Family members have important role in care of older people. In residential long-term settings family members can find themselves in an ambiguous situation: officially, responsibility for provision and quality of care rests with the care provider and staff members, but in practice family members participate in caring. This study explores the role of family members in residential long-term care settings, particularly in supporting their older relatives’ functional ability. Developing and maintaining functional ability lies at the very core of healthy ageing policies and long-term care. The data consist of semi-structured interviews with family members (n=16) from eight long-term care facilities in Finland. Thematic analysis yielded three themes: maintaining personhood, engaging in everyday life and monitoring care. Family members in our study were actively involved in care that supported the functional ability of their older relative. However, family members had also conflicting views about who was responsible for care provision. Some participants willingly accepted their caregiver responsibilities even in residential care, while others described their involvement in care not as a matter of choice but rather as one of necessity in order to ensure good quality care. It is important to see the family members’ viewpoint which, based on the results of this study, emphasizes personhood and continuity of care. If they are willing to participate, family should be able to take part in caregiving together with the care staff and their role should be recognized.


Social support is a well-known protective factor against depressive symptoms and substance use problems, but very few studies have examined its protective effects among residents of permanent supportive housing (PSH), a housing program for people with a history of chronic homelessness. We utilized unconditional latent growth curve models (LGCMs) and parallel process growth models to describe univariate trajectories of social support, depressive symptoms, and substance use problems and to examine their longitudinal associations in a large sample of adults residing in PSH.Participants were 653 adult PSH residents in North Texas  who participated in a monthly health coaching program from 2014 to 2017. Their health behaviors were assessed at baseline and tracked every six months at three follow-up visits.Unconditional LGCMs indicated that over time, social support increased, whereas depressive symptoms and substance use problems decreased. However, their rates of change slowed over time. Further, in parallel process growth models, we found that at baseline, individuals with greater social support tended to have less severe depressive symptoms and substance use problems . Individuals with a faster increase in social support tended to have steeper rates of reduction in both depressive symptoms  and substance use problems , respectively.This study suggests that plausibly, increases in social support, though slowing over time, still positively impact depressive symptoms and substance use problems among PSH residents. Future PSH programs could emphasize social support as an early component as it may contribute to clients’ overall health. People who are homeless are at higher risk for health problems, such as malnutrition, stress, communicable diseases, and violence . The eleThough PSH residents are affected by many of the same issues that homeless individuals experience, PSH may provide the opportunity to begin new and steady social relationships and to gain support through newly formed connections . For manIn general, research suggests that depressive symptoms are negatively related to social support among people with current or past homelessness –24. For Similarly, there is a dearth of information on the longitudinal relationship between social support and substance use problems among PSH population. A randomized trial of a “Housing First” PSH program on substance use problems among homeless individuals in Canada found an inconsistent effect of the housing intervention  on substance use problems . CompareThe existing research on PSH residents has often encountered measurement limitations. Many of the measures used in previous studies were adapted from scales developed for other purposes without empirical validation for applications to this specific population. For example, the measure of social support used by Durbin et al.  consisteThe current study addresses these measurement weaknesses and provides a more rigorous test of the longitudinal relationships of social support, depressive symptoms, and substance use problems. We adapted the existing measures of social support and substance use problems for the PSH population: the Interpersonal Support Evaluation List (ISEL) , 34 and We hypothesized that in the context of monthly health coaching, perceived social support would increase over time, whereas both depressive symptoms and substance use problems would decrease over time. We further hypothesized that individuals with lower levels of perceived social support at baseline would have higher levels of depressive symptoms and substance use problems at baseline and show slower rates of growth in social support over time, which would, in turn, predict slower rates of decline in depressive symptoms and substance use problems.The current study used data from a health coaching program called “Mobile Community Health Assistance for Tenants” (m.chat). This program provided in-person health coaching to PSH residents as part of the Regional Healthcare Partnership (RHP 10) Medicaid Waiver program in the state of Texas (see more details of the m.chat program in ). In addParticipants were 653 PSH residents  recruited from six local housing agencies in Fort Worth, TX, from 2014 to 2017. Table Social support was assessed with a modified version of the Interpersonal Support Evaluation List (ISEL) , 34. TheDepressive symptoms were assessed with the Patient Health Questionnaire-9 (PHQ-9) . The ninSubstance use problems were assessed with a modified version of the Inventory of Drug Use Consequences (InDUC) . The oriWe first conducted a confirmatory factor analysis to examine the structure and performance of social support, substance use problems, and depressive symptoms at each time point. We then used latent growth curve models (LGCM) to estimate changes in social support, depressive symptoms, and substance use problems at baseline, 6-month, 12-month, and 18-month post-baseline . We testWe used the maximum likelihood estimator in the LGCM for social support and depressive symptoms and the robust maximum likelihood (MLR) estimator for the LGCM of substance use problems. The scaled difference chi-square test statistic  was calc2/df), the root mean squared error approximation (RMSEA) [df ratios less than 3; RMSEA scores less than 0.08; and CFI scores greater than 0.90 are generally considered acceptable [We estimated the parameters using all available information under the full information maximum likelihood (FIML) method for missing data . The ade (RMSEA) , 50, and (RMSEA) . Chi-squceptable –56. All ceptable . Figuresnd-order, three-factor model for social support, a single-factor model for depressive symptoms, and a 2nd order, five-factor model for substance use problems; see Additional file Before fitting univariate growth curve models, we separately fitted measurement models for social support, depressive symptoms, and substance use problems at baseline and follow-ups . Table We tested up to the 2nd polynomial term in all growth curve models. Based on our prior experience, we anticipated that participants would have non-zero values at baseline that significantly vary across individuals. We further expected that all three behaviors could be modeled by a linear growth term and a quadratic growth term to capture their change over time. Table rate of increase in social support slowing (− 0.09) over time  over time. We constrained the variances of quadratic growth terms to zero, specifying that any individual differences for the quadratic terms are trivial, which is a common observation in growth analysis [The intercept represents the average baseline level, the linear growth term quantifies the linear rate of change, and the quadratic term quantifies the rate of change in the linear growth rate over time. Social support significantly linearly increased (0.34) over time, with the ime Fig. . For depanalysis . Hence, In testing measurement invariance across time points, the LGCMs of both social support and substance use problems held the strong factorial invariance in which invariance constraints were placed on the configural patterns, the factor loadings, and the intercepts of the measured item variables. The LGCM of depressive symptoms held the configural invariance, in which the pattern of fixed and free factor loadings of measured variables remained the same across time points.The associations between the growth parameter terms for social support and depressive symptoms, and for social support and substance use problems were simultaneously assessed in two separate parallel-process growth models Fig. . By settWe added covariates, including age, sex, race, and length of stay in the current neighborhood, to examine whether the initial levels of depressive symptoms and substance use problems are explained by these covariates see Fig. . Among aThe current study examined the trajectories of social support, depressive symptoms, and substance use problems in a sample of adult PSH residents over 18 months after entering a health coaching program. Over 18 months, significant improvements were observed in all three health behaviors. Specifically, perceived social support increased, whereas depressive symptoms and substance use problems decreased over time, although the rate of positive changes slowed over time. This is an encouraging finding, given that PSH residents have a greater need for physical and mental health services when providing adequate professional care could be costly . With moMore specifically, the findings highlight a sizable variability in perceived social support at baseline, with higher perceived social support associated with less severe depressive symptoms and lower levels of substance use problems. The baseline associations suggest that it may be important for PSH case managers to consider the degree of social support received by their clients. As shown in this study, baseline social support was significantly correlated with the indicators of mental health and substance use problems. This may help case managers to develop need-based customized case management plans.Furthermore, we found that the PSH residents with low social support at baseline showed increasing social support over time. The data show their rate of growth in social support was faster than those with higher levels of social support at baseline. In addition, PSH residents showed steeper rates of reduction in depressive symptoms and substance use problems when their social support increased at faster rates, as we observed a negative association between the slopes of the trajectory of social support and depressive symptoms and the trajectory of social support and substance use problems. The strong cross-domain correlations in the linear slopes suggests that the benefit of improving social support among PSH residents may provide non-disorder-specific overall support for behavior change and improved psychological well-being.To the best of our knowledge, this is the first study to examine the association between rates of social support and depressive symptoms, as well as between social support and substance use problems in a PSH sample using parallel-process growth models. Evidence from this study provides a strong rationale for improving social support for PSH residents with mental health and substance use problems. Because m.chat was not an experimental study, it is difficult to determine if positive changes were due to health coaching, other PSH supportive services, or natural changes over time. Nevertheless, the current study suggests that coaching and increased social support can help initiate positive changes in other areas of health.We also note that this is the most extensive longitudinal study of PSH residents participating in a health coaching program. The results suggest several important implications for the future design of supportive housing programs. First, programs should include regular follow-ups to assess the status of related health behaviors among residents. Since improvements in social support and depressive symptoms may not be consistent over time, frequent regular assessment follow-ups may help case managers identify any emerging challenges or difficulties and offer corresponding services. Follow-up intervals may need to be even shorter than every six months, which we had in this study, to more accurately pinpoint the timing of an inflection where growth slows and consequently plan for an additional coaching session or short booster sessions, delivered briefly over phone calls or text messages, to empower their agency in health-promotive behaviors and further reinforce newly adopted healthy behaviors. Second, future programs could emphasize social support as an early component as it may bring a favorable prognosis on mental health and substance use outcomes. The findings from the current study further emphasize the importance of understanding the status and needs of social support among PSH residents, as stated in the classic work of Cohen and Wills . The neeSimilar directions of correlation between the growth pattern of social support and substance use problems were observed in the other parallel process model. As expected, the statistically significant negative link between the baseline social support and the linear changing rate of substance use indicates that PSH residents who had higher perceived social support at baseline experienced faster improvements in substance use problems. Similarly, the negative link between the baseline substance use problems and the changing rate of social support indicates that PSH residents who had more severe substance use problems at baseline experienced a slower increase in social support. Some researchers have speculated that social support and substance use might be mutually exclusive strategies to cope with stress, such that greater social support might suppress substance use problems . The finOur findings should be interpreted with several cautions. First, there are significant variations among healthcare and case management services, demographic distributions, and their interest in participating in health coaching programs within the PSH population. Because all participants in this study received monthly health coaching, it is challenging to attribute their positive changes solely to health coaching. Second, it is also possible that PSH residents with improving health may report greater social support, although the reported interpretation of the association between social support and health behaviors is more consistent with the literature. Considering the findings from past studies indicating that PSH programs alone may not be sufficient to improve social support among PSH residents , 21, we This study is one of the few longitudinal studies to examine social support, depressive symptoms, and substance use problems among PSH residents, an underserved population. With relatively frequent follow-ups over 18 months, this study reveals the pattern of change in two sets of health behaviors simultaneously using parallel process models. The use of latent variable models, including measurement models and latent growth curve models, allows us to formally test our measurement tools to assure good performance and stable properties across time as well as to maximize the available data to examine the research questions. The findings in this study suggest that the trajectory of social support is very likely to influence the trajectories of depressive symptoms and substance use problems. These findings suggest new ways to integrate these behavioral health targets in the development of intervention and prevention programs for PSH residents.Additional file 1. Supplemental table for measurement model fit indices and diagrams for measurement models and latent growth curve models.


Chen et al.), Developmental Dyslexia (DD), and Autism Spectrum Disorder (ASD), but not Attention Deficit Hyperactive Disorder .Neural timing deficits have become increasingly implicated in neurological conditions, prompting the call for a special issue on this topic. The four papers selected for publication provide compelling new evidence that faulty timing in the interchange of information between magno and parvo-cellular processing streams could be a unifying theme that links Mild Cognitive Impairment , such that inadequate temporal sampling of speech-sounds in people with dyslexia results in poor theta oscillatory entrainment in the auditory domain, and thus a phonological processing deficit which hinders reading ability.Knowledge of oscillatory entrainment and its fundamental role in cognitive and behavioral processing has increasingly been applied to research in the field of reading and developmental dyslexia. Growing evidence indicates that oscillatory entrainment to theta frequency spoken language in the auditory domain, along with cross-frequency theta-gamma coupling, support the phonological processing  which is required for reading. This theory is called the temporal sampling framework (TSF) and can extend to developmental dyslexia  A direct correlation whereby “bottom-up” magnocellular oscillatory entrainment of the visual domain by magnocellular populations phase locks to theta frequency eye movements during reading or (2) an inverse correlation by which attending to text triggers “top-down” low gamma signals from higher-order visual processing areas that organize magnocellular populations to synchronize to the theta frequency, and this drives the timing of eye movements and captures letter images at a higher frequency.Similarly, Chen et al. found that older adults with mild cognitive impairments (MCI) have slower response times when doing direction discrimination relative to congruent and incongruent flankers. They propose that these slower response times result from prestimulus alpha oscillations, conveyed by magnocellular neurons, not being lower for fast patterns, as is found for normal healthy controls. This study also suggests that deficits in focusing attention are due in part to the temporal inaccuracy of magnocellular neurons. Not only do magnocellular neurons provide crucial input to attention networks, but they also provide input for gauging emotional salience, which acts similarly to attention. Mu and Crewther show that negative facial emotional information  is conveyed rapidly by magnocellular neurons to V1 and thence to the amygdala. This activates the pulvinar that projects onto the Thalamic Reticular Nucleus to decrease the activity of magnocellular neurons in the thalamus (McAlonan et al., Mu and Crewther propose that this arrangement mediates a contest between negative feedback and response gain modulation.These papers show that high level cognitive tasks, like reading, face perception, and direction discrimination relative to congruent and incongruent flankers, require both response gain modulation of attention networks by magnocellular neurons and also feedback by coupled theta/gamma and alpha/gamma oscillations, as proposed previously by Lawton  and LawtTL wrote the initial manuscript. JS-T and JS edited the manuscript. JS-T submitted the manuscript. All authors contributed to the article and approved the submitted version.The 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.


This review describes the role of bone resorption in muscle atrophy as well as in muscle protein anabolism. Both catabolic and anabolic pathways involve components of the proinflammatory cytokine families and release of factors stored in bone during resorption. The juxtaposition of the catabolic and anabolic resorption-dependent pathways raises new questions about control of release of factors from bone, quantity of release in a variety of conditions, and relation of factors released from bone. The catabolic responses involve release of calcium from bone into the circulation resulting in increased inflammatory response in intensity and/or duration. The release of transforming growth factor beta (TGF-β) from bone suppresses phosphorylation of the AKT/mTOR pathway and stimulates ubiquitin-mediated breakdown of muscle protein. In contrast, muscle IL-6 production is stimulated by undercarboxylated osteocalcin, which signals osteoblasts to produce more RANK ligand, stimulating resorptive release of undercarboxylated osteocalcin, which in turn stimulates muscle fiber nutrient uptake and an increase in muscle mass. The osteocyte network senses mechanical stress and strain and can sense the weak areas of bone, the areas in which there may be microcracks, and is the largest producer of RANKL in the body. Thus, the osteocyte is capable of stimulating resorption of the weak areas in the bone to be replaced by newly synthesized calcified matrix. The osteoclast is the result of differentiation of the bone marrow granulocytic precursor cells ending up as a producer of enzymatic products, most prominently caspase, that can erode the weakened bone and produce substances called clastokines, most notably sphingosine kinase 1, that can stimulate osteoblast migration to the eroded area and, aided by biphasic calcium phosphates such as hydroxyapatite and β-tricalcium phosphate, aid in new bone formation [Bone resorption, the process by which bone is broken down to liberate products needed by the body’s metabolism, most prominently, but not exclusively, calcium, is incompletely understood. However, what we do know involves most prominently three bone cell types. The osteoblast is the cell which produces new matrix in the form of type I collagen, which undergoes a process of crystal formation ending up as hydroxyapatite matrix. It also serves as a receptor for parathyroid hormone (PTH) and is affected by proinflammatory cytokines, such as interleukin (IL)-1β and IL-6, which stimulate the osteoblast to synthesize the ligand of the receptor activator of nuclear transcription factor ϏB (NFϏB), also known as RANK ligand or RANKL. This product stimulates marrow precursor cell differentiation into osteoclasts resulting in increased bone resorption and serves as one known coupling factor between bone formation and resorption. The ormation  have recA variety of clinical conditions can cause resorptive bone loss. The most common known condition is hyperparathyroidism, as all students learn in medical school that low circulating ionized calcium results in parathyroid calcium-sensing receptor-mediated stimulation of PTH secretion with consequent bone resorption and liberation of bone calcium to normalize circulating calcium.Another condition that results in resorptive bone loss is Paget’s disease. In this condition, the precise cause of the resorptive bone loss is unclear though thought to be genetically linked. It tends to run in families of Northern European ancestry, although a viral etiology has also been suggested.Chronic kidney disease may also cause resorptive bone loss, since the more severe forms are associated with secondary hyperparathyroidism triggered by the kidney’s inability to excrete phosphate.Acute burn injury and cancer metastases to bone, most commonly breast and lung, result in inflammatory reactions which stimulate resorptive bone loss ,4. In adCalcium is the most widely documented factor liberated by bone resorption. While previously thought to exclusively maintain normal circulating calcium concentration, more recent data suggests that it has an effect on systemic inflammation. The earliest report is from Rossol et al.  in 2012,Clinical experience with burn patients is consistent with the inflammatory response being affected by extracellular calcium. Thus, pediatric burn patients can upregulate the parathyroid CaSR in response to both IL-1 and IL-6 ,10,11 rePhosphate and magnesium are also released by resorbing bone. We know this also from experience with pediatric burn patients when muscle protein kinetics were studied using stable isotopes. Typically, pediatric burn patients who receive an intra-arterial infusion of unlabeled amino acids use those amino acids to increase muscle protein synthesis in an attempt to compensate for the increase in muscle protein breakdown that occurs during the systemic inflammatory and stress-induced hypermetabolic response. When an antiresorptive agent such as a nitrogen-containing bisphosphonate was given to these patients, muscle protein synthesis actually fell , suggestAnother important factor produced by resorbing bone is transforming growth factor (TGF)-β. As noted in a recent review by Morikawa , TGF-β1-An additional factor known to be released from bone during resorption is undercarboxylated osteocalcin. Osteocalcin is a protein synthesized by osteoblasts, gamma carboxylated by vitamin K, and found in bone matrix. Originally, it was considered as a marker of osteoblast differentiation until the work in the laboratories of Karsenty and others demonstrated that its metabolically active form, the undercarboxylated osteocalcin, is a potent regulator of glucose metabolism through its Gprc6A receptor on pancreatic islet cells with a specific effect on muscle glucose uptake, as we shall see .All factors so far listed are released from bone on resorption and all have specific effects on muscle protein catabolism or anabolism. These will be discussed in further detail below. Other factors released and their metabolic consequences remain to be described. Thus, the picture of bone factor release with effects on muscle is to date by no means complete. The known factors involved in resorptive release from bone and which play a role in the size of muscle mass are summarized in Of the above-listed resorption-released bone factors, at least two—calcium and TGF-β—have catabolic effects on muscle. The calcium effect is indirect. If the patient suffers from an underlying inflammatory condition, the release of calcium from resorbing bone has the capacity to intensify and/or prolong the inflammatory response, augmenting or perpetuating the production of proinflammatory cytokines. While the complete mechanism for cytokine effect on muscle protein breakdown is still under investigation, the catabolic effect of proinflammatory cytokines on muscle has been well documented .The first report that TGF-β released by bone resorption was catabolic to muscle came from Waning et al.  in the lAt the same time, Borsheim et al.  reportedFurthermore, myoblast cultures containing serum from burned subjects receiving placebo showed significantly increased concentrations of ubiquitin, a key protein in the ubiquitin ligase catabolic pathway, and reduced phosphorylation of the anabolic Akt/mTor pathway, whereas cultures with serum from subjects receiving pamidronate demonstrated significantly reduced concentrations of ubiquitin compared with placebo and significantly increased phosphorylation of the Akt/mTor anabolic pathway. When the myoblast culture experiments were repeated with the addition of anti-TGF-β1-3, myotube size of the placebo controls was rescued to the magnitude of those treated with bisphosphonates, while the myotube size of the bisphosphonate-treated patients did not change significantly in culture with the addition of the antibody. While the study of Pin et al. did not examine muscle protein oxidation or the state of the ryanodine receptor, both the work of Waning et al.  support Moreover, adults with bony metastases from breast or lung cancer, and previously normal healthy children exposed to burn injury are two distinct groups of patients exhibiting a similar mechanism of muscle loss in the face of two distinct conditions with vastly differing etiologies, giving rise to the possibility that this mechanism is basic to the control of muscle mass by bone.Essex et al.  reportedStill another set of studies by Bonnet and colleagues  in GenevA third factor released during bone resorption, undercarboxylated osteocalcin, has an anabolic effect on muscle. These data come mainly from the work of Gerard Karsenty’s laboratory at Columbia. Because osteocalcin concentration in serum falls with age in humans, monkeys, and mice, Mera et al.  studied While this review focuses on factors released by bone resorption that affect muscle wasting, it is important to mention two muscle-derived factors that may influence muscle mass possibly through interaction with bone. These are myostatin and irisin.Myostatin is a member of the TGF-β superfamily but a member of a different subfamily than the TGF-β molecule itself, namely, the activin/myostatin subfamily . It is eAnother hormone primarily expressed in muscle is irisin, a fibronectin-III-containing protein that promotes glucose uptake by muscle fibers as well as the browning of white adipose tissue . In addiA list of bone- and muscle-secreted factors that affect muscle and bone is given in Components of the inflammatory response appear to be active in the three known catabolic and anabolic effects that bone resorption products have on muscle wasting or muscle mass accretion. As shown in These observations raise questions about the nature of factor release from bone. Are specific factors, such as undercarboxylated osteocalcin or TGF-β, specifically targeted for release depending on the original trigger for the bone resorption? Or, does bone resorption indiscriminately release all products of resorption in the same ratio and at the same rate with utilization dependent on the underlying metabolic demands? To cite an example, in pediatric burn injury, circulating osteocalcin is reduced  as bone Nevertheless, knowing what we do about factors released during bone resorption and their effects on muscle mass, it is possible to consider clinical trials to begin to address the problem of muscle wasting together with bone loss, or osteosarcopenia. An example of a clinical trial would be the use of antiresorptive agents to block resorptive release of bone TGF-β, which has been shown to work experimentally in both metastatic bone disease from breast cancer and in pediatric burn injury. A large-scale trial of this potential therapeutic option has not yet been undertaken in pediatric burn injury, partly due to unfamiliarity with the pharmacology of this class of drugs by clinicians caring for burns patients. As we learn more about the release of factors affecting muscle mass from bone and elsewhere in the body, additional therapeutic options may become available.


Although it is now well-appreciated that the increase in hypersensitivity disorders represents a “perfect storm” of many contributing factors, we argue here that two important considerations have rarely been explored. First, the window of microbial exposure that impacts immune development is not limited to early childhood, but likely extends into the womb. Second, restricted microbial interactions by an expectant mother will bias the fetal immune system toward hypersensitivity. Here, we extend this discussion to hypothesize that the cell types sensing microbial exposures include fetal hematopoietic stem cells, which drive long-lasting changes to immunity.Over the last century, the alarming surge in allergy and autoimmune disease has led to the hypothesis that decreasing exposure to microbes, which has accompanied industrialization and modern life in the Western world, has fundamentally altered the immune response. In its current iteration, the “hygiene hypothesis” suggests that reduced microbial exposures during  In this review, we will explore a body of work that demonstrates how maternal exposure to microbes during pregnancy has a significant impact on the development of the immune system in offspring. We will also review a growing body of literature that demonstrates how adult hematopoietic stem cells (HSCs) can sense diverse immune stimuli, thereby impacting the production, and sometimes function, of immune cell progeny. Furthermore, we will reconcile these two bodies of literature to suggest that maternal inflammation and infection are perceived by fetal HSCs to shape the immune system in the neonatal period and beyond. The consequences of these perturbations are underexplored but are likely to impact propensity for hypersensitivity disorders and resistance to certain infections as neonates. Thus, in addition to the passive transfer of maternally derived antibodies, the mother affords a separate mode of immune transfer, one that is driven by inflammation but acts upon a receptive HSC compartment during fetal development. The resultant training, or biased cell output, will have long ranging effects on neonatal and adult immune function. We argue that the hygiene hypothesis should encompass how microbial exposure during pregnancy fosters immune development through “training” immune output at the stem cell level.Thirty years have passed since the inception of the hygiene hypothesis, which attempted to reconcile an inverse correlation between birth order and incidence of allergic disease observed in British families . It was in utero development.The primary focus of the hygiene hypothesis, from a mechanistic perspective, has been on immunological mechanisms that shift CD4 T helper (Th) cell differentiation profiles due to microbial exposures in the first years of life. The original view was that early infection skewed development of Th cell repertoire toward “proinflammatory” Th1 responses, which in general antagonize “allergy promoting” Th2 immunity and allergic disorders –16. The When considering what type of microbial interactions may be missing in hygienic environments of the west, both helminths and the microbiota have garnered the most attention. The inverse correlation between helminth infection rates and hypersensitivity disease intensity in tropical locals has long been suspected as a causal in nature , 28, 29.Toxoplasma gondii, Hepatitis A, and Helicobacter pylori, but not for pathogens acquired by different routes, were much less likely to have atopic disease, especially when seropositive for at least two  scar size and infant pro-inflammatory cytokine production elicited by TLR stimulation  11–13] as compared to late gestation/near term (E17-19), suggesting that a less mature placenta may be more permeable to maternal cytokines  and mononuclear cells , are rapid responders bearing either non-specific, germ-line encoded antigen receptors  or no antigen receptors (ILCs) that release natural antibody or cytokine in response to pathogen. Fetal-derived myeloid cells are mostly “tissue-resident,” and have unique functions in tissue homeostasis within their resident tissues. As these immune cells take up residency in their respective tissues across ontogeny, they both educate and are educated by the tissue microenvironment . For exaDue to the specific functional attributes of fetal-derived immune cells in maintaining tolerance and tissue homeostasis, it is not entirely surprising that their dysregulation is implicated in disorders of tolerance, such as asthma and autoimmunity. For example, dysregulation of innate-like B cells and innate-like marginal zone B-cells has been observed in humans with, and mouse models of, autoimmune diabetes –139. B1-in vivo barcoding experiments have alternately suggested that many fetal HSCs and progenitors are transient, despite possessing multilineage capability  to generate the first blood cells . These “pability , 161. ThOngoing examination of how waves of fetal hematopoietic cell production contribute to adult immune compartments has begun to unravel two critical insights. First, distinct progenitors are responsible for generating parallel “waves” of immune cell production across development. Second, these distinct waves of immune cell production may contribute to functional heterogeneity within adult immune cell compartments. Precisely how developmental waves underlie functional heterogeneity in both normal and abnormal adult immune function is an area of active investigation. The best evidence to date to support the hypothesis that waves of developmental immune cell generation underlie heterogeneity of adult compartments comes from ongoing investigation of the origin of adult tissue-resident macrophages. Elegant studies using a range of fate-mapping and deletion models have revealed overlapping contribution from yolk sac, fetal liver, and adult progenitors across ontogeny –164. Forwhen and how these inputs are interpreted. If subsets of immune cells that persist across the lifespan are produced only from transient fetal progenitors during specific windows of fetal development, the nature and timing of those extrinsic inputs will necessarily influence the trajectory of the immune system. Here we propose that extrinsic inputs could shape the trajectory of the immune system at the progenitor level , and congenital infections, such as Toxoplasma gondii. These observations, along with the concepts reviewed above, underscore the need to expand the Th1/Th2 dichotomy and its role in early immune development through the lens of the hygiene hypothesis. We propose a broader understanding that accounts for the impact of early exposure to both hematopoietic stem cells and immune cells that arise during a critical window of development. Innate-like lymphocytes, immune cells that arise during a critical window of development, including innate-like lymhocytes. By impinging upon their establishment during fetal development, we train or manipulate the immune system in lasting ways.The concept of What are the cellular mediators of fetal immune training by maternal inflammation?What is the mechanism by which maternal inflammation impacts fetal hematopoietic stem cell development?Does fetal immune training lead to fixed or transient changes to the immune system?Does the severity of maternal infection matter?Is the training generalized to all infections, or specific to certain microbes?Can the fetal immune system be trained  to lessen immune hypersensitivity disorders?AB, AA, and KJ wrote and edited the manuscript.The 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.


Cancer therapies should improve patient survival or at least improve the quality of their life as they receive treatment for their disease. This is particularly important in pancreatic cancer, where current treatments often have to balance between limiting tumor growth and minimizing patient toxicity. There has been an increasing appreciation among physicians to capture the patient’s voice using tools called patient-reported outcome measures (PROM). In this article, we describe the available PROMs and their relative strengths and weaknesses to help oncologists make sense of this rapidly growing field. Finally, we present a decision-making tool that can help researchers and clinicians select the ideal PROM that fits their needs.Pancreatic cancer and its treatment often dramatically impact patients’ quality of life (QoL). Given this, as well as increased focus on QoL measures in clinical oncology, there has been a rise in the number of instruments that measure patient-reported outcomes (PROs). In this review, we describe the landscape of different PRO instruments pertaining to pancreatic cancer, with specific emphasis on PRO findings related to pancreatic cancer patients receiving radiotherapy (RT). Twenty-five of the most commonly utilized PROs are compared in detail. Notably, most of the PRO tools discussed are not specific to pancreatic cancer but are generic and have been used in various malignancies. Published findings concerning PROs in pancreatic cancer involving RT are also extracted and summarized. Among the measures used, the European Organization for Research and Treatment Cancer QLQ-C30 was the most commonly utilized. We recommend a careful selection of PRO measures in clinical pancreatic cancer research and care and encourage the use of a combination of symptom-specific and global QoL tools to more fully capture patients’ perspectives. Patient-reported outcomes (PROs) are measures directly obtained from patients, including but not limited to emotional well-being, general quality of life (QoL), and physical and psychological symptoms. Since their introduction in the late 1960s, PROs have gained more attention over the 30 years that followed. The utility of PROs is now widely acknowledged both in clinical practice and research. This trend is particularly striking in clinical oncology, where there are widespread efforts dedicated to improving value-based quality of life for cancer patients. Professional organizations like the American Society of Clinical Oncology (ASCO) and the European Society of Medical Oncology (ESMO) now recommend the inclusion of PROs as central endpoints in modern clinical trials . These gIn oncology, clinicians often assess toxicity and adverse events (AE) using standardized lexicons, such as the Common Terminology Criteria for Adverse Events (CTCAE). While historically adopted and extensively used, CTCAE toxicity measures rely primarily on physician or provider-based assessments of patients’ health and well-being. Such assessments are useful and often essential in order to properly assess and follow-up on patients. However, numerous studies show that CTCAE results and PRO results are not always congruent ,9. ContrThe standard methodology of acquiring patient-reported outcomes is through a questionnaire answered directly by the patient. The tools or instruments used to collect PROs are referred to as patient-reported outcome measures (PROMs). The precise methodology behind the development of PROMs varies. Generally, PROMs are designed based on adverse events that could be appropriate for patients to self-report. Those PROMs test specific domains and are typically developed with input from a variety of multidisciplinary stakeholders, including patients, clinicians, and psychometricians . In PROMs, a domain is made out of a number of measurable items that collectively describe a specific function or perception.Each PROM must be validated in a specific patient population and should be capable of capturing the clinical differences effectively. These tools can either be general or disease-specific. Generic scales have the capability to measure a variety of different outcomes without venturing into a specific disease or outcome, like general physical function, pain, or social function. Disease-specific scales are usually tailored to a particular disease or set of diseases; they have the capability to shed focused attention on specific issues that are relevant to a particular disease process and/or its associated treatment. For instance, patients undergoing radiation therapy for pancreatic cancer may experience gastrointestinal toxicities secondary to radiation fields targeting the abdominal area, with a predominance of symptoms related to bowel dysfunction and toxicity. In such cases, PROMs measuring salivary gland function or respiratory symptoms may yield less useful information. Fortunately, many instruments are readily available now and can allow a researcher to easily capture the PROs for any specific disease site. The use of such PROMs has the potential to improve symptom reporting and management, as well as the establishment of an environment of understanding between patients and physicians. PROs offer the treating physician the opportunity to gain insight into the patient’s experiences while complementing the clinician’s assessment .Understanding the patient experience is critical in the treatment of pancreatic cancer, since patients tend to experience substantial disease-related morbidity compounded by treatment-related toxicity from therapies such as RT; major surgeries ; and potent multi-agent chemotherapies such as FOLFIRINOX  ,13. DespMany PROMs have been studied in pancreatic cancer patients. Each tool was adopted for a specific trial or a subpopulation of patients with this disease. This has led to the creation of multiple excellent PROMs with their own unique scope, domain, and psychometric properties. However, this creates a problem for a trialist or researcher who wishes to implement a PRO metric into their own study—how do you select the right one?The ideal PROM is brief but able to capture the symptomatology relevant to the disease of interest. The use of cancer-specific measures like the EORTC or FACT-G, however, are thought to better capture the symptoms of oncological diseases and their treatments . Both EOThe importance of the patient perspective is growing in clinical trial design and endpoint selection. To that end, the FDA has established a list of core concepts regarding what a PRO measure(s) should assess , which iIn Surgery is the only known way to cure pancreatic cancer, and there have been several studies examining patient experiences with the Whipple procedure. Since surgery is not possible in most forms of pancreatic cancer , RT is the only tested option to achieve local control. Definitive chemoradiation to pancreatic cancer is challenging because these tumors often grow into and around the nearby gastrointestinal tract, which cannot tolerate high doses of radiation. A highly conformal technique called stereotactic body radiation therapy (SBRT) delivers focused doses of radiation to the tumor, which results in better control with less injury to highly susceptible organs near the pancreas. Early SBRT studies for pancreatic cancer yielded unacceptable toxicity, but subsequent refinement of the technique has SBRT greatly improved and better local control and QoL ,46,47,48The potential promise of radiation therapy in pancreatic cancer has been met with concerns over side effects and QoL, which has led investigators and clinicians to incorporate PRO measures in trials where radiation therapy was used. EORTC-QLQ-C30/PAN26 and FACT-Hep were the two most commonly used tools among pancreatic cancer trials  Figure . These tThe importance of assessing the patient’s viewpoints through PROs is now recognized as critical data for patient care and trial design. Thus, increasing efforts are being dedicated toward incorporating PRO measures in clinical studies. The insights that these data provide can have profound effects on therapeutic decisions, since life expectancy with this disease remains dismal. Balancing QoL with therapeutic efficacy will likely have the most impact on patient satisfaction and clinical outcomes by personalizing care for each patient.However, challenges remain in adopting the best practices into clinical trials, since there is no consensus with regards to the best PROM for a given study. Like any individual patient or treatment, the PROM must be tailored to data that would likely be relevant given the known morbidity of the cancer and its associated treatments. Among pancreatic cancer patients, data that can be generalized is of paramount importance, hence the underlying challenge of achieving a standard or benchmark that would allow clinicians and investigators to compare across studies and modalities. Such benchmarks might be the choice of measures, time points assessed, analyses, and interpretational techniques. In the absence of a unifying approach, the best practice for investigators and clinicians aiming to incorporate PRO data into their studies is to be aware of the characteristics of the PRO tools they are selecting and whether a combination might be necessary.While this review focuses primarily on PROMs that can be used in patients with pancreatic cancer receiving radiotherapy, most of these measures can be used in other kinds of interventions in pancreatic cancer  depending on the domains of interest. For trials involving radiotherapy in pancreatic cancer, the combination of a general QoL measure with a disease-specific PROM  should be employed for a comprehensive understanding of the patient experience and symptomatology. The data generated offer insights into the patient’s perspective and can help physicians offer tailored therapies with the patient’s concerns in mind. More importantly, PROs might help bridge the relationship between the physician and patient, leading to better communication and insight, which, in turn, would lead to better outcomes and care satisfaction.


Mesenchymal stem cells are a promising source for externally grown tissue replacements and patient-specific immunomodulatory treatments. This promise has not yet been fulfilled in part due to production scaling issues and the need to maintain the correct phenotype after re-implantation. One aspect of extracorporeal growth that may be manipulated to optimize cell growth and differentiation is metabolism. The metabolism of MSCs changes during and in response to differentiation and immunomodulatory changes. MSC metabolism may be linked to functional differences but how this occurs and influences MSC function remains unclear. Understanding how MSC metabolism relates to cell function is however important as metabolite availability and environmental circumstances in the body may affect the success of implantation. Genome-scale constraint based metabolic modeling can be used as a tool to fill gaps in knowledge of MSC metabolism, acting as a framework to integrate and understand various data types . These approaches have long been used to optimize the growth and productivity of bacterial production systems and are being increasingly used to provide insights into human health research. Production of tissue for implantation using MSCs requires both optimized production of cell mass and the understanding of the patient and phenotype specific metabolic situation. This review considers the current knowledge of MSC metabolism and how it may be optimized along with the current and future uses of genome scale constraint based metabolic modeling to further this aim. In recent years, there has been increasing interest in the possibilities offered by regenerative medicine , a fieldOne important area of regenerative medicine is the use of stem cells to enhance available therapeutic applications and to further the development of new ones . In partFor the past decade, MSCs metabolism has received growing interest due to mounting evidence suggesting that the manipulation of metabolism allows enhanced therapeutic uses of these cells  in cell-based medicine and tissue engineering . Cellulain silico models  metabolism can be used to answer the fundamental question: “How can GEMs be used to optimize MSC therapeutics?” First, we describe the biology of MSCs, their differentiation and immunomodulation properties and their applications and limitations in regenerative medicine. Next, we detail how metabolism affects or can be used to manipulate these functions. We then discuss how mathematical modeling of hMSC metabolism can aid in developing pre-clinical and clinical experiments. Finally, we give our vision for the future of using metabolic modeling to study hMSCs and how the resulting insights could prove transformative for the field of regenerative medicine.Mesenchymal stromal cells comprise non-hematopoietic cells originating from the mesodermal germ layer and are capable of both self-renewal and multilineage differentiation into various tissues of mesodermal origin . These m•Adhere to plastic and develop as fibroblast colony-forming units and differentiate into cells of mesodermal origin . See •in vitro culture expansionExpress the surface markers CD73, CD90, and CD105 during •in vitro culture expansionLack expression of CD11b, CD14, CD34, CD45, CD19, and HLA-DR surface markers during It is likely that this definition will continue to evolve to account for new findings.One of the identifying characteristics of MSCs is their ability to differentiate into cells of mesodermal origin . In addiDifferentiation of MSCs is primarily induced through media supplementation , with different supplements being required for the various differentiations. Adipogenesis, for example, is induced through supplementation with dexamethasone, indomethacin, insulin, and isobutyl methyl xanthine. Osteogenic differentiation is induced by dexamethasone, ascorbic acid, β-glycerophosphate, and sometimes bone morphogenic protein 2 (BMP-2) . The comDifferentiation is controlled by an interlinked set of regulatory molecules forming complex signaling pathways. These pathways are somewhat distinct between differentiation lineages, although there are important areas of overlap. This phenomenon is demonstrated by the inverse relationship that exists between pathways relating to adipogenic and osteogenic differentiation . Most diBeyond their potential for differentiation, hMSCs have remarkable immunomodulatory properties; they possess the ability to inhibit or promote the immune response of the host’s body though mediated immunosuppression. These mechanisms include direct inhibitory effects and other indirect regulatory effects. This regulatory response involves inhibition of B and T cell proliferation, cytokine production inhibition, decreased natural killer (NK) cell activation, and dendritic cell maturation .The immunomodulatory response of hMSCs is activated by inflammatory cytokines  that are secreted by T cells and other antigen-presenting cells . In respIndoleamine 2,3-dioxygenase (IDO) is one of the well-known paracrine factors released by hMSCs and has been shown to promote kidney allograft tolerance . It suppAnother reported soluble factor with immunoregulatory effects is the unstable oxidative molecule nitric oxide (NO), which is generated by NO synthase. Inducible NO synthase (iNOS) is responsible for the immunomodulatory effect of NO. Increased secretion of NO results in modulation of both the proliferation and function of T cells. At very high concentrations, it can lead to the apoptosis of immune cells . A list Mesenchymal stromal cells secrete paracrine factors that promote tissue repair. In response to physical tissue damage, MSCs secrete factors that allow them to navigate to the site of injury, referred to as homing . An examin vitro expansion, and cultural heterogeneity of the hMSCs. Cells derived from different sources seem to express different profiles of the homing molecules  on a host‘s immune system, hMSCs must be mainly in a glycolytic state. Further, it is not well understood to what extent energy metabolism is mixed at different stages of differentiation (early versus late) or whether it is dependant upon the type of differentiation the cells are undergoing. An understanding of how cell source and age affects differentiation and if different states affect the survival and function and, therefore, usability of cells The proliferation, differentiation, and immunomodulatory functions of hMSCs are linked to cellular metabolism . EmerginThere is strong evidence suggesting that, in their undifferentiated state (while undergoing proliferation), hMSCs rely primarily on glycolysis for energy production. This phenotype has been demonstrated in BM-hMSCs, which show a preference for glycolysis during proliferation  but shifThe ability of BM-hMSCs to differentiate has also been shown to be affected by mitochondrial functions . ConsistThe mitochondria of hMSCs seem to be primarily inactive while cells remain in their proliferation stage, during which metabolic pathways related to glycolysis and its associated signaling pathways required for adenosine triphosphate (ATP) generation and general anabolitic activity are most active see . GlycolyThe effect of interferon regulation on hMSC metabolism can be varied. IFN-γ has also been used to inhibit proliferation and alter AD, OD, and neural differentiation (ND) by activating IDO . The kynIFN-β has been shown to enhance immunomodulatory functions of hMSCs in other reports. Overall reports discussing hMSC metabolism seem to, for the most part, agree that during proliferation the cells primarily generate ATP through glycolysis. However, upon initiation of differentiation, cells seem to turn toward mitochondrial metabolism, with reported increases in metabolism and biogenesis indicating the importance of mitochondrial activity when it comes to hMSC functionality .de novo lipogenesis-related genes, and thus de novo lipogenesis produced fatty acid elongation and desaturation) before returning to normal . Their conclusion was that de novo lipogenesis and desaturation comprised the major fatty acid flux during adipogenic differentiation of bovine Ad-MSCs.There is not much known about whether amino acid metabolism is affected during functional progression of hMSCs or what effects they induce if modified through metabolic changes. For example, Ornithine decarboxylase (ODC) and polyamine biosynthesis are important in the proliferation of stem cells . The rolAs suggested in section “MSC Function Is Linked to Metabolism,” active mitochondria are necessary for successful differentiation. Accumulating evidence indicates that mitochondrial enzymes and regulatory pathways are of great importance for MSCs in proliferative and differentiating states . MitochoOther enzymatic activity has been confirmed that further supports the importance of mitochondrial activation for MSC functionality. Creatine kinase (CK) activity, which is involved in buffering and recovery of ATP, has been reported in Ad-hMSCs. It stimulates glycogenolysis by increasing cytoplasmic concentration of inorganic phosphate. Activity of CK was found in both differentiated and proliferating Ad-hMSCs, with more mitochondrial CK activity in AD cells. This further supports the theory of a shift toward oxidative metabolism/mitochondrial metabolism during differentiation of MSCs .Through the reversible mitochondrial nicotinamide adenine dinucleotide phosphate (NADP)-dependent reaction of isocitrate dehydrogenase (NADP-IDH), an anaplerotic pathway exists that forms isocitrate from glutamine through a process called glutaminolysis. Through this pathway, glutamine can compensate for the lack of glucose for both ATP production and anabolic precursor supply . This paReactive oxygen species (ROS) are known to serve as signaling molecules capable of regulating biochemical pathways that are a part of normal cell function. They are particularly important in metabolism and inflammatory signaling . RegulatSince functionality and survival of hMSCs is affected by changes in their metabolism, there is the potential to enhance the efficacy of hMSC therapies through manipulation of metabolism. hMSCs can effectively reconfigure metabolism to respond to the biochemical demands of tissue repair, be it secretion of immunomodulatory factors or integration and differentiation toward tissue specific cell types . CurrentOne such approach is to alter the functional capacity of MSCs through oxygen manipulation, due to the importance of mitochondria and mROS discussed above. 2)] involved in OxPhos. The lower citrate levels indicate a more pronounced metabolic uncoupling of glycolysis and TCA cycle for cells in OD compared to undifferentiated cells  OD of BM-hMSCs may be enhanced and adipogenesis decreased  . HO-1 isMetabolism in MSCs is a complex and dynamic system. We have outlined several gaps in the collective knowledge of MSC metabolism that are actively being addressed by the community. As we gain insights into questions regarding the primary energy-generating pathway(s) utilized during differentiation, we will move closer to manipulating these systems. However, we will need a holistic perspective that integrates knowledge at the various biological levels of MSC differentiation.A bottom-up systems biology approach allows for a mechanistic understanding of a system . Such maThe first step in modeling metabolism at the genome-scale is to reverse engineer the network structure . This reThis ability to integrate information from multiple types of -omic data with previously acquired detailed biochemical data makes metabolic modeling a powerful technique to answer biological questions regarding how phenotypes occur due to genetic mutation or environmental perturbations . As furtVarious community-driven efforts have led to several reconstructions of the global human metabolic network . CompiliRecon3D is the latest update of the Recon family of human metabolic network reconstructions . The keyA base GEM may be utilized to produce general predictions about whole human metabolism, providing useful insights into both human health and predictions of currently unknown human metabolic functions.The various human models have been used to predict biomarkers for inborn errors of metabolism (the accuracy of previous predictions feeds into improving these models) . Inborn By analyzing reactions in Recon1 that were defined as being present due to genome annotation or literature data, but that were not predicted to be active, and then adding surrounding reactions to activate them, predictions have been made about previously unknown human metabolic functions such as that of iduronic acid following glycan degradation, N-acetylglutamate in amino acid metabolism, and the human activity of gluconokinase . Such inOnce a base GEM has been selected, there are a variety of methods available to make the model specific to a particular cell type and circumstance by integrating transcriptomic, proteomic, and metabolomics data. These context-specific models are able to provide more detailed insights into human metabolism in a particular cell type, and comparison of models is particularly useful. Previous context-specific human models have produced useful insights into healthy and diseased metabolism.A cell’s metabolic capabilities are defined by which enzymes it expresses. Transcriptomic and proteomic data provide information about the enzymes expressed under certain circumstances. Both of these data types correlate to enzyme activity, although not perfectly . SeveralAnother way to make models more context specific is to use metabolomic data collected by mass spectrometry or NMR to constrain what the model takes up or secretes to realistic values. By either measuring changes in the concentration of various metabolites in the medium over time or by comparing the relative values of metabolites at different times, the rate of uptake or secretion of a range of metabolites is determined. These rates can be applied to the model as additional constraints that will restrict the model predictions to those consistent with the metabolic dataset . These aModels can also be used to analyze isotope labeling data and this data can, in turn, contribute better constraints to improve the model. Cells may be fed on medium containing glucose or glutamine labeled with heavy isotopes of carbon or nitrogen. The proportions of various metabolites labeled with these heavy isotopes in cells that are sampled and analyzed at different time points after this treatment allows inferences to be made about the production of the labeled metabolites. Sholmi et al. used this technique to elucidate the differences in the TCA cycle during the cell cycle. Further information may be obtained if the cells are fractionated into different organelles before analysis . For exain silico gene deletion screens) that resulted in identification of 52 metabolic drug targets; 8 of these currently correspond to cancer therapeutics. In addition, a set of genes were identified in the healthy cell model network that were downregulated in the cancer model. By inhibiting the genes more highly expressed in cancer cells, targeting could be achieved  , which gachieved .More specific cancer models have also been produced. For example, a model has been created for hepatocellular carcinoma by Many constraint-based metabolic analyses have historically relied upon an objective function, which is defined as a metabolic objective of a cell; flux through this reaction is either maximized or minimized to compute the flux state  across the entire network. For metabolic states that do not have as well-defined objective functions as cancer does , algorithms that are able to create tissue- or cell-specific models without a specific objective function are needed. An algorithm often used for this purpose is the metabolic transformation algorithm (MTA) , an algoObesity has been addressed through the use of the human metabolic reconstruction by identifying pathways implicated in the disease process. As with many diseases, pinpointing a specific genetic or environmental marker as a cause for obesity, making the determination of progression, and deciding on a “treatment” a difficult task. Using the HMR and transcriptomic data from both healthy and obese individuals, a GEM with the objective function defined as acetyl-CoA production and formation of lipid droplets was produced. Through this analysis, two possible drug targets were identified by considering reactions with significantly changed flux values, and a potential biomarker for obesity was identified through reporter metabolites, which is an algorithm allowing the analysis of transcriptomic data in the light of the metabolic network structure to predict highly affected metabolites .Drug toxicity levels and side effects over both short and long periods of usage can also be identified in an easier and more cost-efficient manner using GEMs. It is possible to make predictions on system-wide perturbations using previously determined information on how protein structural analysis can be used to determine off-target binding of drugs, in combination with metabolic networks, as was done by Biological systems often change dynamically over time. Analyzing how these changes occur can be challenging but is being addressed through the integration of time-course experimental data. One approach, dynamic flux balance analysis (dFBA) {ref 10.1016/j.celrep.2017.07.04}, integrates time-course measurements of the major inputs and outputs of the system to provide more accurate flux predictions. dFBA provides a continuous prediction based on these changing inputs and outputs . This method has been applied to murine embryonic stem cells and revealed changes to mitochondrial metabolism and one carbon metabolism during priming . more reA GEM of MSCs, iMSC1255, was recently created to improve understanding of the function of MSC metabolism. This model is based upon publicly available transcriptomic data sets from proliferating, early passage bone marrow MSCs. The data was used with the mCADRE algorithm to generate a tissue-specific version of the global human model Recon1, which was then manually curated by comparison to proteomic data and the literature to ensure that all desirable reactions were included to account for known MSC metabolic functions . FurtheriMSC1255 has subsequently been used to computationally predict metabolic interventions to optimize proliferation and chondrogenic differentiation of MSCs. By analyzing the maximum growth rate predicted FBA with and without allowing the uptake of a range of nutrients, it was proposed that supplementing the MSC medium with the phospholipids phosphoethanolamine and phosphoserine would improve proliferation. This was confirmed experimentally . This pain silico metabolic engineering (gene knock-outs and knock-ins); (2) identifying biomarkers of diseases; (3) predicting drug targets and therapeutic windows; and (4) optimization of cellular functions without the cost of wet lab experimentation. Some success has already been reported, as mentioned in section “Current State-of-the-Art Applications.”Since 2007, when the very first global GEM for humans was reconstructed , researcin silico, the number of experiments needed to optimize growth conditions may be reduced. This may be an interesting avenue to explore for each cell type and cell state, since different responses may be observed. Such testing is more feasible to explore in silico than in vitro. Even though time and money will have to be spent on reconstruction of the GEMs themselves, the savings in experimental time and material costs afforded by the use of validated reconstructions will likely outweigh these costs. Promising in silico outcomes can then be taken further, being validated or explored in vitro and later in vivo. Positive results would further validate the models and perhaps further that particular avenue of cell-based medicine or that particular use of the cells in regenerative medicine.Building on the various uses of existing cell- or tissue-specific human GEMs and the most up-to-date version of the human reconstruction , the potOne potential use for GEMs would be to explore the different metabolic capacities of hMSCs from different sources. There have been some reports, albeit limited, explaining possible differences in the proliferative, differentiation, and immunomodulatory abilities of hMSCs isolated from various tissues . As hMSCThe creation and study of GEMs of MSCs undergoing each of the three classical differentiation  could be approached in various ways. uFBA could be used as a framework to examine MSC metabolomics data collected at different timepoints during each of the differentiation lineages . The stuin silico  also contributed to the writing and editing of the manuscript.The 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.


Based on several novel concepts that are integral to the revised Starling model, we review the clinical features of DSS and discuss a number of implications that are relevant for fluid management. We also highlight the need for context-specific clinical trials that address crucially important questions around the management of DSS.Dengue is the most common mosquito-borne viral infection in the world. The most feared complication is a poorly understood vasculopathy that occurs in only a small minority of symptomatic individuals, especially children and young adults, but can result in potentially fatal dengue shock syndrome (DSS). Based mainly on expert opinion, WHO management guidelines for DSS recommend prompt infusion of a crystalloid fluid bolus followed by a tapering crystalloid fluid regimen, supplemented if necessary by boluses of synthetic colloid solutions. However, following publication of a number of major trials undertaken in other, primarily adult, critical care scenarios, use of both synthetic colloid solutions and of fluid boluses for volume expansion have become controversial. Synthetic colloids tend to be used for severe DSS cases in order to boost intravascular oncotic pressure, based on the classic Starling hypothesis in which opposing hydrostatic and oncotic forces determine fluid flow across the microvascular barrier. However, the revised Starling model emphasizes the critical contribution of the endothelial glycocalyx layer (EGL), indicating that it is the effective oncotic pressure gradient across the EGL not endothelial cells  Dengue is increasingly being recognized as a major threat to global health, especially among people living in endemic areas who are exposed to multiple infections with different serotypes. Although the majority of symptomatic infections manifest as a self-limited illness characterized by fever and systemic symptoms, life-threatening complications can develop. The most feared complication, particularly prevalent in children and young adults experiencing secondary infections, is a poorly understood vasculopathy that presents as a systemic vascular leak syndrome . The vasSevere leakage resulting in DSS is seen less frequently in adults than children , 5, likeIn this review we highlight some of the current controversies relating to DSS fluid resuscitation and consider how evolving knowledge of the physiological principles governing fluid dynamics at the microvascular level can help us to understand the clinical features observed in DSS and potentially be of use in guiding fluid management of shock.In the 1960s, when DSS was first described mortality rates were high, but recognition of the marked intravascular volume depletion and the urgent need for fluid resuscitation led to a notable improvement in outcomes. Great care is needed however to limit iatrogenic complications, in particular development of respiratory compromise due to fluid overload, especially in settings where HDU/ICU facilities with the capacity to provide advanced respiratory support are scarce. The World Health Organization (WHO) dengue management guidelines recommend prompt infusion of 10–20 ml/kg crystalloid fluid boluses for children with established DSS, followed by a tapering crystalloid fluid regimen supplemented by boluses of synthetic colloid solutions as necessary ] or to bThe evidence for the current WHO management guidelines is limited and the recommendations are based primarily on expert opinion and some observational data , 9, althAnother controversial issue is the use of fluid boluses for resuscitation. Rapid correction of profound hypovolemia has always been accepted as an important feature of shock resuscitation, but there is very little formal evidence available in this regard. A large trial in African children (FEAST) with impaired perfusion associated with severe febrile illness of mixed etiology yielded unexpected but unambiguous results, demonstrating that fluid boluses were associated with significantly increased 48-h mortality . HoweverFinally, although blood transfusion is considered life-saving for individuals with DSS and severe bleeding, identification of occult bleeding can be difficult. WHO management guidelines recommend prompt administration of fresh blood as soon as significant bleeding is suspected , but eviIn health, intravascular volume is regulated within tightly circumscribed limits by complex homeostatic mechanisms. Historically, the general principles governing fluid resuscitation developed in parallel with evolving knowledge of human physiology and vascular biology. Starling originally hypothesized in 1896 that opposing hydrostatic and oncotic pressures in the capillaries of the microcirculation determined fluid flow between the intravascular and interstitial fluid compartments . In lineThe revised Starling model emphasizes the crucial role of the EGL in fluid filtration across the “surface glycocalyx endothelial complex.” Although the detailed structure remains to be elucidated, we now know that the EGL consists of an ordered matrix of glycoproteins and proteoglycans anchored in endothelial cells and extending throughout all vascular beds. Plasma proteins such as albumin adhere to and consolidate the structure, resulting in formation of a negatively-charged size-selective barrier that is semipermeable to macromolecules , 33. CelSeveral important consequences of the revised Starling model need to be acknowledged. Originally, plasma was thought to be filtered to the interstitial space at the arteriolar end of capillaries under a dominant hydrostatic pressure gradient, with the majority of the fluid later being re-absorbed at the venular end under a dominant oncotic pressure gradient. Now however, it is clear that the effective oncotic pressure gradient lies within microvessels, i.e., it is the local colloid osmotic pressure difference between free-flowing plasma and the subglycocalyx space that opposes filtration. As the subglycocalyx space is virtually protein free in comparison with interstitial fluid protein levels, the oncotic pressure gradient opposing fluid filtration is greater than expected with the original model, and thus the rate of fluid loss to the interstitial space is much lower than previously thought  31, 35), 3531, 3de novo synthesis. The revised Starling model suggests that a slow plasma leak would be balanced by a proportionate increase in lymphatic return that, together with cardiovascular, renal, and adrenal compensatory mechanisms, should initially serve to ensure that plasma volume remains close to normal. However, over time progressively worsening hypo-albuminaemia would reduce the colloid osmotic gradient between free-flowing plasma and the subglycocalyx space, thereby increasing filtration into the interstitial space to a level that outstrips the capacity of the lymphatic system to return the filtered fluid to the circulation. Eventually plasma volume becomes compromised, but by the time cardiovascular decompensation actually occurs the intrinsic homeostatic mechanisms are maximally upregulated, explaining the counterintuitive rise in diastolic pressure that is observed.Considering the observed clinical features of DSS, a slow sustained plasma leak evolving over several days is consistent with the revised Starling model. Characteristically shock occurs late in the illness episode (day 4–6), heralded by increasing hemoconcentration that is followed by a rise in diastolic pressure with concomitant narrowing of the pulse pressure (PP). Secondly plasma albumin concentrations are often markedly reduced (<20 gm/L) immediately after initial volume replacement counterbalances the effect of hemoconcentration, but rapidly return to normal within 24–48 h as the vasculopathy resolves—i.e., within a timeframe consistent with redistribution rather than consumption and in vivo, but increased levels of several glycocalyx constituents have been documented, with some evidence indicating associations with the timing and severity of leakage  can disrupt the surface glycocalyx both directly and/or through inflammatory-dependent pathways  in the light of new knowledge; what we understand to be the best approach today may not remain so in the future.DT: topic conceptualization for this review, literature review, writing – original draft preparation, writing – review and editing. HT and BW: literature review, writing – original draft preparation, writing – review and editing. All authors contributed to the article and approved the submitted version.The 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.


Dear editor,Septic patients often undergo altered mental status. One possible reason for this brain dysfunction is thought to be related to vascular leakage caused by systemic inflammation. The recent article by Erikson et al.  showed tSince its discovery by Shoichiro Tsukita, a pioneer in the field of tight junction, most of the data in literature about the causal role of occludin in barrier function are from in vitro experiments. However, occludin-deficient mice do not display a perturbation of barrier function, with a complex pathophysiological and inexplicable phenotype  which ev−/− mice may be able to compensate for occludin loss. It also cannot be excluded that the level of occludin plays limited roles under normal physiological conditions but modulates the sensitivity of blood vessels to pathophysiologically relevant stressors. Some questions that need to be answered in the future include the following: Will postnatal endothelial-specific occludin knockdown in mouse brain lead to vascular leak? Are occludin-deficient mice more sensitive to sepsis? Will endothelial-specific occludin overexpression prevent or limit sepsis-induced brain vascular leak in vivo?Therefore, due to the lack of vascular barrier disruption in occludin-deficient mice, additional in vivo animal experiments may be needed to make the conclusions for those studying the role of occludin in vascular barrier function in vivo and those using occludin expression as an indicator of blood-brain barrier function more reliable. However, there is a possibility that occludin


Target-specific anticancer drugs are under rapid development. Little is known, however, about the risk of administering target-specific drugs to patients who have tumours with molecular alterations or other characteristics that can make the drug ineffective or even harmful. An increasing number of randomised clinical trials (RCTs) investigating target-specific anticancer drugs include subgroup analyses based on tumour characteristics. Such subgroup analyses have the potential to be more credible and influential than subgroup analyses based on traditional factors such as sex or tumour stage. In addition, they may more frequently lead to qualitative subgroup effects, that is, show benefit in one but harm in another subgroup of patients . If so, subgroup analyses based on tumour characteristics would be highly relevant for patient safety. The aim of this study is to systematically assess the frequency and characteristics of subgroup analyses based on tumour characteristics, the frequency of qualitative subgroup effects, their credibility, and the interpretations that investigators and guidelines developers report.We will perform a systematic survey of 433 RCTs testing the effect of target-specific anticancer drugs. Teams of methodologically trained investigators and oncologists will identify eligible studies, extract relevant data and assess the credibility of putative subgroup effects using a recently developed formal instrument. We will systematically assess how trial investigators interpret apparent subgroup effects based on tumour characteristics and the extent to which they influence subsequent practice guidelines. Our results will provide empirical data characterising an increasingly used type of subgroup analysis in cancer trials and its potential impact on precision medicine to predict benefit or harm.Formal ethical approval is not required for this study. We will disseminate the findings in a peer-reviewed and open-access journal publication. We will use rigorous methodology including a systematic search for oncology trials published in leading journals, duplicate data extraction by a team involving both experienced methodologists and oncologists, transparent documentation including the collection of verbatim quotes, and use of a formal instrument for assessing the credibility of claimed subgroup effects.The systematic survey will specifically address subgroup claims based on tumour characteristics, which become increasingly relevant for decision making in an era of precision medicine.Potential limitations include a small number of eligible subgroup claims based on tumour characteristics, suboptimal reporting of identified subgroup claims and lack of subgroup analysis plans.The increasing understanding of the biology of malignancies and the availability of new biotechnologies has led to a rapid development of anticancer drugs directed at molecular targets. The hope associated with a target-specific (or biomarker-driven) therapy is to maximise anticancer effects and minimise side effects. Prominent examples include BRAF inhibitors for melanoma,3Target-specific anticancer drugs are designed to directly inhibit tumour growth or enhance immunological antitumour response, by influencing a known—or at least partly understood—molecular mechanism. Typically, the targeted mechanism is complex and spans several steps starting with an interaction of the drug with the target molecule, followed by a signalling cascade, leading to endpoints relevant for tumour growth such as proliferation or apoptosis. Alterations of the molecules involved in this mechanism have the potential to modify the effect of the drug.Anticancer treatments typically have side effectsand are judged acceptable under the assumption that the benefits will outweigh the side effects. Molecular alterations of the tumor could affect this net benefit and render the drug useless or even harmful for certain patients.Investigators of randomised clinical trials (RCTs) increasingly use subgroup analyses to explore effect modifications by tumour characteristics. Those include subgroup analyses based on specific molecular alterations , and also more unspecific tumour characteristics such as measures of mutation burden , tumour grade, or histological subtype. A recent survey of cancer trials showed that 103 of 221 47%) oncology trials published between 2011 and 2013 reported subgroup analyses based on biomarkers.7% oncoloFor instance, an RCT in patients with colorectal cancer addressed the impact of panitumumab, a monoclonal EGFR antibody.Another RCT addressed afatinib, which also targets the EGFR pathway , in patiThese two examples suggest that target-specific agents may be highly beneficial in one but potentially harmful in another subgroup of patients. The authors of the two trials may have underemphasised the potential risk of giving target-specific drugs to the inferior subgroup. Rather than acknowledging the potential for serious harm, they concluded a ‘lack of response’In contrast, findings suggested by subgroup analyses have generally low credibility. The majority of claimed subgroup effects are not confirmed in subsequent researchSome have suggested that subgroup analyses based on tumour characteristics, compared with traditional subgroup analyses , might lead to more credible findings:6No empirical study is available that addresses subgroup effects based on tumour characteristics in target-specific anticancer therapy. Therefore, we will perform a systematic survey of RCTs to determine if the presented examples are isolated instances or a generalisable phenomenon.Specific study objectives areTo assess, in RCTs investigating target-specific anticancer drugs, the frequency ofsubgroup analyses based on tumour characteristics.claims about subgroup effects based on tumour characteristics made by trial investigators.qualitative subgroup effects based on tumour characteristics, that is, suggesting benefit in one and harm in another subgroup.To assess the credibility of claimed subgroup effects based on tumour characteristics.To assess, for claimed subgroup effects, how trial investigators or guideline developers interpreted the results  and how these interpretations relate to the credibility assessment .We will include publications of parallel-group RCTs that (1) enrol patients with a malignant disease; and (2) include a comparison to investigate the effect of a target-specific anticancer drug. We will use an inclusive definition of ‘target-specific anticancer drug’ and consider any drug with a defined mode of pharmacodynamic action, for example, tyrosine kinase inhibitors targeting specific proteins resulting from mutations, all monoclonal antibodies targeting specific proteins or antihormonal drugs Based on a sensitive search strategy that we developed together with a medical information specialist, we searched PubMed for eligible RCTs published since 2014 in the nine oncology and four general medical journals with the highest impact factor in the year 2016. We used free text and medical subject headings for terms related to cancer and combined them with journal indexations and a validated filter for RCTs10.1136/bmjopen-2019-034565.supp1Supplementary dataOur search identified 1119 potentially relevant abstracts of which we already acquired and screened the full texts. Of those, 433 proved eligible RCTs testing a target-specific anticancer therapy and reporting one or more subgroup analysis. Based on previous research,We will develop and standardise extraction forms with detailed explanations for each data point. Teams of two methodologically trained investigators will extract data from publications and associated protocols and resolve disagreements by discussion.We will extract the following information for each trial: population; interventions; primary outcome(s), the overall treatment effect including CIs; whether the trial reported any subgroup analyses  in the main paper, appendices or in secondary publications; number of effect modifiers  type of effect modifiers (categorised by oncologists into (1) tumour characteristics of interest defined as genetic alterations, grade, subtype, or other molecular or histological characteristics; (2) tumour characteristics not of interest such as staging, location, size, or other macroscopic or radiological characteristics; or (3) other effect modifiers such as age or sex), subgroup hypotheses , methods used for subgroup analyses , numerical results of subgroup analyses , and whether authors make no, a weak, a moderate or a strong claim of effect modification.et al.To assess the strength of reported subgroup claims, we will use predefined criteria developed by Sun To assess the credibility of claimed effect modification (based on tumour characteristics), we will apply a recently developed instrument for assessing the credibility of effect modification analyses (ICEMAN).Was the direction of the effect modification correctly hypothesised a priori?Was the effect modification supported by prior evidence?Does a test for interaction suggest that chance is an unlikely explanation of the apparent effect modification?Did the authors test only a small number of effect modifiers or consider the number in their statistical analysis?If the effect modifier is a continuous variable, were arbitrary cut-points avoided?The instrument provides four predefined responses for each question and an overall credibility rating on a continuous scale ranging from very low to high credibility. Teams of two methodologically trained investigators will independently apply ICEMAN to claimed subgroup effects based on tumour characteristics and resolve disagreements by discussion. Items 1, 2, 4 and potentially 5 require a study protocol for optimal assessment. Therefore, for each study reporting a claim, we will search for corresponding study protocols by screening references and trial registry entries.We will use descriptive statistics  to present trial characteristics, including the number and type of subgroup analyses reported per trial, number and strength of claims made, and whether a subgroup claim is qualitative. A qualitative subgroup claim means that (a) authors claim a subgroup difference plus (b) the point estimates suggest benefit in one harm in another subgroup. For all individual subgroup claims, we will present the type of effect modifier (tumour characteristics vs other), the numerical results, the credibility rating and the interpretations presented by primary study authors and/or guidelines. We will also create forest plots showing, for all claimed subgroup effects, point estimates and associated CIs within subgroups, differentiating MA-based and other subgroup effects. We will clearly indicate if a trial reports more than one subgroup claim.A key criterion for the credibility assessment is the result of an interaction test . We know from previous empirical studies that most trials reporting subgroup effects do not include the results of a test of interaction.Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.This study will provide insights into the effectiveness and safety of target-specific anticancer drugs—in particular, in patients subgroups with potentially relevant tumour characteristics. Our study will clarify as yet unresolved issues of whether subgroup analyses based on tumour characteristics performed as part of RCT reports deliver useful results and whether trial investigators and guideline developers draw appropriate conclusions.Several previous studies have systematically assessed the credibility of subgroup effects,34 35We do not anticipate any serious feasibility problems. In previous empirical studies, investigators have successfully used similar methods, including systematic surveys of subgroup analyses in cancer trials,A limitation of quantitative results such as the frequency of subgroup claims based on tumour characteristics in cancer trials will be a high risk of reporting and publication bias. We will therefore make clear that our results will reflect what is reported and not necessarily how often authors consider those analyses. This potential limitation, however, will not weaken inferences made for individual RCTs. Another potential limitation is that, although we know from previous research that subgroup analyses based on biomarkers are included in almost half of oncology trials,In summary, this systematic survey of RCTs will address characteristics, frequency, credibility and impact of effect modification analysis based on tumour characteristics. If our findings support the alarming hypothesis that the risk of harm caused by target-based drugs might be systematically underemphasised, then our results will help to increase the awareness of the issue among patients, oncologists, trial investigators, journal editors, and guideline developers. The results may trigger a re-evaluation of existing trials, inform planning of future trials and influence how trial investigators and guideline developers interpret MA-based subgroup analyses in the context of target-specific anticancer therapy.Formal ethical approval is not required for this study that will be based on published information only. We will disseminate the findings in a peer-reviewed and open-access journal publication.