N-oxide, tryptophan and tyrosine derivatives, and oxidized fatty acids. Such interventions will require modulating either bacterial species or the bacterial biosynthetic enzymes required to produce these metabolites, so we briefly describe the current understanding of the bacterial and enzymatic pathways involved in their biosynthesis and summarize their molecular mechanisms of action. We then discuss in more detail the impact of these metabolites on health and disease, and review current strategies to modulate levels of these metabolites to promote human health. We also suggest future studies that are needed to realize the full therapeutic potential of targeting the gut microbiota.Mass spectrometry- and nuclear magnetic resonance-based metabolomic studies comparing diseased versus healthy individuals have shown that microbial metabolites are often the compounds most markedly altered in the disease state. Recent studies suggest that several of these metabolites that derive from microbial transformation of dietary components have significant effects on physiological processes such as gut and immune homeostasis, energy metabolism, vascular function, and neurological behavior. Here, we review several of the most intriguing diet-dependent metabolites that may impact host physiology and may therefore be appropriate targets for therapeutic interventions, such as short-chain fatty acids, trimethylamine  The symbiotic relationship between mammals and the trillions of microbial cells that reside in their gastrointestinal tracts relies on a complex molecular dialogue, with microbial metabolites acting as major mediators of this dialogue. Essential roles for several microbial metabolic pathways in host physiology have been long established, including in the production of vitamin K, the production of water-soluble B vitamins including biotin, folates, nicotinic acid, pyridoxine, riboflavin, cobalamin and panthotenic acid, the degradation of dietary oxalates, and modification of bile salts . Howcis-12-octadecenoate (HYA); trimethylamine (TMA) and trimethylamine N-oxide (TMAO); tryptophan metabolites such as indole, indole-3-propionate (IPA) and indoxyl-sulfate (IndsS); and tyrosine and phenylalanine metabolites such as hippuric acid, phenylacetylglycine, phenyl sulfate, para-cresyl sulfate (PCS), phenylpropionylglycine, cinnamoylglycine and equol sulfate. Many of the metabolites identified by these studies result from the transformation of specific dietary components by select species of microbes that express the necessary enzymes to act on these components. Thus, the variable presence of microbes utilizing these diet-dependent metabolic pathways may be key to understanding the variable host response to specific dietary components and susceptibility to disease [Elucidating target microbial metabolites that modulate host physiology requires identifying the major metabolites (and their downstream co-metabolites formed by the phase I/II xenobiotic metabolizing enzymes of their host) that differ between healthy and diseased individuals, and assessing the biological activities of these metabolites. A series of landmark metabolomics studies over the past decade have significantly advanced our understanding by using mass spectrometry (MS) or nuclear magnetic resonance (NMR) analysis to identify potentially important microbial metabolites that derive from the gut microbes –10, that disease .Table 1KThis review will focus on several key metabolites formed by the gut microbiota from dietary components that have been revealed recently to produce remarkable effects on host physiology and that are currently being targeted or have high potential to be targeted as treatments for human disease. We will describe briefly the microbial origin of these metabolites and the biological actions of these metabolites on their host. We will then discuss in more detail current and potential therapeutic approaches to manipulate these metabolite levels and broader areas of research that are needed to understand the potential value of gut microbial metabolites.SCFAs constitute the most abundant microbial metabolite, reaching concentrations of 50–130 mM in the proximal colon . The bioA number of molecular mechanisms of action have been ascribed to acetate, propionate and butyrate that may be relevant to their therapeutic potential to promote intestinal health, reduce inflammation, and inhibit cancer Table . All thrA decrease in luminal SCFAs is associated with ulcerative colitis and intestinal inflammation, which can be ameliorated with dietary fiber or administration of SCFAs –50. Redun = 2079) [n = 1429), [ApcMin/+Msh2−/−) [Intestinal inflammation contributes to the development of colorectal cancer, and the contribution of SCFA-producing bacteria to the inhibition of colon carcinogenesis remains unresolved. Besides its anti-inflammatory effects, butyrate also exerts anti-proliferative and anti-cancer effects when tumor cell lines are exposed to it in vitro –63, prim = 2079)  and the = 1429),  showed n= 1429), –77 as we= 1429), ). Butyra= 1429), , which i= 1429),  or in ca= 1429), , butyrat= 1429), , resulti= 1429), . Critica= 1429), , 84. Mor= 1429), . In thisMsh2−/−) . ImportaMsh2−/−) . Thus, wH2-cell-mediated allergic inflammation [Lachnospira, Veillonella, Faecalibacterium and Rothia) and reduced fecal acetate. Inoculating germ-free mice with these four microbial taxa ameliorated airway inflammation in their offspring, demonstrating a causal role in suppressing inflammation [The anti-inflammatory effects of SCFAs extend beyond the gut, such as inhibiting vascular smooth muscle cell proliferation and migration , improviammation . High fiammation . The Canammation . These aammation . TogetheThe landmark study by Turnbaugh and colleagues showing that transfer of microbiota from obese mice into germ-free mice increased adiposity and cecal levels of SCFAs relative to the transfer of microbiota from lean mice  has led A recent meta-analysis of various studies in this field by Byrne and colleagues led them to conclude that increasing SCFA levels had an overall net benefit on obesity due to their effects on satiety and reduced food intake, increased energy expenditure and thermogenesis, and inhibition of lipogenesis and cholesterol synthesis . One molSince, on the whole, increasing the microbial production of SCFAs appears to be a reasonable therapeutic intervention for the treatment of obesity, future studies are needed to determine how to effectively carry out such long-term interventions in humans. For a significant fraction of the human population, consumption of large amounts of non-digestible dietary fibers such as inulin is significantly hampered by undesirable gastrointestinal effects such as bloating, abdominal cramping, flatulence and diarrhea. These adverse effects result in part from the simultaneous formation of hydrogen gas and carbon dioxide during fermentation. Therefore, optimization of the microbiota or substrates to enhance SCFA production and to minimize released hydrogen and carbon dioxide gas will be critical for the wide-spread application of this treatment in the general population.Desulfovibrio desulfuricans, CutC and CutD, allow choline to be used as an energy source [Proteobacteria and Firmicutes, and to a much lesser extent Actinobacteria, suggesting spread via horizontal gene transfer [l-carnitine and choline via an analogous reaction catalyzed by the YeaW and YeaX enzymes originally characterized in Escherichia coli [Acinetobacter baumannii [TMA and its co-metabolite TMAO were identified by screening metabolites associated with cardiovascular disease (CVD), and TMA was shown to require gut bacteria for its formation . Cleavagy source . Recent transfer . TMA washia coli , and by aumannii . After faumannii . Analysiaumannii . Oral anaumannii , 114.l-carnitine to raise TMAO levels can all increase atherosclerosis in Apoe−/− mice [Apoe−/− mice inhibits reverse cholesterol transport from macrophages in vivo [TMAO levels predict risk for atherosclerosis , 115, an−/− mice . The spe−/− mice . Thus fa−/− mice , but TAA in vivo , but tre in vivo . Reducti in vivo . Adminis in vivo . Thus, sl-carnitine would lower TMAO levels, but may have undesirable effects. In particular, supplementation with lower levels of l-carnitine than needed for TMAO formation may improve cardiovascular function [n = 3629) showed that l-carnitine supplementation reduces all-cause mortality by 27 % [l-carnitine diet [Apoe−/− mice [Without identified TMAO molecular targets, interventions to reduce CVD must focus on reducing TMAO levels. Reducing dietary choline or function . A meta- by 27 % . While pine diet . Importa−/− mice . Future Lactobacillus spp. convert tryptophan to indole-3-aldehyde (I3A) through unidentified enzymes [Clostridium sporogenes convert tryptophan to IPA [Tryptophan is an essential amino acid found in a variety of foods such as red meat, fish and eggs. Commensal bacteria expressing tryptophanase catabolize tryptophan to indole, a quorum-sensing compound for bacteria  Fig. 1)1). Lacto enzymes . Clostrin to IPA , likely H17-cell activity, and helps maintain intraepithelial lymphocytes [Citrobacter rodentium infection [E. coli infections). In addition to these effects, recent studies show that indole also modulates GLP-1 release from L cells [Indole and its metabolites affect host physiology via a number of molecular mechanisms Fig. . Indole phocytes . Indole phocytes , 127. Thphocytes , and thephocytes  and respnfection   from Clostridium difficile prevent fermentation of tyrosine or its metabolite hydroxyphenylacetate to para-cresol [Brettanomyces generates 4-ethylphenol from the tyrosine metabolite para-coumaric acid that is present in many foods via cinnamate decarboxylase and vinyl phenol reductase. 4-Ethylphenol also forms from orally administered genistein, a phytoestrogen found in soy, by uncharacterized but presumably microbial pathways [PCS and 4-ethylphenyl sulfate (EPS) are structurally similar uremic toxins formed by hepatic sulfation of the microbial metabolites para-cresol and 4-ethylphenol, respectively. The lack of PCS or EPS in the plasma and urine of germ-free mice demonstrates their microbial origins. Inactivating mutants of the hydroxyphenylacetate decarboxylase operon genes  such as cis-9, trans-11 and cis-15 CLnA, respectively [Lachnospiraceae, Lactobacillus spp. and Bifidobacteria, possess the capacity to generate both CLAs and CLnAs [Lactobacillus, intermediates for the formation of conjugated fatty acids include the oxygenated metabolites HYA and 10-hydroxyoctadecanoate (HYB) [Lactobacillus were recently characterized and include myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase, and acetoacetate decarboxylase [The microbiota of ruminants have long been known to transform the essential fatty acids linoleic acid (LA) and linolenic acid to CLAs such as ectively –163, viand CLnAs –166. In te (HYB) , 168. Thboxylase .Conjugated fatty acids exert many highly beneficial effects, including reduction of adiposity, improved insulin sensitivity, reduced carcinogenesis, and reduced atherosclerosis (reviewed in ). CLAs aIn previous sections, we have touched briefly on potential future studies for individual metabolites, but there are additional developments needed in broad areas of research and understanding to fully realize the potential of gut microbial metabolites for disease treatment. We will conclude by highlighting four of these needed developments.C. difficile infections. For translation to actual treatment, measurements will need to be carried out in clinical laboratories in which immunoassay arrays, rather than the more sophisticated MS or NMR methods available in research settings, will likely continue to be the primary methods available. Thus, identifying the minimal number of biomarker metabolites needed to selectively assess a condition is critical. Similar strategies can be used to determine the efficacy and safety of interventions.First, the development of minimal sets of biomarker microbial metabolites that identify particular disease states or that distinguish between closely related disease conditions. The analysis carried out by de Preter and colleagues for inflammatory bowel disease is proof of principal for this strategy , and simSecond, the development of algorithms to predict personalized responses to dietary and pharmaceutical interventions based on microbial metabolites. An exciting example of this approach was recently reported by Zeevi and colleagues, who demonstrated that the highly variable glycemic response of different individuals to the same foods could be predicted using their gut microbiota and other data . SimilarN-acylphosphatidylethanolamine in commensal E. coli (strain Nissle 1917), leading to inhibition of obesity in mice fed a HFD [Third, the development of readily generalizable methods to increase gut microbial production of beneficial metabolites, either by selectively increasing the abundance of native species that produce that metabolite or by engineering endogenous gut microbiota to produce it in high levels. An example of this latter approach is our study using heterologous expression of the satiety factor ed a HFD . Such stFourth, the development of non-lethal specific inhibitors for various microbial pathways that produce harmful metabolites, similar to work done with CutC/D. In particular, inhibition of the formation of para-creysl and 4-ethylphenol appear amendable to this strategy. This revolutionary approach to controlling harmful bacterial metabolites seems unlikely to result in the rapid evolution of resistance that occurs with standard antibiotics, since there is a much more limited fitness advantage of carrying resistance. If this is the case, then long-term use of such metabolic pathway inhibitors will have great potential benefit in chronic diseases.The past decade has seen remarkable progress in our understanding of the significant role that gut microbial metabolites play in modulating the health of their hosts. MS and NMR studies have identified a significant number of microbial metabolites that differ in disease conditions, and these same methods are now being exploited to better identify subtle differences in closely related diseases. Some of these identified metabolites, such as TMAO, IndS and PCS, appear to directly increase susceptibility to disease, while others, such as SCFA, IPA, CLA and HYA, appear to exert protective effects. Much work remains to fully characterize the physiological effects of these and the many other microbial metabolites that may be important in human health. It seems highly likely that future studies will identify many other disease states in which gut microbial metabolites are significantly enriched or depleted. It is important to keep in mind that by themselves such studies do not demonstrate causality. Thus, it seems there is a considerable need for carefully controlled studies to determine the physiological effects of each identified microbial metabolite and its specific mechanisms of action. Furthermore, in order to fully exploit the potential of the gut microbiota for disease prevention, we need a much greater understanding of how dietary components and host genetics affect the production of various metabolites. Finally, translation of these findings to clinical practice will require the development of widely available clinical chemistry methods to detect changes in an individual’s key metabolites. Despite these tremendous challenges to fully exploiting the gut microbiota for human health, the remarkable progress of the last decade suggests that such approaches have significant potential to revolutionize therapeutic approaches to human disease.


Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system. Different trigger pathologies have been suggested by the primary cytodegenerative “inside-out” and primary inflammation-driven “outside-in” hypotheses. Recent data indicate that mitochondrial injury and subsequent energy failure are key factors in the induction of demyelination and neurodegeneration. The brain weighs only a few percent of the body mass but accounts for approximately 20% of the total basal oxygen consumption of mitochondria. Oxidative stress induces mitochondrial injury in patients with multiple sclerosis and energy failure in the central nervous system of susceptible individuals. The interconnected mechanisms responsible for free radical production in patients with multiple sclerosis are as follows: (i) inflammation-induced production of free radicals by activated immune cells, (ii) liberation of iron from the myelin sheets during demyelination, and (iii) mitochondrial injury and thus energy failure-related free radical production. In the present review, the different sources of oxidative stress and their relationships to patients with multiple sclerosis considering tissue injury mechanisms and clinical aspects have been discussed. Multiple sclerosis is a chronic disease of the central nervous system and is associated with the formation of focal myelin loss and progressive neurodegeneration .Clinically, 80% of patients with multiple sclerosis present with relapsing-remitting multiple sclerosis (RRMS), which refers to clearly defined episodes of neurological dysfunction followed by  recovery. After 15–25 years, RRMS is transformed into secondary progressive multiple sclerosis (SPMS) in a majority of the patients; SPMS is characterised by progressive neurological symptoms. Patients with multiple sclerosis (10–15%) enter this neurodegenerative phase at disease onset, which is referred to as primary progressive multiple sclerosis (PPMS) .Inflammation in the relapsing-remitting and progressive phases is driven by the same mechanisms, but during progressive MS the central nervous system does not respond to currently available anti-inflammatory drugs , which mMicroglia, which are under the control of intact neurons, may become chronically active due to primary neurodegeneration, axonal degeneration, and additional peripheral activation processes such as systemic inflammation –13.Multiple sclerosis may be caused by primarily cytodegenerative processes/infections, which are amplified by inflammation –17.Once patients enter the progressive phase, no currently available drug provides a significant clinical effect. Neurological decline in these patients is caused by chronic and diffuse neurodegeneration. Neurodegeneration is closely associated with inflammation, both morphologically and quantitatively, in all phases of multiple sclerosis , 6. AntiAlthough these models are fundamentally different, they share a common feature; that is, the tissue injury mechanisms are closely related to the production of reactive oxygen and nitrogen species.The central role of oxidative stress has been frequently suggested in the pathogenesis of multiple sclerosis  based onActive inflammation and breakdown of the blood-brain barrier can be detected in the brains of patients with multiple sclerosis as gadolinium enhancing magnetic resonance imaging lesions , 27–29. Macrophage/microglia-derived reactive oxygen and nitrogen species trigger axonal injury . The enzIn physiological conditions, neurons, astrocytes, and oligodendrocytes express molecules that bind to receptors that are expressed on microglia. Signalling via these molecules, such as the fractalkine receptor ligand CX3CL1 , the memIn patients with long disease durations (median > 372 months), the level of inflammatory infiltrates is similar to those found in age-matched controls . AlthougIn the human brain, iron is primarily stored by ferritin  in the mClinically, the duration of the relapsing-remitting phase can vary between individuals ; howeverIn the human brain, the highest iron content is measured in the deep grey matter nuclei, particularly in the basal ganglia , 52, 58.The role of the mitochondria in the pathogenesis of multiple sclerosis was suggested by the observation that patients with Leber's hereditary optic neuropathy, a disease caused by mitochondrial DNA mutations, have an increased risk of developing multiple sclerosis-like Harding's disease . In patiThe detoxification of reactive oxygen species reversed mitochondrial and axonal injury in experimental settings , where tMitochondrial dysfunction and consequently ATP deficiency in multiple sclerosis lesions lead to the failure of sodium removal from the axoplasm into the extracellular space during action potential conduction . In thisThe central nervous system is highly dependent on the continuous blood flow and mitochondrial metabolism that produces ATP . This deSmoking can trigger multiple sclerosis, propagate disease progression, and transiently worsen the motor functions in patients with multiple sclerosis –120. CigEnergy failure, due to mitochondrial injury and oxidative stress, is a key player in the pathogenesis of multiple sclerosis. Early clinical research argued over the “relief by flush” therapy , 128. DrA conclusion, which genetic, experimental, and pathological investigations clearly suggest, is that there is no evidence for a single cause and thus therapeutic target of multiple sclerosis. Instead, multiple amplification steps orchestrate the clinically observed phenotype in susceptible individuals , which i


The use of MSCs in cartilage repair makes it possible to develop single-stage cell-based therapies. However, true single-stage procedures rely on one intervention, which will limit cell sources to fraction concentrates containing autologous MSCs or culture-expanded allogeneic MSCs. So far, it seems both autologous and allogeneic cells can safely be applied, but clinical studies are still ongoing and little information on clinical outcome is available. Further development of cell-based therapies may lead to clinical-grade, standardized, off-the-shelf products with easy handling for orthopedic surgeons. Although as of yet no preclinical or clinical studies are ongoing which explore the use of induced pluripotent stem cells for cartilage repair, a good manufacturing practice-grade induced pluripotent stem cell line might become the basis for such a product in the future, providing that cell fate can be controlled. The use of stem cells in clinical trials brings along new ethical issues, such as proper controls and selecting primary outcome measures. More clinical trials are needed to estimate detailed risk-benefit ratios and trials must be carefully designed to minimize risks and burdens for patients while choosing outcome measures that allow for adequate comparison with results from similar trials. In this review, we discuss the different aspects of new stem cell-based treatments, including safety and ethical issues, as well as provide an overview of current clinical trials exploring these approaches and future perspectives.The evolution of articular cartilage repair procedures has resulted in a variety of cell-based therapies that use both autologous and allogeneic mesenchymal stromal cells (MSCs). As these cells are increasingly available and show promising results both  Cartilage defects in the weight-bearing joint are a severe limitation to the patient and pose a significant burden to society. Symptoms include pain, stiffness, joint effusion and locking, which cause considerable disability and decrease quality of life. It is well understood that cartilage defects need (early) treatment because they have a poor intrinsic healing capacity and tend to lead to osteoarthritis .Cartilage repair strategies have rapidly evolved over time; in 1950 the resection of loose and damaged tissue was the only treatment available. In the late 1980s microfracture was introduced, which involves drilling multiple holes in the subchondral bone to allow an influx of bone marrow that stimulates natural repair. In 1994, the first results on autologous chondrocyte implantation (ACI) were published  and manyin vivo remains unknown: will they survive or disappear in the long-term? Will they all differentiate into chondrocytes or will some remain as MSCs? Current studies are not conclusive on these questions; some have suggested MSCs differentiate and survive in vivo up to 6 months, while others suggest MSCs have a chondroinductive role - that is, stimulate cartilage regeneration through trophic factors while slowly disappearing from the culture [in vivo, MSCs of both autologous and allogeneic origin have increasingly been introduced for cartilage repair in clinical studies.The mid- to long-term results of ACI have been encouraging ,3. Howev culture . AlthougThe development of an off-the-shelf product to treat cartilage defects would rely on autologous MSC-rich concentrates, allogeneic MSCs or induced pluripotent stem cell (iPSC) lines. However, rapid developments in the field make it difficult to evaluate the existing evidence for such cellular therapies in terms of preclinical and clinical safety and early efficacy. The purpose of this review is to provide a concise overview of the available literature on autologous and allogeneic MSCs for cartilage repair of focal defects. Besides clinical studies, the sources of MSCs, safety and ethical issues with respect to allogeneic MSCs, the use of iPSCs and future perspectives are discussed.in vitro [in vitro chondrogenic potential of ATMSCs is lower compared with BMMSCs in vitro, especially when pellet cultures are stimulated with transforming growth factor (TGF)-beta. [Minimal criteria to define expanded multipotent human MSCs, as defined by the International Society for Cellular Therapy, include that they must be plastic-adherent when maintained in standard culture conditions, express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules, and they must be capable of differentiating into osteoblasts, adipocytes and chondroblasts in vitro . Bone main vitro . ATMSCs in vitro ,9. Howevin vitro [in vitro generation of hyaline cartilage tissue-engineered constructs [in vitro-generated constructs showed good repair of cartilage defects in a pig model with SMSCs isolated from both immature and mature pigs [MSCs derived from the synovial membrane (SMSCs) can be harvested through an arthroscopic procedure or from synovial fluid. The amount of SMSCs in synovial fluid is very low; only about 14 cells per milliliter of synovial fluid from healthy donors can form CFU-F colonies. Parts of these colony-forming cells are considered SMSCs as they can differentiate into the adipogenic, osteogenic and chondrogenic lineages. Compared with BMMSCs and ATMSCs, they have a higher rate of proliferation ,13. Sakanstructs . Implanture pigs ,17.MSCs can also be isolated from peripheral blood (PBMSCs) . MSC isoOne of the concerns with using MSCs for cartilage repair is that if they do differentiate into the chondrogenic lineage and engraft the new cartilage, they might undergo terminal differentiation and become hypertrophic, as the default route of chondrogenic differentiation is terminal differentiation . This coArticular cartilage itself, especially the superficial layers, is also a reservoir for progenitor cells with multilineage potential ,24. CartSince Wakitani and colleagues  performeOnly one study compared the use of two MSC-based treatments for cartilage repair . In thisAlthough only two studies directly compared MSC-based treatments with ACI ,39, the Several other studies using autologous MSCs or concentrates are still ongoing, including two studies using ATMSCs to treat cartilage defects  are reported. Pre-differentiated MSCs have not been used as yet. Although MSCs and MSC-rich concentrates are promising for cartilage repair, a lack of comparative studies confines a prediction to what the optimal cell source for MSC-based cartilage repair would be. Moreover, MSCs and BMCs have been implanted using various cell carriers, passages and doses  using a mixture of rapidly isolated autologous chondrocytes with their pericellular matrix (chondrons) combined with allogeneic BMMSCs in fibrin glue ,46 (NCT0The ability to generate iPSCs from somatic cells has created new opportunities for the field of cartilage repair. Just like human embryonic stem cells (hESCs), they show unlimited self-renewal and they can differentiate into all three germ layers , but without having the ethical concerns associated with hESCs. However, there are some differences reported in the efficiency to differentiate towards several lineages, such as neural, cardiovascular and hemangioblastic lineages. iPSCs can be generated by overexpressing transcription factors associated with pluripotency, such as Oct3/4, Klf4, c-myc and Sox2. The genetic reprogramming to induce pluripotency is a limiting factor for clinical use as the most efficient viral transductions lead to integration of viral DNA into the chromosome. Reprogramming without causing genetic change has gained recent interest and several non-viral methods using microRNA, synthetic messenger RNA and proteins have been developed.In vitro studies showed chondrogenic differentiation and cartilage formation by iPSCs derived from human fetal neural stem cells [em cells  and humaem cells . One stuem cells . Differeem cells ,53.Chondrogenic cells were also generated directly from somatic cells by reprogramming with c-Myc, Klf4 and the chondrogenic transcription factor Sox9. The cells were non-tumorigenic and had stable karyotypes, and they formed homogeneous hyaline cartilage ,55.in vitro chondral defect model. As it was reported that iPSCs can differentiate easier along the lineages related to the cell type of origin, iPSCs derived from several chondrocyte donors were investigated for their chondrogenic potential [Diekman and colleagues  generateotential . Indeed,Although safety precautions and new iPSC generation techniques have been introduced, it remains to be shown that cell fate and phenotype can be controlled without having the risk of teratoma formation. Thus, before preclinical and clinical tests can be done, there is a need for reliable control of the cell fate.The design and initiation of clinical trials using stem cells for cartilage repair is ethically challenging . Only a Uniform use of outcome parameters facilitates the comparison of treatments used in various clinical studies. There is still an ongoing discussion whether structural cartilage regeneration, clinical improvement or a combination should be the main outcome measure. Clinical improvement is undoubtedly an important outcome measure, but placebo and nonspecific effects can affect the patient’s perspective and it has been suggested that clinical improvement does not necessarily correlate with cartilage tissue regeneration. A second-look arthroscopy and histological evaluation of a biopsy is the golden standard to evaluate structural parameters of cartilage regeneration, but is relatively invasive for patients. A less invasive, but also less detailed and informative, measure is MRI. However, there is only a weak correlation between clinical and MRI outcomes, so the challenge remains to determine how clinical and structural results may correlate .Another important ethical consideration is the selection of an appropriate control group. For a double-blinded randomized controlled trial, the use of a placebo, or in the case of cartilage repair a sham intervention, could be necessary. In the case of MSC-based cartilage repair, the use of a sham group is unacceptable as there is an alternative treatment that provides medical advantage (ACI) and the risks and invasiveness of sham procedures are disproportionate to the social value. ACI can serve as a control. However, it is impossible to compare the two-stage ACI treatment to a single-stage procedure without introducing a sham intervention. It is also unacceptable to test safety, tolerability, pharmacokinetics and pharmacodynamics of MSC-based cell products on healthy volunteers, as the risks and burdens of the intervention are too high.With respect to the technovolution of articular cartilage repair strategies, it is expected that more single-stage procedures will emerge that utilize a stem cell-based approach as well as procedures using instructive biomaterials that may facilitate the differentiation of MSCs into the chondrogenic lineage. Single-stage cell-based cartilage repair reduces the burden on patients and eliminates a costly cell-expansion phase. As a true one-stage strategy requires only one surgical intervention, additional biopsies apart from the surgery of any kind to isolate chondrocytes or MSCs should be avoided. This suggests that cells should either be isolated during the time frame of one surgery or allogeneic cells should be used.It is common to select MSCs from a heterogeneous starting population based on their ability to attach and expand on plastic. During culture, they overgrow the other cell types, leading to a culture-expansion-driven isolation of MSCs. For a single-stage strategy this would not be possible if autologous cells were to be used. MSCs could also be isolated by fluorescence-activated cell sorting (FACS) based on their cell surface markers. Antibodies used for the FACS sorting should comply with good manufacturing practice (GMP) regulations for clinical use, which at present is quite expensive. Moreover, as the amount of MSCs is relatively low in adult tissues, it is unlikely sufficient MSCs could be isolated in this way for a single-stage approach. What is more, relatively little information is available on freshly FACS-isolated MSCs with respect to their behavior and chondrogenic capacity. This might differ from expanded MSCs as expansion can favor certain clones. To overcome this problem, autologous bone marrow concentrate (containing the mononuclear cell fraction) and the stromal vascular fraction of adipose tissue are being investigated. Just like the cartilage repair capacities of MSCs from different tissue types are not yet compared in clinical studies, there is no real comparative clinical study on concentrated cell fractions versus MSCs. However, several studies confirmed fibrocartilaginous to hyaline-like repair tissue in cartilage defects treated with BMC -37,39,40Allogeneic MSCs have safely been used in clinical studies. The applicability of allogeneic MSCs opens up the possibility to generate an off-the-shelf cell product for cartilage repair. A clinical grade standardized off-the-shelf product with easy handling for orthopedic surgeons would create a considerable advantage. Critical steps in the development of such a product would be to choose the origin of the cells and the cell carrier, as both factors have a pronounced effect on chondrogenesis and cartilage formation. Besides these factors, such a product should contain cells with proper potency from a single cell line to avoid differences in clinical outcomes due to batch variation. Finally, the production process should be performed in a GMP-licensed cell therapy facility with easy access to the treating hospitals. Although as of yet no preclinical or clinical studies are ongoing which explore the use of iPSCs for cartilage repair, a GMP-grade iPSC cell line might become the basis for such a product in the future, providing that cell fate can be controlled. A hESC cell line would also still possess this therapeutic potential, but would bring along some ethical concerns. Thus far, both autologous MSC-rich concentrates such as BMC and the vascular stromal fraction from adipose tissue, and allogeneic MSCs seem promising cell sources that are currently being used for single-stage treatments of cartilage defects in clinics.Implantation of MSCs is a realistic and promising approach for the treatment of cartilage defects, which is increasingly being introduced in early clinical trials. To make optimal use of these different cell types, considerable work remains to be done in terms of finding the optimal cell source, cell dose and carrier along with understanding the (long-term) cell fate and new ethical issues these cell types bring along.Biology and clinical applications of stem cells for autoimmune and musculoskeletal disorders, edited by Christian Jorgensen and Anthony Hollander. Other articles in this series can be found at http://www.biomedcentral.com/series/MSC. This article is part of a thematic series on


Background and Aim: Hormone therapy (HT) has long been thought beneficial for controlling menopausal symptoms and human cognition. Studies have suggested that HT has a positive association with working memory, but no consistent relationship between HT and neural activity has been shown in any cognitive domain. The purpose of this meta-analysis was to assess the convergence of findings from published randomized control trials studies that examined brain activation changes in postmenopausal women.Methods: A systematic search for fMRI studies of neural responses during working memory tasks in postmenopausal women was performed. Studies were excluded if they were not treatment studies and did not contain placebo or blank controls. For the purpose of the meta-analysis, 8 studies were identified, with 103 postmenopausal women taking HT and 109 controls.Results: Compared with controls, postmenopausal women who took HT increased activation in the left frontal lobe, including superior frontal gyrus (BA 8), right middle frontal gyrus (BA 9), anterior lobe, paracentral lobule (BA 7), limbic lobe, and anterior cingulate (BA 32). Additionally, decreased activation is noted in the right limbic lobe, including parahippocampal gyrus (BA 28), left parietal lobe, and superior parietal lobule (BA 7). All regions were significant at p ≤ 0.05 with correction for multiple comparisons.Conclusion: Hormone treatment is associated with BOLD signal activation in key anatomical areas during fMRI working memory tasks in healthy hormone-treated postmenopausal women. A positive correlation between activation and task performance suggests that hormone use may benefit working memory. The influence of hormone treatment (HT) on brain and cognition in postmenopausal women has been a controversial topic. Cross-sectional studies and meta-analyses have promoted the idea that HT might enhance verbal memory performance and decrease the risk for developing dementia in postmenopausal women  method was performed to locate anatomical regions with significant activation differences. The ALE method permits the creation of three-dimensional (3D) probability maps that show the brain regions that are most likely to demonstrate morphometric differences between HT and controls. The frontal lobes are of paramount significance in determining emotions and judgments related to sympathy, which is defined as the ability to perform daily activities, personality manifestations, and decisions  report group comparisons between postmenopausal women who received HT and matched controls, (b) employ fMRI, (c) report their results in a standard stereotactic space , and (d) investigate working memory or working memory-related tasks. Studies that did not fulfill these requirements were excluded. Treatment, gender, and age were not restricted and the HT brain activation studies did not include a control.The data extracted from each study included: a description of (a) each memory task and (b) foci of task-related activation changes in which the active and control states were different between the HT group and the control as well as (c) clinical data concerning hormone dose, years of estrogen use, and the age-matching of the control group. This study examined all foci that were reported as significant based on the above criteria designated in the included studies.Two separate ALE meta-analyses were performed: one on fMRI studies reporting that brain activation was increased in postmenopausal women receiving HT compared to controls and a second on studies reporting that brain activation was decreased.p-threshold for multiple comparisons , and the second analysis pooled the results of a decrease in brain activation  , middle frontal gyrus (BA 9), parietal lobe, precuneus (BA 7), paracentral lobule (BA 5), limbic lobe, and anterior cingulate BA 32) , left parietal lobe, and superior parietal lobule (BA 7)  have not been consistently demonstrated and the brain activation patterns are inconsistent. In this study, we addressed the cognitive effects of hormones in women and performed an ALE meta-analysis on functional brain imaging studies to investigate and aggregate the known data that exist regarding the effects of HT on neural activity in postmenopausal women during working memory tasks. Our results indicate that hormone use is associated with increased regional brain activation during working memory task, with women in hormone-treatment groups exhibiting a more robust neural response than placebo-treated women. Women receiving HT exhibited increases in brain activation in the frontal lobe and the anterior cingulate cortex (ACC) , including left parietal lobe and superior parietal lobule (BA 7) . After accounting for variations in age, education, and age at hormone initiation, long-term users did not have more activation than short-tem users in any part of the brain. Long-term users showed slightly more activation than short-term users in only one small region within the prefrontal cortex. While it is difficult to make conclusions based on one differing region, these results may be consistent with others that have indicated a persisting cognitive benefit to past hormone users , left parietal lobe, and superior parietal lobule (BA 7) were detected. The results are consistent with our hypothesis. Together, the findings of enhanced frontal lobe activation and decreased parahippocampal activation in perimenopausal HT users suggest that HT enhances both state-dependent and recollective processes contributing to cognitive performance.KL designed the study and wrote the protocol. XH and YH managed the literature searches and undertook the statistical analysis, and KL, XH, LY, JL, and DZ wrote the manuscript. JZ and YL took part in the modification and further analysis of the final manuscript. All authors contributed to and have approved the final 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.


Renal colic is the most frequent nonobstetric cause for abdominal pain and subsequent hospitalization during pregnancy. The physio-anatomical changes in the urinary tract and the presence of the fetus may complicate the clinical presentation and management of nephrolithiasis. Ultrasound (US) is the primary radiological investigation of choice. Magnetic resonance urography (MRU) and low-dose computed tomography (CT) have to be considered as a second- and third-line test, respectively. If a study that uses ionizing radiation has to be performed, the radiation dose to the fetus should be as low as possible. The initial management of symptomatic ureteric stones is conservative during pregnancy. Intervention will be necessary in patients who do not respond to conservative measures. Therefore, it is crucial to obtain a prompt and accurate diagnosis to optimize the management of these patients.Teaching PointsIn pregnancy, renal colic is the most frequent nonobstetric cause for abdominal pain and hospitalization.• Magnetic resonance urography should be considered when ultrasound is nondiagnostic.• Low-dose CT should be considered as a last-line test during pregnancy.•  Renal colic is the most frequent nonobstetric cause for abdominal pain and subsequent hospitalization during pregnancy , 2. SympThe physio-anatomical changes in the urinary tract and the presence of the fetus may complicate the clinical presentation and subsequent management of nephrolithiasis .Renal colic has been associated with several pregnancy complications, including preterm labor and delivery, recurrent abortions, hypertensive disorders, gestational diabetes and cesarean deliveries. These potential complications make accurate diagnosis crucial , 7.In a series of pregnant patients with renal colic, 28 % of patients with abdominal pain had an incorrect admitting diagnosis based on clinical evaluation. These diagnoses included appendicitis, diverticulitis and placental abruption .


We report the case of two-year-old girl with hypovolemic shock caused by bleeding from an abdominal cystic lymphangioma. The whole blood was contained within a large omental bag that could be completely removed. There were no associated anomalies. The child progressed satisfactorily. Abdominal cystic lymphangioma is not a common disorder during childhood. It usually presents with abdominal pain or acute abdomen but also presents as an asymptomatic mass.Two-year-old girl, whose father died of an aortic aneurysm in Marfan's disease, showed progressive enlargement of the abdomen, as well as pallor and fatigue of at least two weeks later. She was brought to the emergency department with weakness, apathy, and pallor. AP abdominal radiograph  shows meLymphangiomas are benign tumors of the lymphatic vessels usually located in subcutaneous tissue of head, neck and armpit, and more rarely in abdomen –5, beingThe pathological examination of the masses located in abdomen usually shows thin-walled cavities of different sizes and may contain lymph, pus when infected, or blood as a result of bleeding . Our casCystic Lymphangioma should be considered as a possible cause of massive abdominal hemorrhage.


Obesity and other adverse health outcomes are influenced by individual- and neighbourhood-scale risk factors, including the food environment. At the small-area scale, past research has analysed spatial patterns of food environments for one time period, overlooking how food environments change over time. Further, past research has infrequently analysed relative healthy food access (RHFA), a measure that is more representative of food purchasing and consumption behaviours than absolute outlet density.This research applies a Bayesian hierarchical model to analyse the spatio-temporal patterns of RHFA in the Region of Waterloo, Canada, from 2011 to 2014 at the small-area level. RHFA is calculated as the proportion of healthy food outlets  within 4-km from each small-area. This model measures spatial autocorrelation of RHFA, temporal trend of RHFA for the study region, and spatio-temporal trends of RHFA for small-areas.For the study region, a significant decreasing trend in RHFA is observed (-0.024), suggesting that food swamps have become more prevalent during the study period. For small-areas, significant decreasing temporal trends in RHFA were observed for all small-areas. Specific small-areas located in south Waterloo, north Kitchener, and southeast Cambridge exhibited the steepest decreasing spatio-temporal trends and are classified as spatio-temporal food swamps.This research demonstrates a Bayesian spatio-temporal modelling approach to analyse RHFA at the small-area scale. Results suggest that food swamps are more prevalent than food deserts in the Region of Waterloo. Analysing spatio-temporal trends of RHFA improves understanding of local food environment, highlighting specific small-areas where policies should be targeted to increase RHFA and reduce risk factors of adverse health outcomes such as obesity. Past research has demonstrated that the food environment is an important factor in health outcomes. Several studies have shown that residents with higher access to healthy foods have healthier diets , lower rAcknowledging the role of healthy food access in shaping food consumption and related health outcomes, policymakers have prioritized increasing healthy food access. In Canada, for example, the Ontario Professional Planners Institute has issued a call for action on planning for healthy food systems and engaging planners with food relevant issues . FurtherVarious measures have been developed for assessing the food environment and have been summarized –11 and cThe absolute and relative measures capture different aspects of the food environment . AbsolutCapturing both healthy and unhealthy food outlets in one measure allows for a more comprehensive analysis of different dimensions of the food environment  and enabPrevious research has indicated that changes in the numbers and types of retail food outlets may lead to changes in food purchasing and consumption behaviours , howeverTemporal food access can be considered from supply (retail) and demand (consumer) sides. From the supply side, variations in temporal food access occur across years , seasons , and weekdays  –27. For From the demand side, temporal food access is generally measured for individuals because it is largely determined by consumer time availability . In thiThis study analyses annual spatio-temporal variations of RHFA at the small-area scale for the Region of Waterloo, from 2011 to 2014, complementing past research that analyses only spatial variations and absolute healthy food access. RHFA at a small temporal scale  merits attention given that changes in the number and type of food outlets are slow and it probably takes a long time for the food environment to manifest its health effects . SpecifiThe Region of Waterloo, Ontario, Canada, is composed of three cities, Kitchener, Waterloo, and Cambridge, and four rural townships. It is located approximately 1 h west of Toronto, Canada’s largest city. For this study, rural townships were excluded from the analysis because retail food outlets are primarily located in urban areas. City boundaries were collected from the Region of Waterloo .2, ranging between 2/km2 in a predominantly industrial DA and 16,025/km2 in a DA with many apartment buildings. Population data and geographic shapefiles were obtained from Statistics Canada  assumes that the count of healthy food outlets within 4 km of DA pij is decomposed into parameters measuring purely spatial variation, purely temporal variation, and spatio-temporal interaction at the second level [Model (2)].Using a logit link function, α (average RHFA for the study region), ui (unstructured random effects), and si . Random effects (uiand si) deal with overdispersion (greater variance than expected based on a probability distribution) which occurs when modelling count data at the areal level. Sources of overdispersion in small-area studies include intra-area heterogeneity, which may be due to the presence of missing covariates or measurement errors in covariates . Specifically, i) of δi being less than zero measures the strength that the local trend negatively departs from the regional trend (γ)  and is based on calculated RHFA in a given DA and the average of calculated RHFA’s in adjacent areas [via the spatial random effects in Model (2)]. In this case, estimated RHFA helps to account for the realistic assumption that people could travel beyond DA or buffering zone boundaries to procure food; therefore, the RHFA value is smoothed , study region characteristics , and characteristics of the local population . The buffering size could also be altered accordingly based on food outlet types, which may be linked to the behaviours underlying travel patterns to visit specific healthy or unhealthy stores (and subtypes among them). Second, we applied the most common scheme for classifying healthy and unhealthy food outlets. The NEWPATH survey, from which food outlets were classified, measured in-store characteristics of food outlets and indicated that all non-supermarket and non-superstore outlets , with the exception of specialty stores , should be categorized as unhealthy. Moreover, supermarkets/superstores are also sources of unhealthy food options. We completed additional analyses following in-store classification and counting grocery stores as both healthy and unhealthy, but results of regional and local RHFA trends  were similar. Additional RHFA measures based on consumer nutrition environment, for instance, shelf space devoted to healthy foods divided by the total shelf spaces devoted to healthy and unhealthy foods in accessible food outlets , should Future research should further apply this Bayesian approach in different contexts  to study spatio-temporal variations of the food environment accounting for transportation networks. Of particular interest is the association between changes in public transit and changes to RHFA. Future research could also analyse the association between spatio-temporal patterns of the food environment and health or socio-economic data, when available. Compared to spatial studies that analyse one time period, spatio-temporal analysis clarifies how changes in the food environment influence health outcomes , and how the food environment may be changing in tandem with increasing or decreasing socioeconomic status.This paper explores the spatio-temporal patterns of RHFA in the Region of Waterloo over 4 years, using a Bayesian spatio-temporal modelling approach. This method quantifies regional temporal trend and local spatio-temporal trends of RHFA, which are not available from traditional spatial or descriptive analyses. In particular, this study adds to the literature for investigating relative food access at a small temporal scale .Results of our study are consistent with previous findings in the Canadian context that food swamps are more prevalent than food deserts. While food deserts should be prioritized, food swamps  should not be overlooked by public health practitioners and policy-makers. In general, food swamps have become more prevalent during the study period, given that RHFA has decreased at the regional level and all DAs (most are food swamps in the starting year 2011) at the local level show significant decreasing trend of RHFA. Areas located at south Waterloo, north Kitchener, as well as southeast Cambridge have the steepest RHFA decreasing gradient Fig.  thus are


Mast cells (MCs) have been shown to release their nuclear DNA and subsequently form mast cell extracellular traps (MCETs) comparable to neutrophil extracellular traps, which are able to entrap and kill various microbes. The formation of extracellular traps is associated with the disruption of the nuclear membrane, which leads to mixing of nuclear compounds with granule components and causes the death of the cell, a process called ETosis. The question arises why do MCs release MCETs although they are very well known as multifunctional long-living sentinel cells? MCs are known to play a role during allergic reactions and certain parasitic infections. Nonetheless, they are also critical components of the early host innate immune response to bacterial and fungal pathogens: MCs contribute to the initiation of the early immune response by recruiting effector cells including neutrophils and macrophages by locally releasing inflammatory mediators, such as TNF-α. Moreover, various studies demonstrate that MCs are able to eliminate microbes through intracellular as well as extracellular antimicrobial mechanisms, including MCET formation similar to that of professional phagocytes. Recent literature leads to the suggestion that MCET formation is not the result of a passive release of DNA and granule proteins during cellular disintegration, but rather an active and controlled process in response to specific stimulation, which contributes to the innate host defense. This review will discuss the different known aspects of the antimicrobial activities of MCs with a special focus on MCETs, and their role and relevance during infection and inflammation. Mast cells (MCs) have become famous for their role in type I hypersensitivity reactions. Better known as IgE-mediated allergic reactions, this MC response is induced after multivalent cross-linkage of antigens with antigen-specific IgE, which then bind to high-affinity IgE receptors (Fc∈RI) on the cellular surface –3. For aMCs derive from hematopoietic progenitor cells and circulate in the blood until they reach their destination in the tissues, where they differentiate under the influence of growth factors and cytokines that ultimately determine their mature, long-living phenotype . DiffereStaphylococcus (S.) aureus trigger TNF-α and IL-8 release from cord-blood-derived MCs in a time-dependent manner. Nakamura et al.  activation by the Gram-positive and -negative bacteria Listeria (L.) monocytogenes and Salmonella (S.) typhimurium, respectively, MCs elicit proinflammatory, but not type I IFN responses. In contrast, the response to viral infection is type I IFN dependent. Type I IFN signaling attenuates mast cell-dependent neutrophil recruitment that is required for bacterial clearance. Thus, the fact that MCs are equipped with the ability to release type I IFNs, but mount proinflammatory responses only upon TLR activation by bacteria, illustrates how MCs adjust their responses for optimal antibacterial and antiviral host defenses  and S. aureus infection by direct bacterial killing. Moreover, the presence of cathelicidin in MCs may act to facilitate recruitment of neutrophils, thus indirectly providing enhanced protection against infection.MCs are highly efficient effector cells that do not only release inflammatory mediators but also different antimicrobial peptides (AMPs), such as cathelicidins . These pnfection . The autS. aureus, Streptococcus faecium, Klebsiella pneumoniae, Citrobacter freundii, and Escherichia (E.) coli by scanning and transmission electron microscopy and by quantifying bacterial survival in the presence of MCs compared with human umbilical vein endothelial cells (HUVECs)  . Howevercroscopy .S. aureus without subsequent intracellular killing: the S. aureus strain SH100 was shown to be internalized in vitro by murine and human MCs  and by skin MCs during in vivo infections. MCs are utilized as a vehicle and a safe intracellular niche providing protection against other immune cells. Since the internalization efficiency depends on bacterial viability, the authors hypothesized that S. aureus may actively induce its uptake into MCs  pyogenes, they still can efficiently inhibit the growth by the release of MCETs. MCETs were found to support the extracellular killing of clumped bacteria that were not efficiently phagocytosed. Those MCET fibers are composed of DNA, histones, the MCs-specific protease tryptase, and AMPs such as the cathelicidin AMP LL-37 (Similar to the formation of neutrophil extracellular traps (NETs), MCs have also been shown to release their nuclear DNA and subsequently form antimicrobial MCETs that resemble extracellular dendritic extensions . InteresMP LL-37 .2O2-producing enzyme glucose oxidase, which is another indicator for the ROS-dependent MCET formation  faecalis, and the sensitivity of this pathogen to the antimicrobial effect of cathelicidin LL-37 indicated a potential major role for this AMP in the antimicrobial activity of MCs against E. faecalis  albicans. MCET release appears to be a mechanism of immune defense present in the MC toolbox against fungal pathogens, as both primary cells and HMC-1 release MCETs upon C. albicans stimulation. Interestingly, in contrast to bacteria . Oxygen stress or hypoxia occurs in tissues during an infection, mostly, due to overconsumption of oxygen by pathogens and recruited immune cells. Importantly, HIF-1α activation or stabilization has been shown to support myeloid cell production of defense factors and improved bactericidal capacity of immune cells , 44. In ding MCs –47. Howeding MCs . In goodding MCs .Although, MCETs and NETs share common characteristics, there are several cell type-specific differences. The formation of MCETs can be greatly increased after stimulation of MCs with PMA prior to infection , similarin vivo in a murine S. aureus skin infection model (E. coli and S. aureus (In neutrophils, it has been additionally demonstrated that these cells can release ETs in response to infection, while remaining in a viable status, confirmed on model , 55. For. aureus , 57 and . aureus . HoweverIn summary, the actual literature assumes that MCET formation is not the result of passive release of DNA and granular proteins during cellular disintegration, but rather an active and controlled process in response to specific stimulation. The extracellular structures act antimicrobially through a combination of direct killing of the entrapped pathogen or by its physical immobilization that enables the recruited effector cells to eliminate the pathogen. Both aspects may have a significant impact on the disease outcome. The fact that aggregated NETs limit inflammation by degrading cytokines and chemokines has already been shown . If MCETAll authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Despite intense research, few treatments are available for most neurological disorders. Demyelinating diseases are no exception. This is perhaps not surprising considering the multifactorial nature of these diseases, which involve complex interactions between immune system cells, glia and neurons. In the case of multiple sclerosis, for example, there is no unanimity among researchers about the cause or even which system or cell type could be ground zero. This situation precludes the development and strategic application of mechanism-based therapies. We will discuss how computational modeling applied to questions at different biological levels can help link together disparate observations and decipher complex mechanisms whose solutions are not amenable to simple reductionism. By making testable predictions and revealing critical gaps in existing knowledge, such models can help direct research and will provide a rigorous framework in which to integrate new data as they are collected. Nowadays, there is no shortage of data; the challenge is to make sense of it all. In that respect, computational modeling is an invaluable tool that could, ultimately, transform how we understand, diagnose, and treat demyelinating diseases. The nervous systems of vertebrates are often divided into grey and white matter based on their appearance and corresponding functional roles. While the grey matter consists largely of cell bodies and dendrites, white matter contains mostly axons and gets its name from the lipid membrane sheets called myelin that are wound tightly around those axons . Myelin The electrical insulation provided by the myelin sheets improves axonal function by increasing both the energy efficiency and conduction velocity of action potentials (APs). These two functions may have switched their relative importance during evolution . Myelin The myelin wrapping is interrupted by regularly spaced, un-myelinated stretches known as the nodes of Ranvier. Myelin speeds up conduction by restricting transmembrane charge flow through ion channels located within the nodes. Within the so-called internodes, current flows down the axon with little of it passing across the insulated cell membrane. The AP is regenerated at each node where the density of voltage-gated sodium and potassium channels is very high. This process is called “saltatory conduction” since the AP seems to jump from node to node. Disruptions in this rapid-fire communications system can be associated with an array of nervous systems dysfunctions .In several regards, axons appear to operate at physical limits. One interesting example is that the size of axons seems to be constrained by the thermal noise intrinsic to ion channel proteins; any axon thinner than 0.1 μm would be useless for information transfer due to its high noise levels . Intriguvs. sensory, visual vs. tactile) but not how it will be affected. This highlights the importance of assessing function (in addition to structure) and how it changes following demyelination. After introducing demyelinating diseases, we will discuss how the clinical manifestations of those diseases reflect diverse pathological changes in axon function. We will argue that understanding those changes and fully capitalizing on that understanding for diagnostic and therapeutic purposes can benefit enormously from computational modeling.Demyelination sets in motion functional changes that are important for clinical features but which are not readily explained by immunological or radiological changes. Location of a plaque predicts what system will be affected  and gain-of-function  . Early dIn human diagnostic studies of visual, sensory or motor evoked potentials , only latency or conduction velocity can be measured accurately (with approximately 30%–40% variations between different laboratories). But these measures give little clue about underlying mechanisms that involve conduction slowing or block, or morphological or functional factors such as branching, demyelination, remyelination, axonal tapering , attenuation or regrowth, temperature-related changes in conduction, or malpolarization (hyper or hypo). Nonetheless, the type of demyelinating lesion can yield clues regarding etiology and therefore guide treatment; for example, genetic factors seem to more strongly correlate to internodal disease processes and immunological dysfunctions cause paranodal abnormalities .A number of tests are routinely used to assess the neural function. In electroneurography, a brief electrical stimulus is applied to a peripheral nerve at an anatomically predefined position in order to measure the latency and amplitude of the compound action potential at another location along the nerve. Results need to be interpreted in combination with clinical findings and tests  but, importantly, different diseases exhibit different patterns of electroneurographic changes. This is important not only for diagnostic purposes but can also point to specific pathological changes in axon function which could, in turn, help guide the choice of therapy . Using threshold tracking, excitability has been measured in humans for the several peripheral demyelinating diseases including Charcot-Marie-Tooth Disease Type 1A (CMT1A), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS) and multifocal motor neuropathy (MMN) ,23,24,25Progression from relapsing-remitting MS (RRMS) to the secondary progressive MS (SPMS) is associated with greater involvement of grey matter pathology, although axonal/grey matter involvement can be observed already in early disease stages ,33,34,35The main interventions for MS involve modulation of the immune response with, for example, methly-prednisolone, interferon betas, glatiramer acetate or fingolimod, or by preventing inflammatory cells from crossing the BBB ). Very recently the first two oral agents (fumarate and teriflunomide) as well as the anti-CD52 directed antibody Natalizumab where approved for treatment of RRMS, which can be successfully treated by first-line therapies like interferons, glatiramer acetate, or fingolimod, or by second-line therapies, but progressive forms  still represent an unmet biomedical need . Anti-ne+ channels not only reduces positive symptoms, it may also be neuroprotective  [Disease-modifying drugs are critical for stopping or at least attenuating the demyelination process, but so too is it critical to manage the symptoms arising from whatever demyelination has already occurred. Ion channel modulation is increasingly promising with the advent of new ion channel blockers such as Ampyra (K-channel blockade) ,41. Potatotoxic)   changes in axon excitability, in order to appreciate how neurological function is altered. Those changes are not simple and direct consequences of demyelination but, instead, suggest that axonal physiology itself changes in response to demyelination. Some of those changes are adaptive whereas others are maladaptive, or perhaps adaptive changes can become maladaptive as the situation (myelination status) evolves. If changes in axonal physiology dictate the manifestation of various symptoms, then symptom management will largely fall on treatments that aim to manipulate axon physiology. Strategically developing such treatments require a deep, mechanistic understanding of axonal excitability and its regulation.Axons are profoundly affected by demyelination. Axon morphology becomes irregular or swollen, often with a beaded appearance. Focal accumulation of proteins  is also observed. In chronic active plaques, axonal loss of 20%–80% is apparent within peri-plaque white matter and normal distant white matter . In earlRemyelination is often observed as shadow plaques formed by the recruitment of undifferentiated oligodendrocyte precursors that migrate to and surround the lesions enabling thin layers of remyelination . This pr+ cells [During the disease process, autoreactive lymphocytes and macrophages can cross the blood brain barrier and accumulate in the brain and spinal cord . Regulat+ cells . Release+ cells . This oc+ cells .el al. [Lucchinetti el al.  proposedel al. , whereasBarnett and Prineas  analyzed+, K+ and Ca2+ ions, the dispersal of their corresponding ion channels, a decrease in the efficiency of AP conduction and a resulting metabolic crisis , the system may be less energy efficient. Losing the energy savings afforded by saltatory conduction induces compensatory mitochondrial energy production that can result in oxidative damage and neurodegeneration [The disruption of energy balance in a neuron could also profoundly impact neuron well-being . Indeed,neration ,60,61.Keeping track of this long list of neurobiological changes, understanding the inter-relationships between those changes, and ultimately linking those changes with clinical manifestations and applying effective treatment is no easy task. To this end, computational modeling is an invaluable tool. Simulations not only serve to organize what information is already known, they also identify crucial gaps in knowledge. The judicious use of computational modeling can therefore enable more comprehensive understanding and facilitate the more effective application of that understanding, as discussed below.Especially when paired with traditional experiments, computational modeling is indispensable for making sense of inconsistent data and complex mechanisms. These benefits are exemplified by the application of simulations in other fields, such as epilepsy . Here weThe computational modeling of axons has evolved taxonomically, from squid to mammalian tissues with a corresponding increase in sophistication. The Hodgkin and Huxley (HH) model, which provided the first thorough explanation of AP generation, was derived from experiments in unmyelinated giant axons of squid ,64, but The spatial and biophysical heterogeneity conferred by the addition of myelin, and the consequent formation of nodes and internodal regions, represents a significant increase in axon complexity. The first computational model of a myelinated axon was a one-dimensional model that collapsed the myelin sheath into the underlying passive axolemma, used a uniform spatial step size to form the discrete approximation used in the numerical solution and employed a HH characterization of the nodal membrane . GoldmanNewer models have also improved upon previous simplifications including the anatomical complexity of the node of Ranvier, the distribution of ionic channels in the axon beneath the myelin sheath, the different electrical properties of the myelin sheath and the axolemma, and accommodation of possible current flow within the periaxonal space ,80,81,82Newer models have also considered the non-uniform distribution of ion channels throughout the axon ,88,89,90All of the aforementioned models focus on simulating the change in axon membrane potential but one does not necessarily have experimental access to that variable, which of course complicates efforts to compare simulation and experimental data. Indeed, since extracellular recordings are the primary source of electrophysiological data from human subjects, the mathematical description of the extracellular field potential is of great interest clinically. Mathematical evaluations based on Laplace equations and Fourier transforms are used for calculating these potentials .+ channels in the axolemma of the single multi-segmented internode and treats the remainder as purely passive.Beyond modeling normal axonal function, models can be used to explore particular mechanisms of axonal dysfunction especially when combined with experimental results that might better pinpoint mechanisms . For exaet al. [+ and K+ channels in both afterpotentials and proposed that differences in the AP shape, strength-duration relationship, and the recovery cycle of motor and sensory nerve fibers can be attributed to kinetic differences in nodal Na+ conductances. Richardson et al. [Building on this work, with careful attention to anatomical and electrophysiological details, McIntyre et al.  addressen et al.  also fouThe temperature sensitivity of demyelination effects has also been investigated computationally. Zlochiver  modeled As mentioned above, distinct changes in axon function are likely to manifest certain gain- or loss-of-function symptoms. If one could reproduce those changes in a computational model, the necessary parameter changes needed to convert the model between normal and abnormal operation could be used to predict the underlying pathology. Ideally this can lead to specific experiments in which the suspect ion channel, for example, is directly manipulated to see if its acute alteration is sufficient to reproduce or reverse certain pathological changes. Recent studies from the Prescott lab illustrate this process ,102. ThiIn auditory nerve experiments, Tagoe and colleagues  demonstrWith a myelinated axon multi-layered model Stephanova and colleagues have had on-going success identifying likely anatomical and physiological deficiencies underlying various symptoms and conditions related to demyelination by making comparisons to the threshold tracking measurements from patients including latencies, refractoriness (the increase in threshold current during the relative refractory period), refractory period, supernormality, and threshold electrotonus values including stimulus-response measures such as current-threshold relationships . For exa+ and leak K+ currents increase with temperature, and the accommodation to long-lasting hyperpolarization is greater than to depolarization.Mild systematic and severe focal demyelination correspond to hereditary (CMT1A) and acquired  neuropathies . It was + to leak K+ conductance, g(Na)/g(L), acted as a four-way switch controlling excitability patterns that included failure of AP propagation, normal AP propagation, AD, and spontaneous spiking.Given the temporal dissociation between the manifestation of symptoms and the rates of demyelination and remyelination, homeostatic processes undoubtedly occur within axons, which include the redistribution of ion channels in demyelinated plaques ,107. ButFurther studies with this model suggested the potential for competition or cooperation between different regions of the same neuron . Coopera+ accumulation caused the destruction of the limit-cycle attractor state [Furthermore, these simplified models enabled application of mathematical tools to examine the nonlinear mechanisms by which AD is initiated and terminated ,58,59. Bor state ,109.et al. [Studies mentioned above highlight the importance of ion concentration changes but each of them only considered those changes at a relatively course scale. By comparison, the study by Lorpreore et al.  tackled 2+ dynamics, death complex formation, apoptotic factor release, and inflammatory responses together with three different states: normal, MS disease and treatment. At the atomic-level, a computational model of myelin basic protein (MBP) structure was carried-out because post-translational modifications of MBP may contribute to demyelination in MS [The above example highlights the point that models can simulate more than ion channels and membrane potential. Indeed, models can and must dig deeper into biophysical mechanisms like electro-diffusion and into signaling pathways that ultimately serve to regulate ion channel function and expression. A promising method called Biochemical Systems Theory (BST) may be useful in the future for pre-screening the effects of drugs at the systemic level. Broome and Coleman  demonstron in MS . It is iModels of Immune Factors. While there are numerous computational models of the immune system , those rModels of Mitochondrial Dysfunction. As mentioned above, myelin enables more energy efficient AP conduction along the axon. The increased energy demands placed on the demyelinated axon represents yet another challenge to the afflicted neuron. Beyond the loss of saltatory conduction, there is mounting evidence of a critical role for astrocytes and oligodendrocytes in supplying energy to neurons and this process has also been the subject of computational modeling .2+ signaling within mitochondria, disrupted proton gradients or electron chain, reduction-oxidation imbalance as well as the consequences of reduced ATP availability, locally and globally. Multi-scale models of heart, for example, have been used to link altered mitochrondrial Ca2+ signaling to arrhythmia [There are many ways mitochondrial function can go awry and the compensatory pathways are equally complicated ,60,61. FWithin the field of demyelinating diseases, modeling efforts have traditionally focused on axon models aimed at explaining various aspects of excitability. But as outlined above, those models have undergone tremendous evolution in complexity. In the process, models at different biological scales have begun to coalesce. For instance, models have now begun to address the regulation of ion concentrations and the consequences thereof for slow excitability changes, energy consumption, and toxicity. A computational approach will be necessary for integrating parallel and multifactorial etiologies associated with cognitive decline such as immune system signaling, energy metabolism, grey and white matter interactions, and genetic networks . These cThe normal physiological function of the CNS or PNS relies on a highly regulated interplay of neurons, glia, vasculature and immune cells. This process encompasses and integrates numerous cellular and signaling components that produce a dynamical, computational whole. When any part goes awry, the entire system is forced to compensate. Even when compensation manages to rescue the most obvious consequences of demyelination, certain processes may not return to a completely normal state, which can lead to problems on longer time scales. The resulting symptoms are a confusing mixture of direct and compensatory changes that continuously evolve. The overall complexity has proven to be intractable to efficient experimental dissection. The application of computational modeling techniques represents an invaluable approach to help break the impasse and engender a new era of understanding and discovery.