In recent years, lymphatic vessels have received increasing attention and our understanding of their development and functional roles in health and diseases has greatly improved. It has become clear that lymphatic vessels are critically involved in acute and chronic inflammation and its resolution by supporting the transport of immune cells, fluid, and macromolecules. As we will discuss in this review, the involvement of lymphatic vessels has been uncovered in atherosclerosis, a chronic inflammatory disease of medium- and large-sized arteries causing deadly cardiovascular complications worldwide. The progression of atherosclerosis is associated with morphological and functional alterations in lymphatic vessels draining the diseased artery. These defects in the lymphatic vasculature impact the inflammatory response in atherosclerosis by affecting immune cell trafficking, lymphoid neogenesis, and clearance of macromolecules in the arterial wall. Based on these new findings, we propose that targeting lymphatic function could be considered in conjunction with existing drugs as a treatment option for atherosclerosis. The lymphatic system is part of the human circulatory system and the immune system. It is composed of an extensive branched network of lymphatic vessels that are connected to lymph nodes (LNs). Unlike the closed blood circulatory system, lymphatic vasculature functions unidirectionally. Extravasated fluid called lymph, macromolecules, and leukocytes enter the blind-ended initial lymphatic vessels  and are transported towards the pre-collecting vessels that converge to the larger collecting vessels, pass through chains of LNs, before returning to the blood circulation through the thoracic duct ,2. LymphInitial lymphatic vessels, the absorptive part of the lymphatic vessels, are lined by a single layer of oak leaf-shaped lymphatic endothelial cells (LECs) which are not covered by pericytes or smooth muscle cells (SMCs) . InitialLymphatic vessels have been shown to play essential roles in immune surveillance by, for example, supporting antigen and immune cell transport, dietary fat absorption in the gastrointestinal tract, and maintenance of tissue fluid balance ,9,10,11.In this review, we focus on the functional roles of lymphatic vessels draining medium- and large-sized arteries in atherosclerosis, a chronic inflammatory disease of arteries. We first provide a brief overview of atherosclerosis pathogenesis and then discuss the normal distribution of lymphatic vessels in arteries and their morphological and functional alterations in response to atherosclerosis. Finally, we review recent studies revealing the important contribution of lymphatic vasculature in atherosclerosis progression through the control of immune cell trafficking and lipids.Atherosclerosis is a chronic inflammatory disease of medium-sized to large arteries, and the associated cardiovascular complications including myocardial infarction and stroke represent leading causes of death worldwide. The arterial wall consists of three concentric tissue layers namely: the tunica intima (inner layer) lined by a monolayer of endothelial cells, the tunica media composed of SMCs, elastin, and collagen, and the adventitia, the outer layer formed of fibroblasts, collagen, nerve endings, immune cells, and blood and lymphatic vessels . AtherosAccording to the traditional inside-out theory of atherosclerotic lesion development, the disease is initiated from the luminal side of the artery and involves several orchestrated mechanisms triggered by endothelial activation and subendothelial retention of plasma apolipoprotein in the intima, including monocyte adhesion on the luminal surface, endothelial dysfunction, leukocyte accumulation in the subendothelial space, and subsequent inflammatory responses ,15. The Taken together, infiltration and accumulation of plasma lipoproteins and pro-inflammatory leukocytes in the intima represent the driving force of atherosclerotic lesion initiation, progression, and clinical complications. It is thus not surprising that targeting lipoprotein and inflammatory pathways has been the main approaches in managing atherosclerosis. Statins, 3-Hydroxy-methylglutaryl coenzyme A (HMG Co-A) reductase inhibitors, have been the standard treatment for atherosclerosis-associated diseases . More reAlthough atherosclerotic lesions predominantly localize in the intima, atherosclerosis affects the structure and the functions of all three layers of the arterial wall. Notably, emerging evidence recognizes the adventitia as an active participant in the development and progression of atherosclerotic lesions which have led to a new paradigm of an outside-in theory, in which vascular inflammation is initiated in the adventitia and progressed inwards toward the intima ,36,37. A−/−Apoe), the number of macrophages, T cells, and microvessels was significantly higher in the adventitia of the abdominal aorta and these numbers positively correlated with plaque formation and collagen deposition [−/−Apoe mice employing single cell RNA sequencing to further characterize the cellular landscape of the adventitia at a single-cell resolution confirmed that this tissue harbors inflammatory cells and uncovered subpopulations of lymphocytes, macrophages, and several rare populations such as lymphatic endothelial cells and neuronal cells [−/−Apoe mice, whereas clusters containing T and B cells as well as lymphoid-like structures emerge in non-resolving inflammation in aortic adventitia of older mice, preferentially in the abdominal aorta [−/−Apoe mice [It is increasingly recognized that intimal inflammation is paralleled over time by a distinct inflammatory reaction to adjacent adventitia . Variousposition . A recenal cells . Interesal aorta . T cellsal aorta  and Apoe−/− mice ,47.The progressive organization of cellular infiltrates within non-lymphoid organs into functional ectopic germinal centers or also known as tertiary lymphoid organs (TLOs) is a process called lymphoid neogenesis . Lymphoi−/−Apoe mice. Indeed, TLOs are rarely observed in the adventitia of the aortic arch and its branches, although atherosclerosis is the most advanced. Although locally released soluble mediators such as lymphoid chemokines and lymphotoxin have been involved in the formation of adventitial TLOs in atherosclerotic mice [While adventitial TLOs are associated with atherosclerotic lesions in the intima, suggesting a crosstalk between the intimal lesions and adventitial TLOs via the media, the formation of advanced plaques is not sufficient to promote the formation of adventitial TLOs in tic mice , the tritic mice ,58.−/−Apoe mice, revealed that atherosclerotic lesion formation is associated with neovascularization of the vasa vasorum [Adventitial small blood vessels also named vasa vasorum are small arteries reaching the vascular wall either from the abluminal surface (vasa vasorum externa) or from the luminal surfaces (vasa vasorum interna) and branching out to the outer media . Venous  vasorum ,72 and t vasorum . Notably vasorum . In huma−/−Apoe mice not only reduced atherosclerotic lesion neovascularization, but also lesion growth [The study by Moreno et al. in the human aorta showed that neovascularization is augmented in human atherosclerotic plaques with inflammation, intraplaque hemorrhage, thin fibrous cap, and in ruptured plaques . This fin growth . Infiltrn growth . These dn growth . Since pn growth , pro-athn growth .In conclusion, during the development of atherosclerosis, vasa vasorum from the tunica adventitia may penetrate the thickened media to provide nutrition and oxygenation. Moreover, neovascularization of vasa vasorum together with the endothelial dysfunction of these nutritive vessels may significantly contribute to atherosclerosis by supporting the infiltration of inflammatory cells in the intima and the adventitia.The existence of lymphatics in the arterial wall was studied by G. Hoggan and colleagues more than one hundred years ago when specific lymphatic markers were not available . When HoAs most mechanistic insights depend on in vivo studies using smaller mouse model, it is critical to know the exact topography of aortic lymphatic vessels in a mouse model. Using histological and imaging analysis, characterization and topographic distribution of the lymphatic vessel network in the normal mouse aorta were recently illustrated . Blind-eOverall, an irregular plexus of lymphatic vessels was consistently found in the adventitia and peri-adventitial area, but not in the arterial intima and media, showing a conserved presence and anatomical location in both small and large mammals including humans.Lymphatic vessels are highly dynamic structures that intimately interact with their surrounding tissue microenvironment. Recent research advances in lymphatic biology have revealed that various internal and external stimuli can affect and modulate the growth, structure, and function of lymphatic vessels. Lymphangiogenesis and enlarged lymphatic vessels are associated with chronic inflammation, such as psoriasis  or chron−/−Apoe mice [−/−Apoe and −/−Ldlr mice [−/−Apoe mice [156 by a serine residue), but reduced through VEGFR-3 inhibition, suggesting that hypercholesterolemia may trigger lymphangiogenesis in −/−Apoe mice through a mechanism dependent, in part, on VEGF-C [Similar expansion of adventitial lymphatic network was also observed in hypercholesterolemic −/− mice ,92. The −/− mice . Adventi−/− mice . Furthern VEGF-C . Howevern VEGF-C .It is worth mentioning that lymphangiogenesis and angiogenesis co-exist during atherosclerosis progression and regression. Both vasculature networks are intimately intertwined, even though there is no connection between the two systems. The kinetic of angiogenesis also differs from lymphangiogenesis, with angiogenesis preceding and surpassing lymphangiogenesis during disease progression, which results in the imbalance of the ratio of blood to lymphatic vessel numbers .−/−Apoe mice, it was apparent in the hypertrophic skin draining LNs [Furthermore, a hypertrophy in aorta draining iliac LNs was constantly observed in hypercholesterolemic mice . Changesning LNs . MicroscAfter reviewing the alterations of the lymphatic vasculature observed during atherosclerosis , we willAn artery is formed by three layers, namely: the tunica intima (inner layer) lined by a monolayer of endothelial cells, the tunica media composed of SMCs, elastin and collagen, and the adventitia, the outer layer formed of fibroblast, collagen, nerve endings, immune cells, and blood and lymphatic vessels. Periadventitial adipose tissue is also distributed around arteries. Atherosclerosis is characterized by the accumulation of plaques comprising cholesterol, inflammatory soluble factors, immune cells in the intima, and inflammation in adventitia that can be associated with the formation of adventitia tertiary lymphoid organ (ATLOs) when the disease is more advanced. Similar to lymphatic vessels in other tissues, initial lymphatic vessels in the adventitia converge into larger collecting vessels exhibiting SMC and valves which transport lymph into draining lymph nodes. Studies have shown that atherosclerosis is associated with angiogenesis and lymphangiogenesis. Notably, in the abdominal aorta, initial lymphatic vessels appeared more dilated and lymphatic drainage of macromolecules from the adventitia into the draining lymph node is severely compromised during atherosclerosis. Alterations in collecting vessels such as poor SMC coverage, abnormal valve, and accumulation of extracellular matrix, such as collagen, may account for the impaired lymphatic drainage. These functional defects in adventitial lymphatic vessels may participate in the retention of immune cells, inflammatory cytokines, and lipids in the arterial wall, which in turn favor the progression of atherosclerotic plaque, inflammation, and ATLOs. Restoring lymphatic drainage in the arterial wall may thus be a promising strategy to treat atherosclerosis.Lymphatic vessels, together with their interconnected LNs, are essential for adaptive immune responses and the clearance of inflammatory cytokines, antigens, and cells that may cause harm if not removed from tissues. Through integration with afferent lymphatic vessels, LNs receive antigens, antigen presenting cells such as dendritic cells, and macrophages and lymphocytes for the induction of immune responses . Whereas−/−Apoe mice with a viral encoding apoE which lowers plasma cholesterol and raises high-density lipoprotein, the reduction in plaque macrophage content did not result from migratory egress and did not require CCR7 [−/−Apoe mice, unlike in human arteries burdened with atherosclerotic plaques where phagocytes can be observed in the media at a greater percentage than in normal arteries [−/−Apoe mice with negligible atherosclerotic plaques promotes the intimal accumulation of macrophages, collagen, and SMCs as well as macrophage and dendritic cell infiltration in adventitia layer [−/−Apoe is significantly compromised compared to wild-type mice [Based on the recent literature, several processes have been proposed to account for the accumulation of monocyte-derived cells in atherosclerotic plaque, namely: increased monocyte recruitment into the plaques, alterations in proliferation and/or survival of monocyte-derived cells in plaques, and inability of macrophage and dendritic cells to emigrate from plaques to LNs via adventitial lymphatic vessels ,103. Macire CCR7 . Althougire CCR7 . Indeed,arteries . Notablyarteries . Althougarteries , promotiia layer . Moreoveia layer . Altogetype mice , this su−/−Apoe mice a semi-constrictive collar to induce atherosclerosis after LN and lymphatic dissection [−/−Apoe mice [−/−Apoe mice further support the functional role of lymphatic drainage in limiting aortic accumulation of T lymphocytes in adventitia and in turn the inflammatory response in atherosclerosis. Efferent lymphatic emigration of T lymphocytes from LNs may also be compromised during atherosclerosis. Indeed, we showed that T cell egress from skin draining LNs in hypercholesterolemic −/−Apoe mice is severely impaired, which contributes to LN hypertrophy [−/−Apoe mice [In addition to the control of dendritic cell and macrophage trafficking, lymphatic vessels are also critical for T cell migration from tissue into LNs and T cell egress from LNs into efferent collecting lymphatic vessels. Among the chemokines expressed by lymphatic endothelium that attract T cells, CCL21 is essential for the migration of naïve, memory, and T regulatory T cells to LNs . On the ssection . Interesssection . Consist−/− mice . Moreoveertrophy . The imp−/− mice , and the−/− mice  and incr−/− mice ,118,119.−/−Apoe mice although atherosclerosis is the most advanced [−/−Apoe mice and promotes atherosclerotic plaque development and inflammation in the adventitia [In an excellent review on the mechanisms triggering lymphoid neogenesis during chronic inflammation, Thaunat O et al. proposed, based on their work in the field of chronic rejection of solid organ transplants, that lymphoid neogenesis is a default response of an immune system resulting from both a chronic local antigenic stimulation and a defect in the lymphatic drainage of the tissue harboring the targeted antigen . Accordiadvanced . Notablyadvanced . Moreoveventitia  (Figure Given the primary function of lymphatic vessels in draining fluid, protein, and lipid from peripheral tissues back to the blood circulation , clearanMost clinical and pre-clinical studies demonstrate that an increased lymphatic network is necessary for the resolution of inflammation. The fundamental roles that lymphatics plays in fluid clearance from the periphery make lymphangiogenesis seemingly obvious as a mechanism to alleviate locally inflamed environments by increasing lymph efflux. The lymph contains all transportable tissue macromolecules including cytokines, tissue fragments, hormones, and antigens and is carried along larger collecting lymphatics to the draining LNs. Obstruction of the lymphatics along this path exacerbates unfavorable accumulation of macromolecules, which subsequently leads to architectural tissue changes, oedema, and infiltration of immune cells in the peripheral tissues ,126. It Ldlr−/− mice leads to the attenuation of atherosclerosis [The link between adventitial lymphangiogenesis and atherosclerosis has been postulated for more than a few decades ago, but only a handful of studies investigated the direct association between lymphatic transport and atherosclerosis. Most studies extrapolate the findings obtained from the skin. For example, Milasan et al. concluded that improving dermal lymphatic vessels permeability by intradermal apolipoprotein A-I treatment and promoting contraction frequency by systemic administration of VEGF-C before the onset of atherosclerosis in clerosis ,127. The−/−Apoe mice when lymphangiogenesis is evident and atherosclerosis is well advanced. −/−Apoe aortic lymphatic exhibited more than 50% reduction in transporting macromolecule and was improved during atherosclerosis and inflammation regression in response to cholesterol-lowering drug treatment [−/−Apoe mice, hence highlighting the importance of measuring lymphatic functionality to determine the biological significance of lymphangiogenesis in inflammatory diseases. It might also be important to examine the structure of collecting vessels. Defective lymphatic transport in the skin of −/−Apoe mice is associated with decreased SMC coverage and abnormal valves [It is not surprising that assessment of the functionality of the lymphatic vessels in mouse aorta remains scarce. This is largely hampered by insufficient knowledge of peri-vascular lymphatic anatomy and absence of tools to directly inject molecular tracer in small animal like mouse. Inspired by reports published decades ago, our group developed a functional assay to quantitatively measure lymphatic transport in mouse aorta by injecting fluorescent tracer in the adventitia of the mouse abdominal aorta. Transport of macromolecule was taken up by adventitial lymphatics and travelled to iliac-, renal- draining lymph nodes, cisterna chili, and eventually into the thoracic duct in normal non-diseased aorta . To confreatment . VEGFC-1reatment . Expansil valves  in collel valves . It woull valves .In conclusion, there is little doubt of the essential functional roles of adventitial lymphatic vessels in atherosclerosis including the clearance of immune cells, inflammatory soluble factors, and lipoproteins from the inflamed arterial wall. Present in normal arteries, adventitial lymphatic vessels expand and change their morphology during atherosclerosis . HoweverImpaired lymphatic function and abnormal morphology seem to be characteristic features of diseases with a chronic inflammatory component. Therefore, restoring the defects of the lymphatic vasculature may be a promising therapeutic target for breaking the pro-inflammatory feedback loop in atherosclerosis and other chronic inflammatory diseases. For this reason, it is urgent to identify novel approaches to therapeutically enhance lymphatic function. Interestingly, specialized pro-resolving mediators also known as bioactive lipids have been shown to play a role in controlling the resolution of inflammation in relation to lymphatic vessels ,132. The


Equine herpesviruses (EHV) are a major health concern for domestic and wild equids and represent one of the most economically important disease agents of horses. Most known EHVs are transmitted directly between individuals as a result of direct exposure to exudates and aerosols. However, accumulating evidence suggests that environmental transmission may play a role including air, water, and fomites. Here, we reviewed studies on environmental stability and transmission of EHVs, which may influence viral dynamics and the use of environmental samples for monitoring EHV shedding. Different EHV species present variable clinical manifestations with mild to severe symptoms including respiratory disease, abortion, coital exanthema, neonatal death, immunosuppression, and myeloencephalopathy [Equine herpesviruses (EHV) infect wild and domestic equine species worldwide and represent one of the most economically consequential infections of domesticated horses . EHVs chDuring lytic replication, EHV transmission between hosts occurs frequently by direct contact with nasal mucus of infected animals. However, other sources of transmission include contact with EHV-1-positive aborted fetuses and direct transplacental infection from mother to foal often resulting in multigenerational disease transmission . Equine The majority of research on EHVs has been carried out in veterinary settings, with histology, immunology, and pathology performed on domestic or captive wild equids displaying symptoms associated with EHV. However, recent studies have begun to examine stability and infectivity of EHV in the environment in an effort to explain among-species transmission of EHVs, frequently under conditions where direct transmission of virus can be excluded ,8,9. ForIn this review, we comprehensively summarize research on potential environmental transmission of EHVs from water, air, fomites, and feces,  and how EHV transmission occurs primarily by direct contact between hosts, when EHV is shed in nasal discharge, which can develop into aerosol conveying infectious particles. There is evidence for aerosol transmission for both EHV-1 and -4 . It is pHerpesvirus-contaminated aerosols can travel many meters. Mars et al. 2000 showed that calves 4 m apart could become infected with bovine herpesvirus -1 (BHV-1) via aerosol transmission . They apA fomite can be described as an object that when contaminated with or exposed to a pathogen, can transfer the pathogen to a new host. The first studies examining the stability and infectivity of EHV-1 on different fomites were carried out in 1958 . DiffereAn outbreak of EHV between separated North and South wings of a veterinary hospital is an example of how fomites may be responsible for some EHV-1 transmission. In this case, animals were physically separated and the infections were believed to be the result of exposure of hosts to contaminated fomites. This included equines being exposed to contaminated hands and clothing of personnel , suggestEquus grevyi, E. zebra, and E. quagga). The toys were placed in outdoor enclosures for 7–24 h. Swabs were then taken from the surface of the toys and tested for EHV using a panherpes PCR screen. Four EHV species  were detected by PCR and subsequent sequencing. Host mitochondrial DNA was used to identify individual zebras that had interacted with the toy. The study showed that fomite-derived environmental DNA could be used to monitor the presence of pathogens such as EHV in the environment as well as identify individuals interacting with fomites.A further study has shown that fomites may prove useful for non-invasive monitoring of EHV in both captive and wild equine species . This stIn contrast to herpesviruses of other taxa such as ruminants that may cause enteric diseases , relativThe use of feces for EHV monitoring can facilitate sampling at larger scales and would be useful for screening active virus shedding in wild and domestic equids. In free-ranging and captive zebras, various EHV species were found to occur in feces, with EHV-5 and asinine herpesvirus 5 being most common . HoweverOther considerations need to be taken into account when using feces to monitor EHV shedding. Fecal samples should be collected fresh, as microbial metabolism may decrease the ability to detect viral DNA. The bolus surface typically contains more host intestinal cells than the interior, however, herpesviruses may originate from swallowed nasal exudate and may thus be well distributed within fecal matter; thus, subsamples should be mixed to optimize detection probability. DNA isolation kits for soil/feces typically do not include enzymatic digestion, however, host cells in feces or on swabs which contain herpesviruses may still be intact and thus require proteinase K digestion, which can typically be added to the protocol of non-tissue kits. In order to optimize detection efficiency, samples that likely contain considerable amounts of PCR inhibitors should be tested over a dilution gradient of the template DNA in order to determine the optimal ratio of target DNA and PCR inhibitors .In contrast to other non-invasive methods, fecal sampling has the considerable advantage that it is possible to attribute samples to individual hosts. Moreover, for wild equids, fecal sampling may be the method of choice for noninvasive monitoring of EHV shedding when invasive procedures are impractical or impossible. In addition, fecal sampling allows for matching cumulative endocrine profiles with EHV viremia, as increased glucocorticoid levels can be associated with reactivation of latent herpesvirus infections ,34. ObtaRegarding EHV-3, which is a venereal pathogen, urine rather than feces may be a useful substrate for tracing active viremia as this virus can be isolated from perineal and vaginal swabs of infected equids . Urine fFor some viruses, including influenza, adenoviruses, hepatitis A virus, coxsackievirus, echovirus, reovirus and rotaviruses, stability and transmission in water is a common characteristic ,37,38,39In the case of EHV-1, the virus was recently shown to remain stable and infectious in water under laboratory settings, indicating the potential to infect hosts by water . The stuTo address how EHV-1 behaves in environmental water, a further study investigated EHV presence in environmental water and sediment. Samples were collected from countries known to have large equine populations including Mongolia, Tanzania, and Namibia . Water aSeasonal water restriction leads to animal aggregation, which can be associated with stress in zebras ,17. ThisOne of the factors that may drive the emergence of EHV through seasonal water is climate change . As wateRhinoceros unicornis) [Ursus americanus) [Eudorcas thomsoni) [Cavia porcellus f. dom.) [Ursus maritimus) [Animals in captivity do not face seasonal water restriction. However, their environment poses other opportunities for EHV-1 transmission by water. Shared water sources are a feature of many zoos and are often shared between enclosures of multiple animal species . As difficornis) , black bricanus) , Thomsonhomsoni) , guinea f. dom.) , and polritimus) . The infritimus) . PhylogeIn equids, nasal shedding is pre-dominant during viremia; thus, testing nasal swabs and invasively collected sample material such as blood is considered the most reliable method for monitoring EHV shedding. However, in the case of wild free living equids, collecting invasive samples generally requires physical restraint, which is typically associated with logistic and physical difficulties and may cause severe stress or risk to animal health ,49.As most EHVs are shed via the respiratory passage, active virus excretion can be traced by swabbing surfaces on which virus DNA was shed. Such surfaces include various objects in the immediate environment of equids on which nasal exudate was applied by direct contact or as an aerosol. In captive settings, swabbing of surfaces such as feed troughs and behavioral enrichment toys has been found to be an effective approach for monitoring acute EHV shedding in Grévy’s, mountain, and plains zebra ,19. RegaAt present, very few studies on the effect of physicochemical factors on viability of EHVs in the environment are available ,22,41. TFor swabbing dry surfaces, cotton swabs should be moistened using sterile water or other liquids such as sterile phosphate-buffered saline  so as toThere is a growing body of evidence suggesting that different pathways may affect the transmission of EHV within and among species. Several EHV species are commonly found in the environment , whereas others such as EHV-4, -6, and -8 have not been detected in environmental sample material thus far. This is surprising for species such as EHV-4 that are highly prevalent with over 90% of domestic and wild equid populations infected ,51 and wDirect contact with infected animals and exposure to aerosols, infected respiratory secretions, placenta and uterine fluids, and fetal tissue from aborted fetuses remain the most common and direct pathways for EHV transmission between hosts; however, indirect pathways may play an important role in viral transmission in both captivity and nature. EHV transmission via fomites is of particular concern with the viruses remaining infectious on many different materials for extended lengths of time, which poses a particular risk for cross-species transmission in captive animal facilities where animals that would not be exposed to EHVs in their natural habitats may come into contact through an indirect pathway. Furthermore, studies are need to fully understand how pathogenic EHV-1 strains might transmit through aerosol droplets to infect equids in nearby enclosures. The equine industry would benefit from precise guidelines for distances in which equids can be safely housed without the risk of disease transmission. In addition, fecal matter now needs to be treated as a possible fomite for all EHV transmission until further studies are performed to show if fecal matter contains intact virions or just naked viral DNA. Better understanding of these different transmission pathways is of particular interest to captive animal facilities such as zoos, where exotic animals face high likelihoods of exposure and limited immunity to viruses such as EHVs. Zoo keepers and veterinarians working with equids need to be aware and understand how to isolate sick animals and prevent further spread of infection throughout facilities via indirect pathways, including fomites, aerosols, water, and feces. In natural settings, researchers need to better understand how global climate changes and destruction of habitat will increase direct and indirect contact rates among wildlife. Understanding all transmission pathways of EHV may allow for better prediction of inter and intraspecific viral transmission.


PIK3CA-related overgrowth spectrum (PROS) is a series of congenital, sporadic disorders that are associated with segmental overgrowth phenotypes and postzygotic, somatic gene mutations in the PIK3CA-ATK-mTOR pathway. The variability and overlapping phenotypes between PROS and other complex vascular malformations make the differential diagnosis confusing and challenging. PROS should be considered for the differential diagnosis with other complex vascular malformations and syndromes with a tissue overgrowth phenotype, such as Parkes-Weber syndrome (PWS).Herein, we diagnosed one unique clinically challenging case manifested as capillary malformation (CM), limb overgrowth, as well as increased skin temperature and peripheral venous dilatation of lower limb that indicated a potential fast-flow lesion. The patient was initially diagnosed with PWS. Contrary to the previous diagnosis, based on further MR imaging and digital subtraction angiography (DSA), which ruled out the existence of AVMs and AVFs, and molecular analysis with targeted next-generation sequencing (NGS) revealing a somatic PIK3CA mutation, we ultimately diagnosed that the patient had a unique form of PROS simulating PWS phenotypes. We suggest that it is important to propose the differential diagnosis of PWS and PROS, two diseases that share a common overgrowth phenotype. We recommended radiological diagnosis such as MRI, CT and DSA as well as further molecular diagnosis to provide more information for the assessment of vascular lesions and to further guide clinical treatment strategies. PIK3CA-related overgrowth spectrum (PROS), a series of congenital, sporadic disorders that are associated with segmental overgrowth phenotype and postzygotic, somatic gene mutations in the PIK3CA-ATK-mTOR pathway, was defined to encompass multiple, heterogeneous phenotypes at a National Institute of Health (NIH) workshop in 2013 [RASA1 mutations have been shown to underlie PWS, and genetic study can be essential for differential diagnosis with other vascular overgrowth entities [ in 2013 , 5. At p in 2013 ; CLOVES  in 2013 ; DMEG  that could be identified in association with PROS.In this study, we diagnosed a unique PROS characterized by very subtle phenotypes masquerading as PWS. The patient was initially considered to have PWS because of the bilaterally segmentally distributed capillary malformation (CM) on both the lower and upper limbs, chest and back, the hypertrophic lower limbs associated with longitudinal growth, and the increased skin temperature and peripheral venous dilatation of his lower limbs, suggesting a potential fast-flow lesion. Contrary to the previous diagnosis, based on further MR imaging and DSA ruling out the existence of AVMs and AVFs, and molecular analysis with targeted next-generation sequencing (NGS) revealing a somatic We suggest that in some cases, the diagnosis based on clinical symptoms and signs is restricted. It is important to propose the differential diagnosis of PWS and PROS, two diseases that share a common overgrowth phenotype. We recommended radiological diagnosis such as MRI, CT and DSA as well as further molecular diagnosis to provide more information for the assessment of vascular lesions and to further guide clinical treatment strategies.A 23-year-old male patient visited our vascular anomaly clinic and presented with extensive, bilaterally and segmentally distributed CM on both the lower and upper limbs, chest and back. Bilateral hypertrophy associated with longitudinal growth of the lower extremities presented since birth and became progressively significant with age. Further physical examination revealed peripheral venous dilatation of the lesions on both sides of his lower extremities, and the skin temperature at the lesion was significantly higher (2 °C) than that in the adjacent normal skin Fig. a, b. No Minimally invasive biopsies were performed under local anesthesia for further molecular diagnosis. Tissue specimens of the CM lesion and peripheral blood specimens were collected and preserved for DNA extraction and NGS. We designed the targeted NGS gene panel based on the ISSVA classification .PIK3CA . Integrative Genomics Viewer (IGV) visualization of NGS data for the tissue sample with somatic mutation in PIK3CA  is present in Fig. PIK3CA in the patient’s tissue sample  [Vascular malformations, according to literature, are considered to result from abnormal signaling processes during embryonic development, leading to varying degrees of differentiation vascular plexus cells . In the  (ISSVA) , severalAKT1-activating mutation that comprise with asymmetric tissue overgrowth involving almost the entire body and skin manifestations such as cerebriform connective tissue nevi and epidermal nevi [GNA11 mutation, PWS harbors RASA1 mutations, while KTS is caused by somatic PIK3CA and TEK mutation [Identification of overlapping characteristics including vascular anomalies and overgrowth in syndromes with other specific genetic mutations can be challenging, and PROS can be misdiagnosed. Proteus syndrome is a complex systematic disorder with an mal nevi . Parkes-mal nevi . The idemutation .PIK3CA  mutation. Other vascular diseases associated with PIK3CA mutation such as KTS and CLOVES were excluded due to lack of clinical phenotypes including low-low vascular lesions, congenital lipomatous overgrowth, and epidermal nevus. We diagnosed the patient with a unique PROS masquerading as PWS phenotypes. We hypothesized that the increased skin temperature was caused by chronic phlebitis secondary to venous insufficiency and could be easily overlooked and misdiagnosed as manifestation secondary to fast-flow vascular lesions in this patient.According to the congenital clinical manifestations of the patient described in this case, including bilateral tissue hypertrophy and overgrowth of lower limbs, extensive capillary malformations on both lower and upper limbs, chest and back, significantly increased skin temperature of lesions in both lower limbs than that found in the adjacent normal skin and peripheral venous dilatation suggesting potential fast-flow lesions. Thus, the patient was initially diagnosed with Parkes-Weber syndrome. Subsequent MRI and DSA ruled out the presence of low-flow vascular lesions  or fast-flow malformations (AVM and AVF). A genetic study showed a somatic PIK3CA-related vascular anomalies, such as venous/lymphatic malformations (VM/LM) and PROS, by offsetting the progression of the malformations [Detailed medical history and physical examination, imaging  to identify the characteristics of the vascular lesion, and possible skin biopsy as well as molecular diagnosis can be helpful in establishing a detailed “clinical-pathological-molecular” diagnosis for complex vascular anomalies associated with tissue overgrowth. Chemotherapy, such as sirolimus and alpelisib, inhibitors of the PI3K/AKT/mTOR pathway –24, has rmations , 26.PIK3CA  mutation. We found that PROS can present with clinical phenotypes similar to PWS, which may lead to misdiagnosis. Further imaging diagnosis and molecular diagnosis can be valuable to propose a differential diagnosis between PROS and PWS, to fully understand the condition and to assess the potential risks, complications and prognosis of the disease. Molecular targeted therapies such as sirolimus and alpelisib, targeted inhibitors of mTOR and PI3K, respectively, will be viable treatment options for these conditions in the early stages of development.Herein, we report one unique clinically challenging case with bilaterally segmentally distributed CM on both the lower and upper limbs, chest and back, and hypertrophic lower limbs associated with longitudinal growth. Initially, the patient was thought to have PWS because of the increased skin temperature and peripheral venous dilatation of his lower limbs; however, we diagnosed the patient as PROS later based on molecular analysis with a somatic


The number of therapeutic antibodies in development pipelines is increasing rapidly. Despite superior success rates relative to small molecules, therapeutic antibodies still face many unique development challenges. There is often a translational gap from their high target affinity and specificity to the therapeutic effects. Tissue microenvironment and physiology critically influence antibody-target interactions contributing to apparent affinity alterations and dynamic target engagement. The full potential of therapeutic antibodies will be further realized by contextualizing antibody-target interactions under physiological conditions. Here we review how local physiology such as physical stress, biological fluid, and membrane characteristics could influence antibody-target association, dissociation, and apparent affinity. These physiological factors in the early development of therapeutic antibodies are valuable toward rational antibody engineering, preclinical candidate selection, and lead optimization. In 1984, the first therapeutic monoclonal antibody was approved by the U.S. Food and Drug Administration (FDA). In 2021, the 100th antibody was approved just 6 years following approval of the 50th . This trBringing a therapeutic antibody to market requires a team of scientists across multiple disciplines closely collaborating in all stages of development. At the early stage, after the therapeutic target for an indication is selected, decisions must be made regarding the design format, affinity requirement, feasibility of efficacious doses, and candidates for subsequent stages. Rational lead optimization and candidate selection are critical tasks in early drug development and can differentiate success and failure in clinical stages. Antibody engineering provides means for controlling a candidate’s half-life, affinity, and biological activity . Computain vivo interactions by integrating local physiological factors beyond those commonly considered in physiologically based pharmacokinetic (PBPK) models could enhance model prediction fidelity and boost confidence in early-stage decisions  is a label-free technique to measure the kinetics of molecular interactions and has become the standard for  binding . An exte binding . Major a binding . In addi binding .The slow dissociation rate of antibodies from their target necessitates relatively long incubation times to reach equilibrium compared to small molecule drugs. Equilibrium states are, by definition, invariant with time; thus, determining accurate estimates requires the demonstration of negligible change in product and reactant amounts over time. For therapeutic antibodies with pM or nM affinities, it takes hours, even days, to reach binding equilibrium with their targets. However, nearly 90% of reported incubation times for equilibrium constants in a survey by Jarmoskaite et al. were an hour or less . JarmoskD) are used, in essence, to summarize ligand-receptor engagement. While an affinity summary metric is theoretically useful, understanding both the association (kon), and dissociation (koff) rates are essential for in vivo characterization of antibody-target interactions. These rate constants describe the microkinetic relationship between individual antibody domains and corresponding binding domains on the target. The “intrinsic” value to these rate constants may be estimated through in vitro techniques, such as SPR. However, these values do not reflect binding under physiological conditions. Understanding antibody-target interactions under physiological conditions is necessary for developing accurate foresight into the potential efficacy of preclinical antibody candidates; yet remains largely uncharacterized at the very early stage. A schematic representation of concepts depicting how the microenvironment and local physiology can influence antibody-target interactions is shown in Equilibrium rate constants  result in faster complex formation while decreases to koff (slower off-binding rates) result in a more stable complex. The ratio between these rates (koff/kon) is used to describe the overall affinity  of the interaction. Notably, affinity is determined by the relative difference in these rate constants, not by their individual magnitudes. While affinity can be easily estimated from in vitro techniques, in vivo affinities can be profoundly different  and further restricted by other macromolecules, membrane rigidity, tight junctions, etc. The diffusion rate of bound antibodies on lymphocytes has been estimated to be approximately 10–10 cm2/s , efficacy may not be directly related to the fraction of receptors engaged, but rather to the successful initiation of subsequent effector mechanisms . RituximAssociation and dissociation kinetics influence target engagement and, thereby, influence subsequent initiation of effector functions. Effector cell cytotoxicity can be mediated through a multitude of mechanisms including: ADCC, antibody-dependent cellular phagocytosis, or initiation of the complement cascade . ImportaThe diversity in antibody mechanisms of action continues to increase with increasing use of novel design formats, such as bispecific or trispecific antibodies . Novel don beyond this theoretical rate limit will not increase the probability of species interaction. Engineering efforts should therefore focus on optimizing koff to promote stable target binding.Conventional PK models typically assume uniform drug distribution within a given tissue as well as proportional uptake and loss from the tissue with respect to plasma concentrations. However, antibody distribution within tissues, a process affected by transvascular permeability, local target expression, target affinity, cellular internalization, and the extracellular environment , is knowIf the target exists exclusively within densely packed physiological spaces , only a small fraction of antibody molecules may access the area . Howeveron, dense microenvironment). Collectively, heterogeneous antibody distribution within tumor tissues could affect treatment outcomes by promoting survival and resistance of unexposed cells.The binding site barrier effect is a widely acknowledged concept in which high-affinity antibodies show strong perivascular distribution within tumor tissues . The theTargeting soluble, pathologically relevant targets  have been a common strategy for therapeutic antibodies, particularly for treating autoimmune diseases . Solubleoff becomes increasingly challenging because rapid fluid turnover may prevent antibodies from reaching equilibrium with their targets. Tissues with higher ISF turnover rates generally experience more robust target suppression, partly due to greater antibody convections into these tissues (on (fast binders) are preferred to promote engagement of as many targets as possible before the species are washed away. Lowering koff beyond the ISF turnover may become futile, and an affinity ceiling exists. Simulations demonstrating the relative efficacy of adalimumab, etanercept, and infliximab on TNF-alpha target suppression under various ISF turnover conditions is shown in When ISF turnover in the diseased tissue is relatively fast, the time for antibodies and targets to engage before being washed away is limited. Maximizing target suppression through lowering k tissues . Additiooff) demonstrate greater target suppression than fast binders (high kon). However, the reaction equilibrium may shift toward dissociation due to antibody-target complex accumulation  with monoclonal antibodies has been challenging. Antibody concentrations in the cerebrospinal fluid (CSF) and brain ISF are typically on the order of 0.1% of plasma concentrations . Given tAntibody-target engagement in the brain is further complicated by dynamic fluid exchange and a diverse extracellular environment . Past esMany therapeutic targets are transmembrane proteins . These molecules face spatial and diffusional constraints, unlike soluble targets . The binon and koff between antibody and target  and epidermal growth factor receptor (EGFR), by up to an order of magnitude . The disd target . Moleculd target . In addid target . Notablyd target . When mad target .When an antibody binds a transmembrane target, the molecule becomes anchored to the membrane creating an effective local concentration of antibody at the cell surface. This regional concentration promotes subsequent interaction with additional targets on the cell surface . This loon and koff  is critical to overall antibody selectivity, distribution, and effector function. The relative concentrations of antibodies and targets critically influence the likelihood of bivalent binding . When taoff,  for monovalent-bound complexes because bivalent binding events must compete with free antibodies for unoccupied targets. In their study, the apparent koff for both rituximab and obinutuzumab differed approximately threefold in tested concentration ranges despite similar antibody affinities. The authors posited that obinutuzumab’s increased koff, relative to rituximab, led to more dynamic bivalent target binding than for rituximab, demonstrating an important point: antibodies with similar affinities can display significant differences in their predominant binding mode depending on the relative magnitude of their kon and koff. The implications for these concentration-binding-mode relationships are varying bivalent-monovalent ratios of bound antibody on cells/tissues depending on the relative concentration of antibody and target and antibody-target binding characteristics. If bivalent binding stability is advantageous, engineered reductions in koff may be used to promote increased bivalent binding.Work by Bondza et al. demonstrates the influence of free antibody concentration on bivalent binding stability . Increasoff has been recognized as a strategy to promote selective binding to cells upregulating targets, such as HER2  products have been approved. However, over 85 bispecific agents were in clinical development in 2019  suggesting a potential influx of BsA formats in coming years. BsA can be used to bind two targets on the same cell (cis-) or different cells . The ability of BsA to modulate multiple targets may prove advantageous for addressing multifactorial diseases, such as cancer, where target pathway dysregulation, upregulation of alternative pathways, and crosstalk between pathways can lead to treatment resistance . For tarBsA can also be used to facilitate cell-cell interactions. For example, blinatumomab, the first in class bispecific T-cell engager molecule is used to promote interaction between T cells and CD19-expressing tumor cells . EfficacWhen used to mediate a bridging strategy between a ligand and receptor attached to cell surfaces, the reaction kinetics will be much different compared to the two species in solution. In this situation, bivalent binding rates are not reflective of interaction affinities, but more generally, the relative rates of cross-linking and intercellular encounter since only adjacent cells can facilitate bivalent binding. Mathematical modeling and experimental interpretation of these reactions is complicated by quantifying the likelihood of cell-cell interaction and potential for additional molecular interactions  between cells contributing to bond avidity .in vivo target engagement. Additionally, identifying lead drug candidates with optimal target engagement within the tissue/cellular context is paramount to minimizing futile resource allocation in drug development programs. Extensive evidence indicates that the engagement dynamics for antibody-target interactions in living systems differ considerably from that observed in vitro. Insight into how the native microenvironment and local physiology influence antibody-target interactions could improve preclinical evaluation, lead optimization, and translation of preclinical candidates to clinical development. Notable takeaways from this work include 1) SPR technologies can serve as a rational basis for antibody screening, but affinity estimates should be used with caution in modeling and simulations depicting target engagement; 2) implementation of local tissue/cellular microenvironment and physiology in preclinical antibody-target engagement models could improve our understanding of in vivo antibody-target interactions; 3) antibody physical characteristics, microenvironment, and antibody-target interactions influence the predominant antibody binding mode and can be leveraged to modulate antibody selectivity, distribution, and effector function. Here we briefly reviewed how the interplay between physiological factors and the kinetics of association/dissociation for an antibody-target interaction can influence their engagement in vivo. We hope to draw attention to the knowledge gap surrounding the characterization of antibody-target interactions in living systems and demonstrate the relevance of this information to preclinical candidate selection and optimization processes.The number of antibodies and other protein-based therapeutics on the market is increasing rapidly . Despite


Life on Earth has evolved continuously under Earth’s 1 G force and the protection of the magnetosphere. Thus, astronauts exhibit maladaptive physiological responses during space travel. Exposure to harmful cosmic radiation and weightlessness are unique conditions to the deep-space environment responsible for several spaceflight-associated risks: visual impairment, immune dysfunction, and cancer due to cosmic radiation in astronauts. The evidence thus reviewed indicates that microgravity and cosmic radiation have deleterious effects on the cardiovascular, lymphatic, and vision systems of astronauts on long-duration space missions. The mechanisms responsible for the decline in these systems are potentially due to cytoskeletal filament rearrangement, endothelial dysfunction, and muscular atrophy. These factors may alter fluid hemodynamics within cardiovascular and lymphatic vasculatures such that greater fluid filtration causes facial and intracranial edema. Thus, microgravity induces cephalad fluid shifts contributing to spaceflight-associated neuro-ocular syndrome (SANS). Moreover, visual impairment via retinal ischemia and altered nitric oxide production may alter endothelial function. Based on rodent studies, cosmic radiation may exacerbate the effects of microgravity as observed in impaired endothelium and altered immunity. Relevant findings help understand the extent of these risks associated with spaceflight and suggest relevant countermeasures to protect astronaut health during deep-space missions. Long-duration spaceflight subjects astronauts to unique conditions not present on Earth, generating altered physiological responses to environmental stress. Microgravity and exposure to cosmic radiation ultimately contribute to a decline in cardiovascular, lymphatic, and ocular health systems, which may pose significant risks to astronauts both acutely and chronically. On Earth, gravity-dependent hydrostatic forces maintain body fluid equilibrium, and the magnetosphere protects organisms from harmful cosmic radiation. Weightlessness in space eliminates the typical hydrostatic pressure gradients resulting in facial edema and headward fluid shift, a possible contributor to spaceflight-associated neuro-ocular syndrome (SANS). The purpose of this review is to summarize the current understanding of physiological changes induced by space travel and to explore potential countermeasures against maladaptive responses to spaceflight.The loss of Earth’s gravitational pressures and forces cause many adaptations and maladaptations regarding blood circulation, vasculature, and headward fluid shifts in space. Previous studies have highlighted the physiological responses to microgravity, such as decreases in venous pressures, loss of plasma volume, and orthostatic intolerance, among other symptoms . In spacAnother impact on venous adaptations in microgravity is increased coagulation in the cephalad venous system . As demoExposure to various forms of ionizing radiation during spaceflight also negatively affects the venous system, as the cardiovascular system may be prone to defect when exposed ,11. MorePotential countermeasures against physiological impacts on the cardiovascular system that are currently being considered include pharmacological methods or employing a protective water shield within the walls of the spacecraft ,15. WateThe lymphatic system is important to help control immunity in the peripheral tissues ,17 and tLymph flow depends on local tissue deformation and gravity-dependent hydrostatic gradients . IntrinsThe lymphatic vasculature is lined by the endothelial glycocalyx (GCX) , a carboMicrogravity and cosmic radiation acutely and chronically affect the CNS and immune system in mice . Space rTo date, most microgravity studies pertaining to the lymphatic system consist of rodent (rat and mouse) models subjected to HDT as the ground-based analog for gravitational unloading. It is important to note that these studies have limitations in human application, as the animal model and absence of actual microgravity are insufficient to understand many aspects of human physiology in space. The development of countermeasures against lymphatic and immune dysfunction in space has not yet been studied.Visual impairment via spaceflight-associated neuro-ocular syndrome (SANS) is currently a high-profile risk associated with spaceflight. It is likely caused by microgravity-induced headward fluid shifts that lead to retinal endothelial dysfunction and damage. Some efforts to counteract these fluid shifts and prevent SANS include using lower body negative pressure, a promising countermeasure of which there are a variety of potential devices.Visual impairment of astronauts is a major concern with spaceflight and is related to headward fluid shift, a potential contributor to SANS. SANS is a collection of structural ophthalmologic and neurologic changes found in astronauts that are evidenced by visual impairment ,12,35. MThe etiology of SANS is unknown but is probably primarily attributed to a mild but chronic elevation of intracranial pressure (ICP) due to cephalad fluid shift in weightlessness, which has adverse impacts on the optic nerve ,41. RetiThe optic nerve sheath (ONS) is a compartment ,43 that 12-dependent one-carbon transfer pathways, which may cause more significant ICP increases as fluids shift headward in microgravity [In terms of the impact of SANS among individual astronauts, pre-existing biochemical differences may play a role in predisposing some astronauts to long-term optic nerve damage. These differences include dissimilarities in the folate- and vitamin Bogravity ,45,46. Iogravity .With respect to the effect of cosmic radiation on ocular health, a mouse study indicated that mice subjected to a combination of proton irradiation and HDT displayed increased retinal endothelial apoptosis, causing retinal endothelial dysfunction . These pIssues concerning astronauts’ ocular health are currently of utmost importance as they present a major risk that currently prevents long-term space missions, such as a mission to Mars, which entails consecutive years in microgravity or 3/8 Earth gravity. Currently, lower body negative pressure devices  are usedAstronauts are currently using and investigating a variety of LBNP devices. The Russian Chibis suit is one such device that is currently being used on the International Space Station to prepare astronauts for their return to Earth. The device is a pair of rigid LBNP trousers worn while standing that generates negative pressure using an external vacuum. The negative pressure creates a force that must be opposed by muscular contraction of the legs; therefore, the Chibis suit bears a load on the bottom of the feet and stimulates the expansion of lower body vasculature and tissue ,49. AnotElectricity and volume are limited resources in space. A device currently under investigation that does not require either resource is the self-generated (SELF) LBNP device . It consThe evidence thus reviewed indicates that microgravity and cosmic radiation have deleterious effects on the cardiovascular, lymphatic, and vision systems of astronauts. The loss of hydrostatic pressures due to weightlessness causes fluid shifts resulting in facial edema and mild but chronically increased ICP. Thus, microgravity and cosmic radiation may combine to cause a range of maladaptive physiological responses to spaceflight. The mechanisms driving dysfunction are potentially cytoskeletal, as endothelial damage to cardiovascular and lymphatic vasculatures may cause hyperpermeability that increases capillary filtration into tissues and consequent edema. Headward fluid shifts in microgravity may be the basis for visual impairment associated with SANS, by which retinal ischemia potentially predisposes astronauts to long-term visual impairment. Harmful cosmic radiation probably exacerbates the effects of microgravity as observed in endothelial damage, altered immune cell count, and promotion of retinal endothelial apoptosis. Future studies are needed to determine the chronic effects of microgravity and cosmic radiation on the systems reviewed, especially because animal studies and the absence of actual microgravity in Earth-based models are significant limitations to understanding human physiological adaptations to the deep-space environment. Lower body negative pressure simulates Earth-like gravitational forces and is a promising countermeasure against cephalad fluid shifts and, by extension, SANS. However, further investigations of LBNP are required to determine its efficacy for long-duration spaceflight. With considerable progress made toward understanding the risks associated with space travel, future studies promise improved physiologic countermeasures to protect future space crews.


Strenuous and unaccustomed exercise frequently lead to what has been coined “delayed onset muscle soreness” (DOMS). As implied by this term, it has been proposed that the associated pain and stiffness stem from micro-lesions, inflammation, or metabolite accumulation within the skeletal muscle. However, recent research points towards a strong involvement of the connective tissue. First, according to anatomical studies, the deep fascia displays an intimate structural relationship with the underlying skeletal muscle and may therefore be damaged during excessive loading. Second, histological and experimental studies suggest a rich supply of algogenic nociceptors whose stimulation evokes stronger pain responses than muscle irritation. Taken together, the findings support the hypothesis that DOMS originates in the muscle-associated connective tissue rather than in the muscle itself. Sports and fitness professionals designing exercise programs should hence consider fascia-oriented methods and techniques  when aiming to treat or prevent DOMS. Delayed onset muscle soreness (DOMS) is a frequent post-exercise phenomenon that typically manifests after strenuous and unaccustomed loading. Its symptoms, comprising pain and stiffness of the affected soft tissue, occur hours following activity, reaching maximal levels after one to three days . While aOne of the oldest theories describing the possible mechanism of DOMS focuses on the accumulation of lactic acid. Lactate is a substance produced during glycolytic metabolism and has been reported to induce pain when injected into skeletal muscles with ,8 or witAlmost 120 years ago, Hough  first prA third complex of theories focuses on inflammatory processes. Contrarily to tissue damage, a delayed and sustained activation of type-III afferents may be induced via inflammation-associated intramuscular swelling, which exerts pressure on the mechanosensitive receptors. This is supported by the observation that maximal swelling coincides with the post-exercise DOMS peak . In addiThe above assumptions fit with Armstrong’s early theory of the mechanisms underlying DOMS ,32. He hFinally, free radicals are also repeatedly cited as a reason for damage to the muscle fiber membrane ,36. The In summary, the classical pathogenic models focusing on the muscle tissue are not able to explain the symptoms of DOMS commonly observed after unaccustomed eccentric contractions.A recent stream of research suggests that the collagenous connective tissue could represent the pathogenic substrate of DOMS. Based on the available evidence, both the architectural features and the sensory capacity of fascia may be causal for the perceived post-exercise discomfort . Agten et al.  examinedBesides helping to clarify the pathophysiology underlying DOMS, focusing on the collagenous connective tissue could open new avenues for its treatment. According to a systematic review with meta-analysis from Dupuy et al. , active Foam rolling, an intensive form of self-massage with polypropylene tools, represents another promising method to address fascial alterations after strenuous exercise. Krause et al.  demonstrWhile both collagen supplementation and foam rolling represent relatively passive strategies to trigger adaptations of the soft tissue, conditioning coaches and health professionals may consider designing specific exercise paradigms aiming to increase the tolerance of fascia to loaded lengthening. Besides regular eccentric training and plyometrics, this may particularly include multidirectional dynamic stretching at varying velocities. However, although the connective tissue has generally been demonstrated to substantially adapt to mechanical stimuli , studiesAlthough theories explaining the pathogenesis of DOMS have long focused on the skeletal muscle, there is accumulating evidence suggesting a prominent role of the collagenous connective tissue. Based on the available literature, strain forces associated with eccentric contraction may cause micro-ruptures and inflammation of the deep fascia. As experimental research clearly demonstrates that fascia is more pain-sensitive than muscle following chemical, thermal, electrical, and mechanical irritation, we propose that delayed onset soft tissue stiffness (DOSS) is a more precise descriptor of the post-exercise phenomenon. Currently, there is still a lack of studies examining fascia-specific approaches for the prevention and treatment of DOSS. However, initial evidence suggests that foam rolling and supplementation with collagen peptides may represent promising options to alleviate the post-exercise-discomfort. Sports and fitness professionals may hence modify previous approaches designed to prevent and treat DOSS, now providing more specific stimuli for the deep fascia.


Traumatic brain injury (TBI) represents a significant health crisis. To date, no FDA approved pharmacotherapies are available to prevent the neurological deficits caused by TBI. As an alternative to pharmacotherapy treatment of TBI, ketones could be used as a metabolically based therapeutic strategy. Ketones can help combat post-traumatic cerebral energy deficits while also reducing inflammation, oxidative stress, and neurodegeneration. Experimental models of TBI suggest that administering ketones to TBI patients may provide significant benefits to improve recovery. However, studies evaluating the effectiveness of ketones in human TBI are limited. Unanswered questions remain about age- and sex-dependent factors, the optimal timing and duration of ketone supplementation, and the optimal levels of circulating and cerebral ketones. Further research and improvements in metabolic monitoring technology are also needed to determine if ketone supplementation can improve TBI recovery outcomes in humans. Traumatic brain injury (TBI) occurs when an insult to the head disrupts normal brain function. The Centers for Disease Control and Prevention reports that 2.87 million TBIs are diagnosed each year, with age adjusted TBI emergency room visits increasing 54% in recent years , 2. ThesTBIs are significant injuries that may result in long-term consequences, including neurodegeneration, cognitive dysfunction, and comorbid neuropsychiatric illnesses. It is estimated that 5.3 million individuals in the United States have a TBI-related disability, which places a sizable socioeconomic burden on patients, healthcare systems, and society , 3. DeveThe consequences of TBIs can be classified as either primary or secondary injuries. Primary injuries result directly from mechanical forces, including shearing and direct impact forces, at the moment of the traumatic event. Preventative measures, such as using protective equipment, can reduce the severity of the injury. However, brain tissue deformations from primary injuries are largely irreversible. Secondary injuries occur indirectly as a response to primary injuries and are characterized by interconnected cascades of biochemical, cellular, and molecular events that cause further damage but have the potential to be minimized and reversible , 6. SecoThe cascade of secondary injury mechanisms begins to occur immediately following the primary injury. The force of the primary injury triggers the release of glutamate in neurons and disrupts the integrity of the plasma membrane resulting in an efflux of potassium and influx of sodium and calcium , 8. SustA hypometabolic stage begins around 6 hours post-injury with its duration being dependent upon injury severity . This stThe impairment of oxidative phosphorylation leads to the production of reactive oxygen and nitrogen species that exceed the cell's antioxidant defenses , 15. As The observed impairments at numerous points in the glycolytic pathway and oxidative metabolism of glucose suggest that a more optimal substrate could be used as fuel after a traumatic brain injury. Considering that secondary injury processes are interconnected and have downstream consequences, interventions targeting specific secondary injury mechanisms could improve TBI treatment outcomes. Because the reduction of cerebral energy has significant adverse consequences and is a strong, independent predictor of poor outcomes associated with neuropathology, cognitive deficits, and functional decline, establishing treatment methods to target cerebral energy deficits could help improve TBI recovery –23.via lipolysis of free fatty acids in hepatic mitochondria and locally within astrocytes -3-hydroxybutyl (R)-3-hydroxybutyrate . It is h2 in the TCA cycle, which disrupts the ability of the electron transport chain to generate ATP  cycle. Second, β-hydroxybutyrate bypasses the need to use the pyruvate dehydrogenase complex to enter the TCA cycle. In TBIs, the pyruvate dehydrogenase complex is inhibited . The pyrrate ATP . Inhibitrate ATP . BecauseKetone bodies also consume less NAD+ than glucose to produce ATP. Both glucose and β-hydroxybutyrate pathways produce two molecules of acetyl-CoA, which subsequently enter the TCA cycle to generate ATP. Although glucose generates a higher final yield of ATP and reduces four molecules of NAD+, β-hydroxybutyrate reduces only one molecule of NAD+ during acetyl-CoA synthesis . The decIn addition to being an efficient energy substrate for the injured brain, ketone bodies also have various neuroprotective effects. In TBIs, oxidative stress has been linked to many aspects of secondary injury mechanisms including lipid peroxidation, protein oxidation, DNA damage, electron transport chain inhibition, ionic dysregulation, and protease activation , 45. KetKetone bodies have also been shown to regulate inflammatory responses. In TBIs, inflammatory responses can be both detrimental and beneficial. Inflammatory processes are crucial to recovery because these processes help clear debris and facilitate regeneration mechanisms. However, excessive inflammation can be harmful and contribute to the development of cerebral edema, a breakdown of the blood-brain barrier, and neurodegeneration , 48. In Another way ketone bodies can reduce inflammation and neurodegeneration is through the inhibition of the NLRP3 inflammasome . In TBIsKetone bodies may also improve global and regional cerebral blood flow , 57. In Ketone bodies can also aid in closing the mitochondrial permeability transition pore (MPTP). The MPTP is a protein complex embedded in the inner and outer mitochondrial membrane that is linked to apoptotic and necrotic cell death. Following a TBI, the MPTP opens as a result of calcium overload and decrease in the ratio of ATP/ADP , 60. It Although evidence suggests ketones may be beneficial in TBI treatment, several aspects of treatment need to be investigated before exogenous ketones can become a valid therapeutic approach. More research is needed to understand differences in ketone metabolism due to age and sex, the optimal timing of ketone administration, and how to translate results from experimental models to human patients. See Before exogenous ketones can be used in TBI treatment, differences in ketone metabolism across different ages must be considered. Studies with pediatric and adult rats fed a ketogenic diet following a TBI indicate that the efficiency of ketone metabolism is age dependent –69. PediConsidering the rapid pathological progression following TBI, alternative approaches, such as incorporating a period of fasting to increase monocarboxylate transporter expression, could help resolve issues of age-dependent effects . HoweverOur current understanding of TBI pathophysiology is also limited because it is currently uncertain whether TBI pathophysiology differs between males and females. Historically, in TBI studies, fewer females have been recruited to clinical trials, and male rodents have been the predominant experimental injury model . BecauseRecent studies suggest that neuropathological, physiological, social, and behavioral outcomes in TBIs are sex dependent . In a reIn respect to TBI and ketones, only one study to date has examined TBI sex differences in combination with ketone therapy . In thisThe timing and duration of exogenous ketone administration could also have significant consequences on TBI outcomes and warrants further investigation. Following a TBI, changes in cerebral energy metabolism occurs in two stages. The first stage is an acute period of hypermetabolism associated with excitotoxicity and increased glucose utilization . The secWhile research has shown that ketone metabolism can be beneficial during the acute phase of TBI, more research is needed to understand the effects of long-term ketosis in TBI recovery , 81. In Moderate-to-severe TBIs present an additional challenge in studying the timing of ketone administration due to the presence of additional complications. Although the secondary injury mechanisms of excitotoxicity, ionic disturbances, mitochondrial dysfunction, inflammation, and hypoglycemia still occur in moderate and severe TBIs, these issues are not the most pressing concern in early treatment. Early treatment of moderate-to-severe TBI primarily focuses on managing neurocritical care issues, such as multiple organ dysfunction syndrome, paroxysmal sympathetic hyperactivity, hemorrhagic progression, and coagulopathy . The admOne barrier to studying the long-term effects of ketosis is that ketogenic diets are somewhat controversial, and high-quality clinical trials are lacking. Critics of the ketogenic diet, especially when it is employed primarily to lose weight, argue that the diet is a fad because conventional wisdom holds that high-fat diets are harmful, consumption of fiber and whole grains are essential, and ketogenic diets depend upon the consumption of animal products . AdditioTo fully understand whether ketones are beneficial in treating TBIs, experimental models of TBI must be improved. Thus far, animal models of TBI have played an important role in characterizing TBI pathophysiology and in developing treatment strategies . HoweverWhile early TBI research took place in rodent models and provided a fundamental understanding of TBI mechanisms, differences in rodent and human TBI pathophysiology exist. Because studying animal models with similar pathophysiological responses to humans is crucial to improving TBI treatments, the pig has become an increasingly prevalent TBI model . The pigThus far, the majority of TBI and ketone research has used rodent models. No studies to date have examined ketone administration in TBI pig models. However, a study examining mitochondrial function in response to cyclosporine administration in a TBI pig model showed that intravenous cyclosporine administration improved brain metabolism and mitochondrial function by closing the mitochondrial permeability transition pore . These rThe majority of TBI research in animals has used a controlled cortical impact (CCI) model, shock tube model, lateral fluid percussion injury (FPI) model, or weight drop model to replicate the mechanisms of TBI pathophysiology. The CCI model best replicates focal injuries with localized tissue deformation resulting from a blow to the head, such as in sports-related TBI or violent assaults , 90. TheMost evidence regarding the benefits of ketones in treating TBI has come from animal studies. Ketone therapy research in human TBI is only beginning to look at the safety and efficacy of ketone supplementation. The most recent research includes a phase II clinical trial that found an enterally administered ketogenic formulation can be safely administered to TBI patients while improving glucose control and an ongoing phase I clinical trial investigating the safety, feasibility, and effectiveness of an enteral ketogenic formulation for severe TBI , 92. AnoAlthough not yet widely studied in human TBIs, ketones have been used more frequently in other health conditions with similar pathophysiological consequences. In type 2 diabetes, where cognitive impairment is associated with cerebral glucose hypometabolism, intravenous administration of ketone salts improves working memory performance . In the The studies examining ketones in the treatment of various health conditions involving disruptions in cerebral metabolism focus on outcomes and symptoms that differ from TBI treatment measures. However, the underlying mechanisms behind why ketones help improve such outcomes provides support for why and how ketones can address similar mechanisms present in TBIs. Support for use of the ketogenic diet and forms of exogenous ketones in other health conditions involving disruptions in cerebral metabolism offer promise for future TBI and ketone studies in humans.18F is attached to deoxyglucose and can quantify the proportion of glucose uptake and phosphorylation, and 11C is attached to acetoacetate to quantify the proportion of ketone body uptake  is a common, non-invasive imaging approach used for various neurological problems, including TBI . In PET,y uptake , 108. Why uptake , 107. Thy uptake . The dety uptake . While sy uptake , 110.An image-derived input function is a non-invasive alternative to an arterial input function that can accurately quantify absolute physiological values. The most accurate method of calculating an image-derived input function uses a fully integrated positron emission tomography-magnetic resonance imaging (PET-MRI) system . Until rvia CMD  is an alternative approach to monitor cerebral metabolism. CMD allows semi-continuous bedside monitoring of cerebral metabolism by sampling parenchymal extracellular fluid with a probe to obtain concentrations of specific biomarkers . Variousvia CMD , 29, 81.via CMD , 115. Hovia CMD . While tvia CMD . AnotherOptions for monitoring ketone metabolism in milder forms of TBI and outpatient settings are limited because cerebral ketone concentrations cannot be determined with the methods currently available. These options include urinalysis, breath analyzers, and blood meters. Urinalyses methods predominately use a urine ketone stick test, which can give a semi-quantitative and inexpensive measure of acetoacetate . HoweverTo translate experimental data supporting the benefits of ketone metabolism in TBI into clinical practice, the optimal level of cerebral ketone bodies must be determined. Thus far, most ketone research has focused on plasma levels of ketone bodies, and limited data exists about ketone body levels in the brain. A study exploring cerebral ketone metabolism following TBI in humans indicates that plasma ketone bodies are the primary source of cerebral ketone bodies . HoweverAchieving optimal cerebral ketone body levels is complicated by ketone body transport into the brain that requires monocarboxylate transports to cross the blood brain barrier. Even if plasma ketone body levels are high enough to theoretically reach optimal cerebral ketone body levels, ketone transport into the brain is limited by monocarboxylate transporters. Further complicating this issue is that monocarboxylate transporters are expressed significantly more in pediatric TBIs compared to adult TBIs . TherefoIn addition to understanding the level of plasma and cerebral ketones needed to target TBI mechanisms, the optimal method of increasing cerebral ketone body levels must be determined. A ketogenic diet enriched with medium chain triglycerides provides only small increases in plasma ketone body levels and may not be sufficient to increase cerebral ketone body levels , 91. OthOur understanding of TBI pathophysiology has drastically improved in the past 20 years. However, more than 30 large clinical trials have failed to identify targeted pharmacological therapies in TBI patients that improve recovery outcomes. The heterogenic nature of TBI presents challenges in both TBI research and treatment. Because disruptions in cerebral metabolism are a commonality across all forms of TBI, ketone therapy may be a valid therapeutic approach to improve recovery by boosting cerebral energy metabolism, mitigating secondary injury mechanisms, and exerting neuroprotective effects against acute and progressive neurodegenerative processes. The mechanisms of ketone therapy for TBI are beginning to be understood, however more research is needed in both animal models and humans to elucidate how to effectively utilize ketones and translate pre-clinical findings into clinical practice. Specifically, age-dependent differences in ketone metabolism, sex differences in the response to ketone administration, the timing and duration of ketone administration, and optimal level of cerebral ketone levels needs to be explored. Advances could also be made to improve TBI modeling techniques and methods of monitoring cerebral metabolism. Overall, ketone therapy holds significant promise as an option for TBI treatment, and preliminary evidence strongly warrants further investigation of ketones in TBI treatment.SD confirms sole responsibility for the conception and design and manuscript preparation of this review.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objective: To characterize regional brain metabolic differences in patients at high risk of sudden unexpected death in epilepsy (SUDEP), using fluorine-18-fluorodeoxyglucose positron emission tomography (18FDG-PET).Methods: We studied patients with refractory focal epilepsy at high (n = 56) and low (n = 69) risk of SUDEP who underwent interictal 18FDG-PET as part of their pre-surgical evaluation. Binary SUDEP risk was ascertained by thresholding frequency of focal to bilateral tonic-clonic seizures (FBTCS). A whole brain analysis was employed to explore regional differences in interictal metabolic patterns. We contrasted these findings with regional brain metabolism more directly related to frequency of FBTCS.Results: Regions associated with cardiorespiratory and somatomotor regulation differed in interictal metabolism. In patients at relatively high risk of SUDEP, fluorodeoxyglucose (FDG) uptake was increased in the basal ganglia, ventral diencephalon, midbrain, pons, and deep cerebellar nuclei; uptake was decreased in the left planum temporale. These patterns were distinct from the effect of FBTCS frequency, where increasing frequency was associated with decreased uptake in bilateral medial superior frontal gyri, extending into the left dorsal anterior cingulate cortex.Significance: Regions critical to cardiorespiratory and somatomotor regulation and to recovery from vital challenges show altered interictal metabolic activity in patients with frequent FBTCS considered to be at relatively high-risk of SUDEP, and shed light on the processes that may predispose patients to SUDEP. Sudden unexpected death in epilepsy (SUDEP) is the most common cause of premature death in people with epilepsy, and is second only to stroke as a neurological cause of years of life lost in the general population . PatientThe underlying mechanisms of SUDEP are unclear, but a landmark study of physiological changes immediately prior to SUDEP identified a consistent pattern of cardiorespiratory collapse: a bilateral tonic-clonic seizure is followed by a short, variable period of normal cardiovascular and respiratory patterning, and then a combination of central apnoea and bradycardia, ultimately evolving to asystole . The absBiomarkers for SUDEP risk are urgently needed. Sudden unexpected death in epilepsy is a tragic outcome in epilepsy, but sudden death remains sufficiently rare that it is difficult to conduct prospective mechanistic studies in this population. One option is to use a surrogate marker of risk to stratify patients, but no validated risk prediction tools exist. Several SUDEP risk factors have been identified, with frequent convulsive seizures, anti-seizure polytherapy, and increased duration of epilepsy emerging as the strongest predictors . However18FDG) uptake in patients at high and low risk of SUDEP, and sought to identify regions that demonstrate metabolic differences between these groups.Patients who have died of SUDEP demonstrate changes in brain volumes within regions that are key for regulating breathing and cardiovascular functions , 12. In 18FDG-PET) scans as part of their pre-surgical workup. Patients were excluded if original PET data could not be retrieved, if there were significant structural abnormalities seen on MRI, or if there were missing demographic or clinical data critical to the analyses. Patients enrolled between April 2015 and December 2018 provided written informed consent to participate in a prospective study evaluating autonomic, respiratory, and imaging biomarkers of SUDEP. This study was approved by the relevant ethics committees .We enrolled 135 patients aged >18 years with medically refractory focal epilepsy who were being evaluated in the video telemetry unit at the National Hospital for Neurology and Neurosurgery, London, UK, and who had undergone fluorine-18-fluorodeoxyglucose positron emission tomography (n = 56) vs. zero FBTCS per year “low risk,” n = 69)]. In our previous studies, this approach distinguished >80% of SUDEP cases .All clinical data used for risk assessment and/or as covariates were extracted from multidisciplinary team meeting notes via chart review, and from a local database capturing autonomic, respiratory, and imaging biomarkers of SUDEP. Mann-Whitney-Patients were asked to fast for a minimum of six hours prior to scanning. All scans were acquired at the Institute of Nuclear Medicine, University College London Hospitals, on either a Discovery VCT  or Discovery 710 PET/CT  . Patients were injected with a target activity of 250 MBq and an uptake time of 30–45 min. Scans were acquired for 15 min per bed (slice thickness: 3.27 mm) and reconstructed . Time-of-flight imaging was used for those scans acquired on the GE Discovery 710. Reconstructed images were exported as DICOM files, and converted to NifTi format for further pre-processing.Images were processed and analyzed using SPM12 (Wellcome Center for Human Neuroimaging) implemented in MATLAB 2018b (Mathworks). Details of our adapted voxel-based morphometry methodology  have pre18FDG-PET images. As the final step prior to statistical analysis, images were smoothed with a 6 mm FWHM Gaussian filter.We were primarily interested in the gray matter signal; thus, a binary optimal-threshold mask was created to include only consistently high-intensity voxels in the spatially-normalized scans. Compared to the standard SPM method for creating binary masks, this strategy minimizes the risk of excluding regions with very low intensity that may bear some physiological relevance, particularly in the context of brain atrophy . This ma18FDG-PET images, many of which have no physiological bearing. These factors include the individual pharmacodynamics of the 18FDG levels, and the timing and dosage of 18FDG relative to the imaging acquisition. To account for these variations, we estimated the background 18FDG-PET signal per scan for later use in our statistical models, using voxel intensities extracted from high-intensity areas, ventricular compartment, and white matter compartment. High-intensity activity was indexed as the summed intensities of all raw 18FDG-PET counts within the optimal-threshold mask, ventricular and white matter activity as the summed intensities of the voxels falling within the CSF and white matter tissue compartments, respectively (based on the Neuromorphometrics atlas included with SPM).Several scanning and patient factors can introduce between-subject signal variation in 18FDG uptake between the groups . An automated meta-analysis of human functional neuroimaging studies . BW would like to acknowledge support for his research time through a Samuel R. McLaughlin Fellowship and the University Internal Medicine Research Fund , as well as a Detweiler Traveling Fellowship .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In 2017, we received funding form the Patient-Centered Outcomes Research Institute to conduct a large, state-wide, randomized controlled trial to test the effectiveness of a home-based palliative care (HBPC) program within accountable care organizations. Participants were randomized to either HBPC or enhanced usual care, where physicians were provided added training and support in core palliative care practices. Originally, we planned to obtain patient referrals to the trial from primary care physicians, however we were unable to engage primary care physicians in patient identification processes. In this session we will describe the numerous trial modifications made to our trial recruitment methods and the success of each approach. Ultimately, after 20 months of trial recruitment, we had recruited just 28 patients and 10 of their caregivers. Findings from this terminated trial may inform other researchers in development of participant recruitment methods.