed a similar pattern, with a 2:1 difference between audiovisual and auditory-only substitutions. Three-year-olds, however, showed no significant difference in visually salient substitution errors for auditory-only compared to audiovisual conditions. This suggests that by 4 years of age children may have reached a point in development at which visual phonetic processing is mature enough to benefit audiovisual word recognition in noise. The same 3-years-old children are sensitive enough to salient differences between visual consonants that they can use them for discrimination benefit see , but theAudiovisual effects on some early-stage components of electrophysiological responses, such as the auditory N1, are related to visual speech’s ability to temporally cue the onset of acoustic speech ,72,73,74Kaganovich et al.  examinedEvidence suggests that infants are sensitive to audiovisual phonetic cues ,107,108.Like infants, 3- and 4-year-olds are sensitive to the correspondence between auditory and visual speech cues. They can match acoustic vowels to the correct facial articulations with greater than chance accuracy in the absence of any temporal cues to distinguish between the two . Adults Differentiating between the use of phonetic and lexical cues for audiovisual benefit is perhaps even more difficult than differentiating between phonetic and temporal cues. Nevertheless, a couple of studies have been completed that speak to the use of lexical cues. To test children’s use of visual speech to activate lexical representations, Fort et al.  tested fSensitivity to the correspondence between auditory and visual speech cues is apparent shortly after birth  and is oFirst, infants and children often use different mechanisms than adults to benefit from visual speech. An important distinction—supported by both behavioral and neurophysiological evidence—has been drawn between general perceptual audiovisual benefit resulting from the use of temporal cues and speech-specific audiovisual benefit resulting from the application of visual/multimodal phonetic knowledge. The use of temporal cues for general perceptual benefit emerges early and does not improve with development. At 6 months of age, infants can use temporal cues to the onset of speech to better detect and discriminate syllables in noise . Adults Second, different testing methods target different underlying audiovisual mechanisms. Infant matching studies measure whether infants are sensitive to auditory-visual correspondences, whereas child studies measure whether children can use this correspondence to benefit from visual speech cues. Techniques compatible with testing infants typically cannot differentiate between sensitivity to the correspondence between auditory and visual speech cues and use of these correspondences for audiovisual benefit. Further, the level of perceptual processing required by a task and its emphasis on phonetic processing and lexical access affect the types of cues that are used at any given stage of development ,51,110.Finally, the complexity and salience of the particular temporal or phonetic cues needed to perform an audiovisual speech task affect whether an infant or child can use it. Development of general sensitivity to audiovisual temporal cues during the first year of life is sequential and hierarchical; sensitivity to onset cues develops first and bootstraps later development of sensitivity to more complex temporal cues . This suGiven these three facts, careful experimental design is necessary to determine what cues are used on any particular audiovisual speech perception task. Stimulus manipulations that decrease the auditory or visual cues available to observers help to disambiguate what cues are being used, as do comparisons across tasks differing in complexity. A caveat about using these stimulus manipulations is that they decrease ecological validity. A more cohesive account of the development of audiovisual speech perception may follow from a more thorough understanding of the development of sensitivity to and use of various temporal and phonetic cues.