The discovery of the neurotrophins and their potent survival and trophic effects led to great enthusiasm about their therapeutic potential to rescue dying neurons in neurodegenerative diseases. The further discovery that brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) had potent survival-promoting activity on motor neurons led to the proposal for their use in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). In this review we synthesize the literature pertaining to the role of NGF, BDNF, CNTF and GDNF on the development and physiology of spinal motor neurons, as well as the preclinical studies that evaluated their potential for the treatment of ALS. Results from the clinical trials of these molecules will also be described and, with the aid of decades of hindsight, we will discuss what can reasonably be concluded and how this information can inform future clinical development of neurotrophic factors for ALS. Ntrk1) is the receptor for NGF, TrkB (Ntrk2) is the receptor for BDNF and NT4, and TrkC (Ntrk3) is the receptor for NT3 (via association with the two transmembrane receptor components gp130 and LIF receptor beta (LIFRβ), which engage the cytoplasmic JAK tyrosine kinases , brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4 (NT4). The receptors that communicate these functions are the receptor tyrosine kinases known as the Trks. TrkA  and vascular endothelial growth factor (VEGF), this review will predominantly focus on BDNF, GDNF and CNTF. This review will also discuss the transmembrane receptor p75 and its role in ALS.Shortly after the discovery of the potent survival and growth effects of neurotrophic factors, they began to be evaluated as therapeutic molecules for the treatment of nervous system injuries and diseases. One of the first diseases to be considered was amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND). ALS is a rapidly progressing, invariably fatal disease that causes the rather selective degeneration of upper and lower motor neurons, leading to fatal paralysis. While 10–15% of ALS cases are inherited, known as familial ALS (FALS), with many of the gene mutations identified, the remainder of ALS cases are sporadic (SALS), with no known genetic cause . Moleculin vitro, and their regenerative activity in injury models, even before the physiologic functions of the neurotrophins had been fully evaluated, there was enthusiasm for their potential use in the treatment of ALS , brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4 (NT4). Their survival and growth promoting activities are communicated by the receptor tyrosine kinases known as the Trks; TrkA is the high affinity receptor for NGF, TrkB is the high affinity receptor for BDNF and NT4, and TrkC is the high affinity receptor for NT3 . BDNF wat of ALS .Trkb demonstrated a significant loss of facial motor neurons, as much as 70%, a moderate loss of lumbar motor neurons, approximately 30%, but no loss of sacral motor neurons  to more slow (S) and fatigue resistant (FR) motor units , and not affect other tyrosine kinases  has long been known to play a neuroprotective role in several regions of the nervous system. First identified as a driver for ciliary ganglion neuron survival in chick embryos, several studies subsequently demonstrated CNTF expression in different types of neurons and glial cells, including reactive astrocytes and myelinating Schwann cells . Signaliin situ hybridization and northern blotting concluded that CNTFR-α expression in muscle is regulated by innervation, as expression was increased in atrophied fibers but not dystrophic ones  as a potential treatment for ALS . In a phin vitro and is more effective than either BDNF or CNTF . GDNF has also been delivered using stem cells that were engineered to express and secrete GDNF. Regardless of how GDNF was ultimately delivered, studies have evaluated application into the spinal cord directly, into muscles in the periphery, or into the motor cortex to trigger the anterograde transport and release of GDNF into the spinal cord. Application of GDNF into the spinal cord to directly preserve motor neuron cell bodies has had variable results. Injection of lentivirus encoding Gdnf into the spinal cord of Sod1G93A mice did not significantly alter the survival of motor neuron cell bodies, although injection into the facial motor nucleus did protect facial motor neurons  displayed a significant slowing of neurodegeneration in the Sod1G93A model, but not GFAP-Gdnf mice that express Gdnf in astrocytes  showed a modest protection when crossed with Sod1G93A mice, but male mice were not protected , insulin-like growth factor 1 (IGF-1), granulocyte-colony stimulating factor (G-CSF) and hepatocyte growth factor/scatter factor (HGF) have all been evaluated preclinically for ALS, and all of these molecules have undergone Phase I trials in humans. VEGF has been investigated in significant depth, partly because polymorphisms in the n others . In addihenotype . The del vectors . Just re vectors . Another muscles . There hd safety . For botIgf-1, and intrathecal injection of IGF-1, observed a slowing of disease progression in mouse ALS models, raising the possibility that a different delivery method could be explored further (There was significant interest in IGF-1 for the treatment of ALS, and numerous preclinical studies were conducted and Phase I trials were performed. This led to a Phase III trial that included 330 ALS patients that were treated with subcutaneous IGF-1 daily for 2 years. Unfortunately, this study concluded that there was no clinical benefit to IGF-1 treatment for ALS . Neverth further . While G further . G-CSF ( further . A recen further . WhetherThe initial enthusiasm for the use of neurotrophic factors in the treatment of ALS led to a series of clinical trials that showed little or no clinical benefit and, in the case of systemic administration of CNTF, some patients fared worse. These failures slowed further development of neurotrophic factors for the treatment of ALS, even though newer and more precise delivery methods are available. The preclinical development of GDNF has continued over the years, culminating in new clinical trials that were just recently initiated, which take advantage of transplantation of hNPCs engineered to express GDNF . This saSod1G93A mouse model of ALS. While this is an exceptionally well-characterized model, the etiology likely differs from other ALS gene mutations. Analysis of the neuroprotective activity of BDNF, CNTF and GDNF in other available mouse models, such as Tardbp, Fus and Ubqln2 mutation models that display motor neuron degeneration, would be prudent. In studies evaluating the effectiveness of neurotrophic factors in slowing the progression of the Sod1G93A model, the most successful strategies only increase lifespan by 2–3 weeks. In addition, many of these studies began treatment at pre-symptomatic age in Sod1G93A mice, which does not generally match when human ALS patients are diagnosed and begin treatment. Importantly, recently developed human iPSC motor neuron cultures, which allow for the evaluation of SALS motor neurons, should also be evaluated. Interestingly, BDNF, GDNF and CNTF all promote the survival of motor neurons derived from human iPSCs and are often included in the culture medium at the end stage of the differentiation process (When these many studies on the preclinical development of BDNF, CNTF, GDNF and the non-traditional neurotrophic factors are considered in total, there are still many unresolved questions related to their potential effectiveness as ALS therapies. Nearly all of these ALS studies utilized the  process .Sod1G93A and TardbpM377V mouse models (On a more positive note, the therapeutic use of neurotrophic factors for ALS is more likely to be broadly effective for both FALS and SALS patients because it takes advantage of the intrinsic responsiveness of motor neurons to these factors, and does not rely on any one particular aspect of disease etiology. Indeed, when these factors, GDNF in particular, were successfully delivered to motor neurons in sufficient quantities, they were potently neuroprotective. Interestingly, the receptors themselves have been directly targeted for other conditions, such as the highly effective activating TrkB antibody , which re models . Anothere models , the phae models . In concWS and BP wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.This work is supported by grant R01 NS089585 from the National Institutes of Health. WS is supported by NIH T32 AG071444 and BP is supported by NIH R01 NS089585. Schematic diagrams were made with BioRender software.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.


Compelling evidence indicates that Epstein Barr virus (EBV) infection is a prerequisite for multiple sclerosis (MS). The disease may arise from a complex interplay between latent EBV infection, genetic predisposition, and various environmental and lifestyle factors that negatively affect immune control of the infection. Evidence of gene-environment interactions and epigenetic modifications triggered by environmental factors in genetically susceptible individuals supports this view. This review gives a short introduction to EBV and host immunity and discusses evidence indicating EBV as a prerequisite for MS. The role of genetic and environmental risk factors, and their interactions, in MS pathogenesis is reviewed and put in the context of EBV infection. Finally, possible preventive measures are discussed based on the findings presented. Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system (CNS), with both genetic and environmental factors contributing to its development. While significant progress has been made in identifying genetic risk factors for MS, these genetic variants explain only a proportion of the heritability, which is estimated to be around 50% . This miEBV lytically infects epithelial cells and B lymphocytes. The virus has a double-stranded DNA genome enclosed by an icosahedral capsid, a tegument layer, and an outer lipid envelope containing the viral attachment and entry proteins. Infection of B lymphocytes involves the attachment of the viral envelope glycoprotein gp350/220 to the complement receptor CD21 on B lymphocytes, followed by the interaction of viral gp42 with MCH class II molecules . This inPrimary EBV infection is usually asymptomatic in childhood. Subsets of natural killer (NK) cells become activated within one or two days as a first-line defense, playing a crucial role in limiting viral spread. EBV-specific CD8+ T cells are responsible for eliminating proliferating and lytically infected B cells while EBV-specific antibodies neutralize viral infectivity by binding to free virus , 15. DurWhen primary EBV infection is delayed beyond childhood, it often manifests as acute IM, indicative of a reduced capacity of the CD8+ T cell response to rapidly control the infection . The symIn IM, the number of latently infected memory B cells can rise to half of the peripheral memory B cell compartment. Latent infection is only achieved after a massive expansion of CD8+ T cells that target EBV proteins from the early stages of the lytic cycle. The CD8+ T cell expansion coincides with the onset of symptoms. In addition, cytotoxic CD4+ T cell populations are also amplified and contribute to the symptoms of acute IM by massive cytokine production . AlthougEBV has a profound influence on the immune system, and recent studies have suggested a causal role of EBV in MS. There is consistent evidence that EBV infection precedes MS onset. EBV-specific antibodies are present in nearly all patients with MS whereas the risk of MS in seronegative individuals is extremely small . In a sePresence of EBV in latency has been found to be considerably more prevalent in MS brain tissue than in non-MS brain, and lytic EBV infection has been detected in chronic MS lesions , 26 and Generally, patients with MS have higher levels of EBV-specific antibodies than control populations , which sSeveral hypotheses have been put forward to explain the role of EBV in MS , 43, butThe risk of MS is increased more than two-fold following IM , 6, suggPast IM does not correlate with EBNA-1 antibody levels , 47 and The high prevalence of EBV seropositivity in the general population and the low incidence of MS indicates that a potential pathogenic effect of the virus is influenced by other risk factors for the disease. According to the sufficient cause model by Rothman , the devInteraction between two exposures is present when the effect of one exposure on the outcome depends on the other exposure. Statistical interaction evaluates whether the combined effect of the exposures deviates from either additivity or multiplicativity, depending on the scale of the model . AlthougIn addition to EBV, several other viruses have been implicated in MS, such as human endogenous retroviruses (HERVs), human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV), which all have the capacity of establishing lifelong latent infections . The undHHV-6, associated with increased risk of MS , is capaIn seroepidemiological studies, CMV has been inversely associated with MS , 62. CMVThe genetic contribution to MS is complex, with hundreds of common genetic variants, identified by genome-wide association studies, contributing to MS heritability . The impConsidering the role of HLA class II for the infection of B lymphocytes, variations in HLA may account for the differences in EBV susceptibility . EBV serOnce established, both class I and II-restricted antigen presentation are involved in the control of the EBV infection. Genetic differences in the HLA class I locus have been associated with both the development and outcome of primary EBV infection as well as with viral persistence , 68. TheCarriers of DRB1*15:01 have higher EBNA-1 antibody levels compared to DRB1*15:01 negative individuals, both among MS patients and healthy controls , 68, indThe findings presented may contribute to explain the interaction between HLA alleles, encoding molecules regulating T cell adaptive immunity, and measures of EBV infection, in the development of MS –85. BothThe increasing MS occurrence with distance from the equator , 87 has in vitro and in vivo studies show that vitamin D results in increased production of the anti-inflammatory cytokine IL-10  and then in the kidney to its active form . Active vitamin D acts as a transcription factor by binding to the vitamin D receptor (VDR) and modulating the expression of over 200 genes. Vitamin D influences the immune system in multiple ways, including enhancing innate immunity, modulating adaptive immunity, and regulating cytokine production. It increases the differentiation of hematopoietic stem cells into NK cells and may enhance their function , 91. It ne IL-10 . The effivo data .Exposure to UVR also has effects on the immune system that are independent of vitamin D, including induction of interferons that inhibit viral replication and enhancement of innate immune responses . Sun expIn patients with MS, an inverse relationship has been observed between EBNA-1 antibody levels and serum vitamin D levels , 101, suThe influence of vitamin D on MS risk has been suggested to be important during a time window. While low vitamin D levels during the first trimester resulted in increased risk of MS in the offspring , studiesThe latitude gradient in MS occurrence and the association between increased risk of MS and both IM history and vitamin D deficiency have led to the hypothesis that the season of IM could influence MS risk . If vitaThere is strong epidemiological evidence for an association between smoking and other lung-irritating agents and risk of MS. Smoking increases the risk of MS in a dose-response manner , 113. A Tobacco smoking and its oxidative agents results in airway inflammation and increased levels of inflammatory markers –124. AlvSmoking may act synergistically with high levels of EBNA-1 antibodies to increase the risk of MS  –128, Ta, Ta128,Smoking-associated pulmonary inflammation could contribute to the activation of tissue-resident immune cells within the lung including potentially autoreactive effector and memory cells. In experimental studies, CNS-directed cells in the lung become activated by local stimulation, whereby they assume a migratory pattern and traffic to the CNS . SmokingWhile some aspects of childhood or adolescence seem to be critical regarding the impact of several environmental factors on MS risk, smoking increases the risk of MS regardless of age at exposure . FollowiSeveral observational studies have observed that obesity in adolescence and young adulthood confers an approximately doubled risk of MS, compared to normal weight individuals , 137. ThObesity induces a chronic low-grade systemic inflammation due to increased numbers of adipose tissue macrophages that undergo a phenotypic switch with increased production and secretion of inflammatory mediators . Several2 interacted with both past IM and high EBNA-1 antibody levels regarding risk of MS  possess potent immunomodulatory effects . SeveralAlthough studies have not been consistent –167, sibShift work, poor sleep, and psychological stress, all of which can negatively affect the immune response to infection, have also been associated with increased risk of MS –170.Evidence strongly suggests that EBV is a prerequisite for MS and the most effective way to prevent the disease would be to prevent the primary EBV infection through vaccination. Various immunogens, including both lytic and latent proteins, have been explored for inclusion in prophylactic EBV vaccines , and sevSeveral modifiable factors have consistently been associated with increased risk of MS that could be targeted through preventive measures. Based on the presented findings, one approach could be balanced information regarding the risks and benefits of sun exposure. The ultraviolet index can be useful for guiding sun exposure behavior in accordance with WHO guidelines to minimize the risk of skin cancer , but senMS may arise from a complex interplay between latent EBV infection, genetic predisposition, and various environmental and lifestyle factors that negatively affect immune control of the infection. Increasing evidence from various research disciplines emphasizes the crucial involvement of EBV in the development of MS. EBV infection as a prerequisite for MS is further supported by evidence indicating that other established risk factors for MS act synergistically with EBV in the development of the disease. However, while numerous genetic variants and environmental factors have been identified that predispose to MS, their biological significance remains uncertain, and their effects are often small or modest. Certain environmental factors could have multiple biological effects that are significant in the pathogenesis, whereas other exposures may impact a small set of shared pathways, leading to comparable biological effects. Despite recent progress, gaining a comprehensive understanding of the various risk factors for MS onset remains a significant challenge. A multidisciplinary approach could provide further insight into the mechanisms of disease pathogenesis and provide important information for disease prevention that could be implemented.The author confirms being the sole contributor of this work and has approved it for publication.


ALES is a rare subtype that demonstrates the EWSR1-FLI1 translocation characteristic of ES and demonstrates complex epithelial differentiation including diffuse cytokeratin and p40 expression. It has predominantly recognized in the head and neck and is common in middle-aged population. This case is the first case of ALES reported in the pancreatic tail, sharing some morphological characteristics with ALES in the head and neck, including monotonous cytology, infiltrative growth pattern, and complex epithelioid differentiation, but ALES in the head and neck often has high-grade histological features , and sudden keratinization can also occur, but these features were not reflected in this primary pancreatic tail ALES. Although ALES arising in the pancreatic tail and in the head and neck sites share the immunohistochemical and molecular profile, our case can provide new ideas in differential diagnosis of ALES arising in pancreatic tail and promote increased recognition and understanding of ALES. In the latest version of the WHO classification of soft tissue and bone tumors, Ewing sarcoma (ES) is defined as a malignant small blue round cell tumor that occurs in bone and soft tissue, with common genetic characteristics, including fusions of FET  gene family members with transcription factor ETS  family members. The most commonly EWSR1-FLI1 fusion accounts for approximately 85% of all confirmed ES cases . TherefoThe patient was a 43-year-old male who had dull-aching epigastric pain without apparent reason for one month. On radiological evaluation: The pancreatic body and tail lesion with a size of about 65*36mm was highly suspected diagnosis of pancreatic cancer Fig. A. The boGross evaluation revealed a tumor in the tail of pancreas where is 2 cm away from the pancreatic stump, with a size of 65*50*40 mm, grayish white appearance, tough texture, and involving adrenal gland tissue. The histological morphology showed that the tumor cells were distributed in the form of infiltrative lobules, trabeculae, or irregular nests of basaloid cells embedded in a prominent hyalinized or fibromyxoid stroma Fig. A and B CImmunohistochemistry revealed tumor cells were diffusely positive for cytokeratin (AE1/AE3), P40, and CK5/6  alternating with the IE plan (ifosfamide and etoposide). At present, the patient has completed the chemotherapy of CAV/IE regimen for 3 times. The patient tolerates it well and remains disease-free by imaging 4 months after diagnosis Fig. B.Looking back at all the reports on ALES, almost all of them reported in the head and neck, including salivary glands, thyroid gland , nasal cHistological appearance is characterized by nests of small round blue cells embedded in the hyalinized or fibromyxoid stroma with an infiltrative growth. The leading differential diagnostic consideration in this case was desmoplastic small round cell tumor (DSRCT) . DSRCT pSince radiology description indicated that this case appears as hypoattenuating masses involving body and tail of the pancreas, tumors arising from the pancreas need to be ruled out. The morphologic appearance should firstly trigger consideration of pancreatic neuroendocrine tumors , especiaAt the same time, imaging studies also suggest that there is a solid mass in adrenal gland, and the histomorphology also shows that there is multiple tumor nests infiltration in the adrenal gland, so the primary adrenal gland tumor also needs to be identified. Uniform small round blue cells distributed in nests and the formation of rosette-like structures can be seen. Therefore, the sex cord-stromal tumor arising in adrenal gland needs to be identified firstly, especially granulosa cell tumor . This tuAside from primary tumors, metastasis of surrounding epithelial tumors needs to be considered. According to morphological appearance, the first differential diagnosis is the metastasis of invasive urothelial carcinoma, especially nested urothelial carcinoma , 22. It In summary, we reported this case of primary pancreatic tail ALES firstly. It is invasive growth involving in solid organs including the pancreas and adrenal gland. As a rare tumor, ALES was usually demonstrated in the head and neck, and it has many potential diagnostic pitfalls when it is arising in the pancreatic tail. Therefore, it is important to recognize the morphological, immunohistochemical, and molecular genetic diagnostic criteria of ALES, so that an accurate diagnosis can be made. This report will provide new insights into the diagnosis of small round cell tumors in solid organs, and help facilitate the recognition of ALES and further insights into this rare tumor.


Glomerulonephritis are renal disorders resulting from different pathogenic mechanisms . Clarifying details of the pathogenic cascade is basic to limit the progression from starting inflammation to degenerative stages. The balance between tissue injury, activation of protective systems and renal tissue repair determines the final outcome. Induction of an oxidative stress is part of glomerular inflammation and activation of protective antioxidant systems has a crucial role in reducing tissue effects. The generation of highly reactive oxygen species can be evaluated in vivo by tracing the inner-layer content of phosphatidyl ethanolamine and phosphatidyl serine in cell membranes. Albumin is the major antioxidant in serum and the level of oxidized albumin is another indirect sign of oxidative stress. Studies performed in Gn, specifically in FSGS, showed a high degree of oxidation in most contexts. High levels of circulating anti-SOD2 antibodies, limiting the detoxyfing activity of SOD2, have been detected in autoimmune Gn(lupus nephritis and membranous nephropathy) in association with persistence of proteinuria and worsening of renal function. In renal transplant, high levels of circulating anti-Glutathione S-transferase antibodies have been correlated with chronic antibody rejection and progressive loss of renal function. Annexins, mainly ANXA1 and ANXA2, play a general anti-inflammatory effect by inhibiting neutrophil functions. Cytosolic ANXA1 is decreased in apoptotic neutrophils of patients with glomerular polyangitis in association with delayed apoptosis that is considered the mechanism for polyangitis. High circulating levels of anti-ANXA1 and anti-ANXA2 antibodies characterize lupus nephritis implying a reduced anti-inflammatory effect. High circulating levels of antibodies targeting Macrophages (anti-FMNL1) have been detected in Gn in association with proteinuria. They potentially modify the intra-glomerular presence of protective macrophages  thus acting on the composition of renal infiltrate and on tissue repair. Glomerulonephritis (Gn) constitute a heterogeneous group of renal disorders characterized by glomerular inflammation resulting from different pathogenic noxae such as autoimmunity, activation of complement cascade and other factors linked with innate and adaptive immunity. Infiltration by immune cells  is commonly detected in affected areas of the kidney; the proliferation of intrinsic renal cells  occurs in selected conditions. The tubulo-interstitial compartment is also interested in most severe cases, often representing a negative predictor of outcome in terms of progression to renal failure. Different causative triggers usually result in the homogeneity of inflammatory responses and, as direct consequence, the classification of Gn integrate causative elements with pathological findings. Among others, membranous nephropathy-MN, Lupus nephritis-LN and ANCA-associated Gn represent valid examples of renal involvement in autoimmune disease. On the other hand, post-infective, membrane-proliferative and IgA Gn are characterized by renal deposition of immune complexes, not related to autoimmune disease.In this respect, Idiopathic Nephrotic Syndrome represents a separate entity, where glomerular inflammation resulting in focal areas of segmental sclerosis (FSGS) characterized the late stages of the disease. Common pathological elements justify the administration of similar treatments based on steroids to reduce inflammation and immune-suppressive agents to modulate native and adaptive immune response.Clarifying details of the pathogenic cascade leading to glomerular and tubulo-interstitial inflammation is fundamental to speculate new therapeutic scenarios that can improve the efficacy of current therapies and limit the progression to advanced stages characterized by glomerulosclerosis and tubulo-interstitial fibrosis. Blocking autoantibody generation through immunomodulatory drugs represent the current therapeutic strategy; targeting specific pathways or key molecules of the inflammatory cascade is may represent a complementary approach.Activation of the oxidative system follows the deposition of autoantibodies and represents a pro-inflammatory and additive mechanism of renal injury, that should be considered as second potential therapeutic targetIn many Gn, antioxidative systems are activated in response to oxidants stress that frequently occur. Components of the Annexins family, are also of interest because they play a direct anti-inflammatory role or mediate the effects of drugs such as corticosteroids . AnnexinAt any stage and in all pathological conditions we should consider elements that limit the effects of protective systems or molecules  as key molecules participating in disease evolution and should consider the balance between tissue injury, activation of protective systems and renal tissue repair as a determinant of the final outcome ,4,5 and The description of mechanisms involved in renal tissue protection and repair in Gn and the analysis of factors counterbalancing their effects is the topics of the present review that will consider, in a progressive order, the following points: (a) the equilibrium between oxidative stress and antioxidant systems ,7, (b) qThe reason for describing such different aspects of Gn is that they contribute synergistically to counterbalance tissue injury pathways and limit their negative effects on renal function ,4,5.Oxidative stress is part of glomerular inflammation in Gn and activation of protective antioxidant systems has a crucial role in counteracting renal injury. Several techniques have been utilized to evaluate both the oxidative and the protective systems.Ex vivo studies on erythrocyte membrane composition in phosphatidyl ethanolamine and phosphatidyl serine content and circulating malondialdehyde levels, a stable product of lipid peroxidation, have been considered as indirect markers of oxidative stress in the past [Cell membrane phospholipids are the major target of reactive oxygen species (ROS) deriving from mithocondria. the past ,9.3−) and, as consequence, albumin acquires an higher molecular weight (+48 Da) for the adding of three molecules of Oxygen  and diatomic oxygen, Catalase decomposes H2O2 into water and oxygen and completes the detoxifying cycle [Other antioxidant molecules are involved during systemic inflammation and the reduction of circulating levels for consumption may be correlated with the grade of oxidative stress. Glutathione (GSH) is the key intracellular antioxidant molecule that is almost completely reduced in normal condition: the ratio of oxidized/reduced GSH is considered to be a reliable indicator of intracellular oxidation ,9. Glutang cycle . Upon acng cycle ,14.The status of both oxidant and antioxidant systems has been analyzed in Gn and in FSGS. Overall, studies confirmed that oxidation is part of the inflammatory cascade and also demonstrated that antioxidant systems are rapidly saturated, thus suggesting that loss of their protective effect may contribute to the gravity of the process.Studies in humans and in animal models have consistently shown that the phosphatidyl ethanolamine and phosphatidyl serine content of erythrocytes is decreased in Gn whereas malondialdheyde, an end product of peroxidation, is high, especially in patients with lupus nephritis, IgA Gn, focal segmental glomerulosclerosis (FSGS) and membranous nephropaty(MN) ,8,9; botImportant observations that confirmed the high impact of oxidation and the difficulty of limiting its effects in vivo were carried out in FSGS utilizing serum oxidized albumin as reliable marker of oxidative stress activation. Results were of particular interest since they showed that circulating albumin is almost completely oxidized in this condition, thus indicating its high oxidative activity . They alOther indirect observations supporting activation of oxidative pathways in Gn come from studies on detoxifying enzymes. In renal biopsies of patients with MN, Catalase co-localizes with IgG4 along the glomerular basement membrane, clearly showing an extra-cellular localization that is an indirect sign supporting its activation . In MN aThe protective effect of SOD2 may be reduced by anti-SOD2 antibodies. High levels of circulating anti-SOD2 antibodies have been reported in either MN and Lupus nephritis, two common forms of autoimmune Gn ,15,16. IDevelopment of anti-GST antibodies is a part of antibody-mediated rejection (AMR) in recipients of renal graft and is correlated with a loss of renal function, suggesting a role in inducing graft damage ,19. It h+-regulated phospholipid-binding proteins that play an important role in the cell life cycle, exocytosis and apoptosis [Annexins are Capoptosis . Two comANXA1 is a 37KDa protein expressed in cytoplasm of innate immune cells, such as neutrophils, monocytes, mast cells, macrophages, eosinophils and in minimal amounts in T-cells; ANXA1 is also expressed by some renal components, mainly by Bowman’s capsule, cells of macula densa and medullary/papillary collecting ducts ,22. ANXAOf interest, ANXA1 expression is induced by glucocorticoids through the Annexin A1 Lipoxin A receptor (ALXR) and the glucocorticod-induced leucine zipper gene, suggesting that their anti-inflammatory effects may be mediated by this protein ,31. In lPrevious findings emphasized the anti-inflammatory role of ANXA1 in either renal and extra-renal pathologies in humans. The role of ANXA1 in renal conditions, such as acute kidney injury, metabolic renal diseases and renal fibrosis is reported in the literature (for a comprehensive overview see ). We wilAnxa2−/−) develop two important phenotypes linked with the functions of the protein: in respect to inflammation, Anxa2-/- manifests a more severe phenotype [Anxa2-/- mice accumulate fibrin in several organs, including the kidney, and have an impaired removal of vascular thrombi similar to what observed in plasminogen, uPA and tPA deficient mice [ANXA2, the second molecule of interest, is expressed by several cells and has distinct functions. Its main role is the maintenance of vascular integrity that regulates chemotaxis of neutrophils. By binding the p11 factor , ANXA2 interacts with circulating plasminogen and tPA and activates plasminis, thus having an important fibrinolytic effect . As suchhenotype ; in respent mice .There are at least three different modalities according to which the protective role of both ANXA1 and ANXA2 may be modified in Gn. The first is quantitative reduction of protein expression in relevant cells , the second is correlated with post-translational modifications such as deimination occurring in ANXA1 of neutrophil extracellular traps (NETs) in SLE and the third is the formation of autoantibodies that target both ANXA1 and ANXA2.In kidney, granulomatosis with polyangiitis  are charStudies in RA documented high levels of Annexin A1 in fibroblasts recovered from synovial fluids  where thAnti-lipocortin 1 antibodies, homologous of ANXA1, have previously been proposed as a marker  of SysteAs previously described, ANXA2 is a profibrinolytic endothelial cell surface receptor that binds beta(2)-glycoprotein I (beta2GPI), the main antigen for antiphospholipid antibodies. Caserman-Maus et al.  describeMore recently, Ye et al.  describeMacrophages (Mφs) are mononuclear cells characterized by sub-phenotypic heterogeneity that reflects a variety of functions. Mφs (CD68+/CD11b+) originates in the bone marrow  and migrEvidence for the involvement of macrophages in Gn is scarce and mostly based on the characterization renal infiltrates in human and in animal models of Gn. The grade of infiltration is not directly related to the grade of activity, therefore our knowledge is limited to a mere description of the presence/absence of the different monocytic cell phenotypes detected in the Gn. Moreover, only in the last two decades was CD68 recognized as the main marker of macrophages and relied essentially on anti-FM32 and anti-Leu 32 for monocyte characterization ,56,57 , Mφs are to be considered as fundamental regulator of inflammatory response in the kidney.We recently reported circulating anti-Mφs antibodies in serum of patients with autoimmune MN ,68. SuchWe need to better explore the correlation between serum levels, kidney inflammation in terms of quantity and quality, and renal function. Preliminary data in patients with LN are confirming the direct correlation between the high serum levels of anti-FMNL1 antibodies and proteinuria. In vivo experiments blocking the effect of anti-FMNL1 antibodies would be useful to better understand such correlations.While it is widely accepted that autoimmunity, inflammation, oxidants and other molecules are involved in the pathogenesis of Gn, it is less considered that other mechanisms limit the deleterious effects of all pathogenetic triggers active in Gn. The balance between the proactive and the limiting factors determine the final outcome of renal injuries in many Gn. Loss of albumin (the main antioxidant molecule) in urine and almost complete oxidation of serum albumin in patients with nephrotic syndrome are probably deleterious for the kidney. The presence of antibodies against major antioxidant enzymes or against proteins involved in the immune response such as ANXA1 and ANXA2 have been reported in different Gn. Antibodies targeting macrophages in patients with MN have been described as well, implying also a potential limitation of the role of these cells in the inflamed kidney. It is time to consider that factors limiting the immune response or the tissue repair that follows inflammation may be considered in the pathogenesis of auto-immune and inflammatory conditions with the same importance as other causative mechanisms.


HRAS was also observed in both mediastinal lesion and lung lesion tissues. We also evaluated the intratumor heterogeneity of non-silent mutations. The results showed that the mediastinal lesion tissue has higher degree of heterogeneity and the lung lesion tissue has relatively low amount of variant heterogeneity in the detected variants. Through pathology and genomics sequencing detection, we initially revealed the genetic differences between mediastinal thymoma and ectopic thymoma, and clonal evolution analysis showed that these two lesions originated from multi-ancestral regions.Thymomas and thymic carcinomas are rare and primary tumors of the mediastinum which is derived from the thymic epithelium. Thymomas are the most common primary anterior mediastinal tumor, while ectopic thymomas are rarer. Mutational profiles of ectopic thymomas may help expand our understanding of the occurrence and treatment options of these tumors. In this report, we sought to elucidate the mutational profiles of two ectopic thymoma nodules to gain deeper understanding of the molecular genetic information of this rare tumor and to provide guidance treatment options. We presented a case of 62-year-old male patient with a postoperative pathological diagnosis of type A mediastinal thymoma and ectopic pulmonary thymoma. After mediastinal lesion resection and thoracoscopic lung wedge resection, the mediastinal thymoma was completely removed, and the patient recovered from the surgery and no recurrence was found by examination until now. Whole exome sequencing was performed on both mediastinal thymoma and ectopic pulmonary thymoma tissue samples of the patient and clonal evolution analysis were further conducted to analyze the genetic characteristics. We identified eight gene mutations that were co-mutated in both lesions. Consistent with a previous exome sequencing analysis of thymic epithelial tumor,  This result revealed genetic differences between type A ectopic pulmonary thymoma and mediastinal thymoma with high tumor heterogeneity. The mHRAS. In addition, we evaluated the intratumor heterogeneity of non-silent mutations by PyClone, and clonal evolution analysis showed that these two lesions originated from multi-ancestral regions.In this study, we reported a rare case of type A mediastinal thymoma and ectopic pulmonary thymoma. We performed whole exome sequencing (WES) of the 2 ectopic thymoma nodules in the ectopic thymoma patient and revealed a set of cancer-related genes that are recurrently mutated in thymomas, including 22.1The sequencing data were first tested for quality control analysis using FastQC. Next the data were aligned to the human reference genome (NCBI build 37) using BWA , and thehttps://software.broadinstitute.org/gatk/documentation/topic?name=tutorials). We used the MapScape R package to analyze the interactive visualization of spatial clonal evolution of tumor tissue (https://bioconductor.org/packages/release/bioc/html/mapscape.html).Somatic mutation calling was performed using Mutect2, VarDict, and Strelka2 . Somatic2.2A 62-year-old male patient was admitted to our hospital with “dizziness, nausea, and vomiting” in December 2018. Blood routine examination, liver and kidney function, electrolyte, myocardial enzyme profile, thyroid function, lymphocyte subsets, cytokines, pre-transfusion examination, tumor markers, and inflammatory indicators were performed at the initial diagnosis. No obvious abnormality was found in the physical examination. Positron emission tomography (PET) and computed tomography (CT) indicated that the patient had soft-tissue density lesions with calcification in the left lower lobes of the thymus  and multAccording to the proportion of positive cells and the degree of staining, the immunohistochemical results were divided into five grades of “+ (the proportion of positive cells and the degree of staining >60%),” “Partial + (the proportion of positive cells and the degree of staining is about 50%),” “Local + ,” “Few + (the proportion of positive cells and the degree of staining <10%),” and “– (negative).” Immunohistochemical results (lung puncture biopsy under the guidance of CT in the left lung) showed that the expression of phosphoenolpyruvate carboxykinase (PCK) and P40 in tumor cells revealed positive and the expression of CD5, CD20, CD117, terminal deoxynucleotidyl transferase (TdT), and Ki-67 revealed negative as shown in HRAS mutation was observed in both mediastinal lesion and lung lesion tissues. We compared results from ten canonical signaling pathways and cancer driver genes with frequent genetic alterations in our study, using recently published mutational profiles of multiple samples from TCGA publications [To obtain insights into the genetic alterations that characterize the tumor heterogeneity of thymomas, we performed WES analysis of mediastinal lesion and lung lesion tissues and a paired peripheral blood samples from the patient. We identified 383 and 269 somatic non-synonymous single-nucleotide variations (SNVs) in the coding regions of mediastinal lesion and lung lesion tissues, respectively (Tables S1 and S2). Direct comparison between the mediastinal lesion and lung lesion tissues revealed overlapping frequencies of commonly mutated genes, a total of eight somatic mutant genes were shared (Table S3). Consistent with a previous exome sequencing analysis of thymoma and thymic Carcinoma ,11,12, Hications , 18 geneications , there aPIK3CA was clustered together in subclone-1. Mutation of MUC17 was clustered in subclone-3. BRCA2, ALK, and PIK3R1 mutations were clustered together in subclone-6. Driver gene mutation was not found in subclone-2 and subclone-4. Two clusters in ectopic pulmonary thymoma were detected by PyClone. Mutation of MTOR was clustered in subclone-5, and ZNF440 mutations were clustered together in subclone-7. The results showed that the mediastinal lesion tissue appeared to have higher degree of heterogeneity in detected variants whereas the lung lesion tissue showed a relatively low amount of variant heterogeneity.To gain further insight into the accumulation of genetic alterations, we constructed phylogenetic trees of disease evolution taking all mutations in mediastinal thymoma and ectopic pulmonary thymoma . We usedInformed consent: Informed consent has been obtained from all individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the Ethics Committees of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.3Thymomas and thymic carcinomas (TCs) are rare and primary tumors of the mediastinum, which is derived from the thymic epithelium . ThymomaIn addition, this concept readily explains thyroid, paratracheal, and pericardial thymic tissue, there is no explanation for the presence of thymic rests or neoplasms in the lung or pleura. As the larynx and pharynx develop, the pulmonary system is much more advanced than the thymus . Before Although the cytologic features of ectopic thymomas are identical to those of mediastinal thymomas, the accurate diagnosis is extremely challenging due to the rarity and diversity of histology patterns found in such unusual location. Diagnosis becomes more difficult because of the lack of typical features of the thymoma or thymic carcinoma, which affects pathologists’ understanding of this entity. Ectopic pulmonary thymoma is identical to the characteristic histological features of mediastinal thymoma based on the definitive diagnosis of histologic findings . HoweverHRAS mutation was identified as one of the recurrent somatic mutations in thymic carcinoma, which is the most aggressive type of TCs [GTF2I  mutation in the type A thymoma but not in the aggressive subtypes. GTF2I mutation was not found in either ectopic pulmonary thymoma or mediastinal thymoma. To further understand the function of the identified gene mutations, we reviewed the literature reports addressing these mutations and found that most of these mutations have not been reported to be related to human tumors at present [Thymomas rarely metastasize, whereas TCs exhibit a more aggressive behavior, with distant metastases in liver, bones, or lymph nodes, and are frequently symptomatic due to the extent of local invasion. Type A thymoma is usually characterized by a low grade of malignancy, and we found genetic differences between mediastinal thymoma (type A) and ectopic pulmonary thymoma in our case. A total of eight genes were found to be co-mutated in both mediastinal lesion and lung lesion tissues, and e of TCs . Brieflye of TCs  analyzed present . Spatial present . AnalysiThe following are the implications and clinical value of the study for future research. The results of the genetic analysis in this study suggest that the two lesions, type A mediastinal thymoma and ectopic pulmonary thymoma, may be derived from multi-ancestral regions, laying the foundation for the polyclonal evolution of the disease. In addition, this study reveals that IHC staining of spindle cells, epithelial cells, and mature adipocytes can suggest the occurrence of ectopic thymus tissue, which provides a new perspective and reference value for clinical practice. Moreover, HRAS, as a commonly mutated gene, could be a potential therapeutic target in the future due to its gene function and high mutation rate in thymoma. More importantly, this study illustrates the pivotal role of genetic analysis and molecularly targeted therapy in the identification and treatment of ectopic thymoma.This study has several limitations. First, postoperative therapy is limited and not standardized due to the low incidence of this disease. Complete resection, followed by chemotherapy and radiotherapy, is the generally accepted strategy. In this patient, the surgery was performed without radiotherapy or chemotherapy. To date there is no sign of disease recurrence after surgery. Second, myasthenia gravis is the most common adverse effect in patients with thymoma, and it is logical that the study should follow the patients’ hormone levels over time. Although this study was a case report, we believe the results of the analyses of large sample were convincing.4In conclusion, our results revealed genetic differences between type A ectopic pulmonary thymoma and mediastinal thymoma with high tumor heterogeneity.


The present systematic review aimed to determine the chronic effects of the combination of transcranial direct current stimulation (tDCS) and exercise on motor function and performance outcomes. We performed a systematic literature review in the databases MEDLINE and Web of Science. Only randomized control trials that measured the chronic effect of combining exercise (comprising gross motor tasks) with tDCS during at least five sessions and measured any type of motor function or performance outcome were included. A total of 22 interventions met the inclusion criteria. Only outcomes related to motor function or performance were collected. Studies were divided into three groups: (a) healthy population (n = 4), (b) neurological disorder population (n = 14), and (c) musculoskeletal disorder population (n = 4). The studies exhibited considerable variability in terms of tDCS protocols, exercise programs, and outcome measures. Chronic use of tDCS in combination with strength training does not enhance motor function in healthy adults. In neurological disorders, the results suggest no additive effect if the exercise program includes the movements pretending to be improved . However, although evidence is scarce, tDCS may enhance exercise-induced adaptations in musculoskeletal conditions characterized by pain as a limiting factor of motor function. Noninvasive brain-stimulation techniques have gained attention in neuroscience and clinical research due to their ability to modulate cortical excitability and influence various cognitive and motor functions. Among these techniques, transcranial direct current stimulation (tDCS) involves the application of low-intensity 1–3 mA) constant electrical currents to specific cortical areas through two electrodes on the scalp –3 mA con. This saThe versatility of tDCS has sparked research interest across various fields, such as cognitive enhancement  or pain However, most of the research conducted to date has primarily focused on the acute effects of a single tDCS session ,13,14,15However, evidence about the potential benefits of systematically  incorporating tDCS into exercise protocols is scarce and controversial. Two studies did not report any effect of tDCS on motor performance after 12 sessions of tDCS combined with exercise in stroke survivors ,17. HoweTherefore, the aim of the present systematic review is to determine the chronic effects of combining tDCS with exercise (comprising gross motor tasks) on motor function and performance outcomes. We hypothesize that the immediate effects of tDCS before or during exercise, when used systematically during a training or rehabilitation period, would chronically enhance motor function and performance to a greater extent than exercise alone. We addressed this aim through a systematic literature search that identified three main different populations where the effects on motor function of chronic tDCS in combination with exercise have been investigated: (a) healthy population, (b) neurological disorder population, and (c) musculoskeletal disorder population.This systematic review adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement .A systematic literature search was conducted on the US National Library of Medicine (PubMed) and Web of Science databases up to 28 March 2023. The search strategy employed the following terms: (exercise OR “resistance training” OR endurance OR aerobic OR “strength training” OR running OR cycling) AND . The search was performed by D. M.-F. In cases of uncertainty, a second author (G. M.) was involved in the process until a consensus was reached. The authors of the selected articles were contacted to request any relevant missing information.After the removal of duplicates, the titles and abstracts of the remaining articles were screened. Subsequently, the full texts of the obtained reports were evaluated. The following inclusion criteria had to be met in the studies: (a) published in English, (b) adult population (aged over 18), (c) randomized control trials, (d) the intervention protocol combined exercise with tDCS, (e) the protocol mainly comprised gross motor tasks , (f) measurement of any type of motor function or performance outcome before and after the intervention , and (g) at least 5 sessions conducted during the intervention. Studies involving cognitive interventions were excluded.After study selection, the essential characteristics of the trials were reported in three tables, including the main author and year of publication, sample , tDCS protocol, exercise protocol, and outcomes. Only outcomes related to motor function or performance were collected. If any study had more than two intervention groups, only data from the sham stimulation plus exercise group and the real stimulation plus exercise group were collected. The participants’ inclusion criteria were reported in the tables for neurological and musculoskeletal disorder populations.p-value was recorded. Whenever possible, the level of significance from the magnitude of change comparison or effect x time interaction was extracted to compare the tDCS effect with the sham group.For significant results, the corresponding The risk of bias and the methodological quality of the included studies were evaluated using the Physiotherapy Evidence Database (PEDro) scale . StudiesAll the included studies obtained a PEDro score between 6 and 9 points, indicating a “high methodological quality” (mean score: 7.59 ± 0.91). The most frequently omitted items were the “intention to treat” analysis (16 studies), blinding of therapists (12 studies), blinding of assessors (7 studies), and allocation was concealed (6 studies) . Notably2) or at Cz for HD-tDCS . All studies, except one   . Wong et [2) [2]  applied The exercise programs varied across the studies (session range 6–36). The main components of the protocols involved gait ,31,34,37The combination of tDCS with exercise in patients with neurological disorders shows varying effects across different outcome measures. In stroke patients, no significant additional effects were observed in dexterity ,17, spasRegarding strength in stroke patients, tDCS enhanced the effect of exercise on knee flexion and extension peak torque of the paretic limb but not on the nonparetic limb, further reducing extension but not the flexion bilateral deficit . HoweverAdding tDCS to exercise improved the gait speed of stroke patients in two studies ,29 but nCardiovascular fitness, measured as external work performed at maximum oxygen consumption or at the gas-exchange threshold, showed greater improvements in the tDCS group in one study . HoweverIn multiple sclerosis patients, balance improved more in the tDCS group in one study . RegardiIn Parkinson’s disease patients, the only study included did not report significant improvements in bradykinesia, Timed Up-and-Go Test, gait speed, or balance in the real tDCS group compared to the sham group .In old people with mild cognitive impairment, the only study included did not report significant improvements in normal gait speed or in motor dual-task walk speed .2) [2) [Most studies applied conventional a-tDCS or HD-tDCS . The tar2) ,41 or Cz2) [2) ,42. Two In individuals with chronic ankle instability, balance improved more in the tDCS group in one study , but notFor individuals with knee osteoarthritis, the only included study reported a greater improvement in the tDCS group for the Western Ontario and McMaster Universities Osteoarthritis Index Physical Function Subscale .In the only study that included women with patellofemoral pain, the tDCS group exhibited a significant improvement in strength compared to the sham group .The purpose of this systematic review was to examine the chronic effects of combining tDCS with exercise on motor function and performance. We hypothesized that systematic application of tDCS before or during exercise over a training or rehabilitation period would chronically enhance motor function and performance to a greater extent than just exercise alone. Our findings suggest that combining tDCS with exercise, compared to just exercise, (a) did not demonstrate greater chronic effects on performance in healthy individuals; (b) the effects on function and performance in neurological disorder populations varied depending on the task tested, but overall results suggest modest or null additive effects when exercise is specific enough to the motor function that wants to be improved; and (c) enhanced the effects of exercise over function in musculoskeletal conditions characterized by pain as a limiting factor of motor function .To the best of our knowledge, this is the first systematic review focused on the chronic effects of combining exercise with tDCS in healthy adults. Previous acute studies have demonstrated the positive effects of tDCS on various aspects of motor function, such as strength or endurance ,10,11. TStroke patients have lower corticospinal excitability in the affected hemisphere, which usually correlates with chronic poor motor function . Given tRegarding multiple sclerosis patients, a sense of fatigue is one of the most commonly reported symptoms and is known to interfere with daily activities . BecauseIn Parkinson’s disease, patients experienced a reduction in motor cortical excitability, decreased excitatory signalling from the thalamus to cortical areas, degeneration of dopaminergic neurons, and abnormalities in motor cortical region connectivity . Given tAge-related changes in motor cortical properties include a decreased corticospinal excitability that may affect motor function . AlthougFollowing a ligament injury, the individual experiences cortical abnormalities in the somatosensory, motor, and frontal cortex, such as increased motor thresholds and smaller motor-evoked potential amplitudes in the musculature surrounding the injured joint . ConsideAs tDCS could modulate pain , combiniThe results of the present systematic review suggest that the effectiveness of tDCS to enhance exercise-induced adaptations may depend on the specific combination of treatment modalities and individual patient characteristics. Specifically, chronic use of tDCS in combination with strength training does not seem to enhance motor function in healthy adults. In neurological disorders, the results are contradictory, but, overall, the results suggest that the additive effects may be null if the actual exercise program already includes the functional movements pretending to be improved. However, although evidence is scarce, tDCS may enhance exercise-induced adaptations in those musculoskeletal conditions characterized by pain as a limiting factor of motor function. Notwithstanding, these conclusions should be interpreted with caution due to several limitations derived from the included manuscripts. The studies included in this systematic review exhibited considerable variability in terms of tDCS protocols, exercise programs, and outcome measures. Other studies which combined tDCS with exercise were not included in this review due to a lack of specific exercise protocol descriptions , although their results were mixed, as found in this review. Also, many populations include a few or even a single study or had relatively small sample sizes, limiting the statistical power and the generalizability of the findings.Therefore, the chronic clinical application of this technique needs further investigation to determine if the presumed acute effects on several motor-function capacities have any priming effect over chronic exercise-induced adaptations. This review highlights the need for future investigations with standardized and detailed protocols and larger sample sizes, together with long-term follow-up assessments to enhance the quality of evidence and provide more robust conclusions regarding the chronic effects of tDCS combined with exercise on motor function and performance outcomes.


A 3D scaffold developed for inguinal hernia repair has been designed to overcome the multiple incongruences of currently used flat meshes. Conventional hernia implants, positioned in a high-motile environment, are static, need fixation and, instead of healing the degenerative source of hernia disease, produce a granuloma of low-quality scar tissue. On the contrary, the new 3D scaffold, endowed with centrifugal expansion, avoids fixation, reacts dynamically to the motile impulses of the groin and attracts tissue growth factors, promoting the development of newly formed tissue structures. Despite being made of the same polypropylene material as conventional flat meshes, the dynamic behavior of the 3D scaffold that moves in tune with inguinal movements proves that modifying the design and kinetic attitude turns the biological response into tissue regeneration.Surgical repair of groin protrusions is one of the most frequently performed procedures. Currently, open or laparoscopic repair of inguinal hernias with flat meshes deployed over the hernial defect is considered the gold standard. However, fixation of the implant, poor quality biologic response to meshes and defective management of the defect represent sources of continuous debates. To overcome these issues, a different treatment concept has recently been proposed. It is based on a 3D scaffold named ProFlor, a flower shaped multilamellar device compressible on all planes. This 3D device is introduced into the hernial opening and, thanks to its inherent centrifugal expansion, permanently obliterates the defect in fixation-free fashion. While being made of the same polypropylene material as conventional hernia implants, the 3D design of ProFlor confers a proprietary dynamic responsivity, which unlike the foreign body reaction of flat/static meshes, promotes a true regenerative response. A long series of scientific evidence confirms that, moving in compliance with the physiologic cyclical load of the groin, ProFlor attracts tissue growth factors inducing the development of newly formed muscular, vascular and nervous structures, thus re-establishing the inguinal barrier formerly wasted by hernia disease. The development up to complete maturation of these highly specialized tissue elements was followed thanks to biopsies excised from ProFlor from the short-term up to years post implantation. Immunohistochemistry made it possible to document the concurrence of specific growth factors in the regenerative phenomena. The results achieved with ProFlor likely demonstrate that modifying the two-dimensional design of hernia meshes into a 3D outline and arranging the device to respond to kinetic stresses turns a conventional regressive foreign body response into advanced probiotic tissue regeneration. The conventional concept of modern prosthetic inguinal hernia repair implies the deployment of flat meshes to cover the herniated groin . Flat meThe Ethics Commission of the Medical Board of the Land Hessen—Germany ethically approved the research . The study was performed in accordance with the Declaration of Helsinki for experiments involving humans. All participants taking part in the study signed informed written consent.The study involved patients who had already undergone primary groin hernia with the 3D hernia scaffold ProFlor that for different reasons required re-intervention in the same inguinal region. In particular, the biopsies were removed in patients that needed surgery in the groin for various pathologies, e.g., hydrocele in the groin already operated for inguinal hernia, hernia protrusion overlooked in the primary repair or recurrence. In these patients, biopsy samples were removed from the 3D scaffold for histological examination. The investigation utilized the same biopsy samples used for previous studies removed from 15 individuals between 2010–2015 ,22,23,24The tissue samples gathered for the investigation were excised from 3 patients between 3 and 10 weeks post implantation (short-term—ST); from 5 patients between 3 and 5 months post-operation (mid-term—MT); from 4 patients between 6 and 12 months post implantation (long-term—LT); and in the last 3 patients more than one year after the operation (extra-long-term—ELT). All biopsy samples were removed from the anterior aspect of ProFlor, which faces the fascia of the external oblique muscle and has no connection to other inguinal structures B,C. TherAll excised tissue samples, fixed in neutral-buffered 10% formalin, were immersed in paraffin wax. Then, 4-μm-thick slices were prepared and kept until usage at room temperature. Histological analysis, focused on the plain detection of muscle, vascular and nervous structures, was performed with hematoxylin and eosin (H&E). Azan Mallory stain  was utilized to highlight the connective structure and the presence of muscle tissue in the 3D scaffold.w/v hydrogen peroxide in methanol for 30 min was utilized to block endogenous peroxidase activity. Slides were processed for 15 min with Background Sniper. Then, all sections were incubated with primary antibodies  at room temperature (RT) for 1 h. Details on the immunohistochemical antibodies utilized for the research are shown in Following dewaxing, for antigen retrieval, a solution of Tris EDTA (pH 9.0) heated the slides for 20 min at 96 °C. A solution of 3% After incubation, the slides were washed out three times for five minutes with phosphate-buffered saline (PBS), then treated at room temperature for 30 min with biotinylated immunoglobulin . After twice cleansing with PBS for 5 min, the slides were incubated at room temperature for 60 min with streptavidin, horseradish peroxidase conjugate. After washing out 3 times with Tris-buffer saline (TBS), the slides were incubated with chromogen 3-3′-diaminobenzidine tetrahydrochloride . Sections were then cleansed in running water and counterstained with Mayer’s hematoxylin, following dilution for 1 min in 0.035% TBS. In the case of positivity, the DAB-reaction established a brown precipitate. Specific primary antibodies were replaced in tissue sections with normal goat serum or PBS and were used as negative controls. Tissue specimens with specific tissue markers  were used as a positive control. All immune-stained slides were examined at a 100× magnification by means of Leica DMR microscope equipped with a Nikon DS-Fi1 digital camera and image capture software (NIS Basic Research Nikon software).Monoclonal primary antibody NSE (LSBio) was applied at 1:100 dilution for 60 min to highlight evidence of newly developed nervous structures in the 3D device.Biopsy samples and related histologic specimens were blindly examined by two pathologists in respect to the timing of excision. The series of figures included herewith, shown according to the postoperative stages of the biopsies, are ordered, first showing the specific tissue growth factors found followed by microphotographs, which highlight the stage of development of the related tissue elements. Details of the histological and immunohistochemical evidence are summarized in Concerning the evidence of vascular tissue components, slides of the specimens removed after 3–5 weeks post-implantation (short-term—ST) showed clearly detectable vascular endothelial growth factor (VEFG)-modulated neoangiogenesis A and a nConcerning the presence of myogenetic growth factors in the biopsies removed in the short-term after placement of the 3D scaffold, a striking number of nerve growth factor (NGF)-positive cells in the polypropylene fabric of the 3D scaffold were reported A. As a cConcerning the presence in the short-term (ST) of the neurogenic growth factor NGFRp75, this nerve development mediator, unlike NGF, was seen in limited quantities throughout the structure of ProFlor A. At thiProsthetic reinforcement of the herniated groin with flat meshes mostly made of polypropylene is considered the gold standard for the cure of inguinal hernia disease ,32,33. CAiming to improve such controversial treatment results, a new concept has been conceived for a more physiological and pathogenetic coherent method. It concerns a 3D scaffold, ProFlor, designed with a multilamellar shape, available for open and laparoscopic approaches and made of polypropylene, similar to conventional hernia implants ,38,39. CThe regenerative scaffold ProFlor has been developed to dynamically interact with the motile environment of the groin, compressing and relaxing in compliance with the inguinal musculature, being therefore fully in line with the physiology of this area of the body. While being produced with polypropylene material, its biological response is in no way comparable to the foreign body granuloma typical of flat hernia meshes made of the same material . Indeed,On the other hand, development of skeletal muscle is subject to stimulation, development and differentiation of myocytic ancestors. Cytokines and specific growth factors constituting the microcellular environment of myogenic precursors activate the development of myofibrils . In the Nerve growth factor receptor (NGFR) is also referred to as p75(NTR) due to its molecular mass and capability to bind not only NGF, but also to other neurotrophins, including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NTF3), and neurotrophin-4 (NTF4) at low affinity . During In light of the above, in all probability, the proprietary regenerative behavior that characterizes ProFlor is in compliance with the steady movements induced by the physiologic cyclical load of the groin muscles. As previously mentioned, while being made of the same material, ProFlor differs in design from traditional hernia meshes. This difference gives rise to a distinctive reaction to dynamic stresses: conventional static meshes are flat and, being passive, do not interact with the recipient; on the contrary, the 3D scaffold responds in tune to dynamic impulses of the inguinal barrier where it is firmly positioned. Briefly, conventional hernia flat meshes do not move and produce low-quality foreign body granulomas while ProFlor moves in synchrony with the groin and allows for the regeneration of the tissue components wasted by the degenerative injury of hernia disease. A limitation of this research, which warrants attention in future studies, pertains to the limited patient cohort. Although the findings are statistically significant, the number of biopsies (15) could have been increased with additional samples. However, it took several years to acquire enough biopsies to proceed with the study, as such opportunities were rare.For inguinal hernia repair, in contrast to the mainstream, old-fashioned and controversial treatment concept finalized to “reinforce” the groin with the stiff fibrotic incorporation of flat static meshes, the dynamic responses of ProFlor to the physiologic cyclical load during inguinal movements likely demonstrate that, using the same material, it is sufficient to modify the design and physical aspect of the mesh to achieve a totally different biological response. Dynamic compliance, regenerative behavior, fixation-free placement and effective obliteration of the hernia opening characterize this novel device. First in the field of inguinal hernia therapy, ProFlor would appear to embody an innovative technology that combines resistance to pathogenetic insult with a new type of a regenerative effect able to restore the integrity of a fully functional inguinal barrier. The unique technology of ProFlor likely represents a turning point for an advanced cure of inguinal hernias that aims to reduce adverse events and improve patients’ quality of life.


Heart Failure (HF) has been one of the leading causes of death worldwide. Though its latent mechanism and therapeutic manipulation are updated and developed ceaselessly, there remain great gaps in the cognition of heart failure. High morbidity and readmission rates among HF patients are waiting to be addressed. Recent studies have found that myocardial energy metabolism was closely related to heart failure, in which substrate utilization, as well as intermediate metabolism disorders, insulin resistance, oxidative stress, and mitochondrial dysfunction, might underlie systolic dysfunction and progression of HF. This article centers on the changes and counteraction of cardiac energy metabolism in the failing heart. Therefore, targeting impaired energy provision is of great potential in the treatment of HF. And shifting the objective from traditional neurohormones to improving the cellular environment is expected to further optimize the management of HF. HF is characterized by impaired ventricular filling and ejection function, which is the ultimate destination of numerous cardiovascular diseases The heart is a high-energy-consuming organ, accounting for approximately 12% oxygen consumption of the whole body The uptake of cardiac glucose is determined by the concentration of glucose, the number and intrinsic activity of Glucose Transporter 4 (GLUT4) KBs include Acetylacetic Acid (AcAc), β-Hydroxybutyrate (β-HB), and acetone. KBs provide only about 5% needs of the whole body, and the contribution of KBs can rise up to 20% during fasting The process of HF is accompanied by poor energy production and metabolic remodeling. Metabolic remodeling involves increased neurohormonal stimulation and impaired calcium processing that exacerbate energy expenditure. These changes disrupt the balance between energy supply and demand, challenging the cell environment and promoting cardiomyopathy.Metabolic changes seem complex in failing hearts, not only depend on the severity and type of HF, but also highly correlated with comorbidities, such as obesity and Type 2 Diabetes (T2D). Although the exact metabolic changes and substrate preferences in HF remain controversial, current studies believe that FAO is unchanged or slightly increased in the early stage of HF and significantly decreased in later  and 2. SIncreased glucose uptake and glycolysis are conspicuous marks of a failing heart, but glucose oxidation is not synchronized with the increase in glycolysis ,29. WhenEmerging evidence suggested that under the condition of low cardiac output, increased lipolysis in HF patients augmented the availability of ketone bodies, and overexcitation of the sympathetic nervous system promoted the generation and utilization of KBs  and 2. I+. Hyperacetylation inhibits the activity of the malate-aspartate shuttle which is the limitation of the transport of NADH from cytoplasm to mitochondria, and then disrupts the cytoplasmic REDOX state of the failing heart Mitochondria are in charge of the homeostasis of energy metabolism. The latest study found that the expression of Dual-Specificity Tyrosine-Regulated Kinase 1B (DYRK1B) which mediated cardiac hypertrophy and fibrosis by damaging mitochondrial bioenergetics was upregulated in HF. DYRK1B increases its phosphorylation and nuclear accumulation by directly binding to STAT3, leading to the down-regulation of PGC-1α level and subsequent cardiac insufficiency Failing hearts exhibit significant metabolic remodeling, and there is a debate about whether these alterations contribute to the development of heart failure. A study involving 2623 patients found that there were increased wall thickness and LV mass with the deterioration of glucose intolerance among patients +/+ mice −/− mice and mice treated with antibodies specific for VLDLR In heart failure, the chaos of uptake and utilization of FA gives rise to the accumulation of lipid intermediates, such as ceramide and diacylglycerol. The study has found that the overexpression of GSK3α boosted the uptake of FFA in the failing heart leading to fibrosis and cardiac hypertrophy The extra glucose entered pathways, mainly Pentose Phosphate Pathway (PPP), and Hexosamine Biosynthesis Pathway (HBP). Glucose 6-Phosphate Dehydrogenase (G6PD), the rate-limiting enzyme of PPP, is essential to maintain the REDOX state of cardiomyocytes. And elevated expression and activity of G6PD are observed in both humans and canines ,52. Stud2+, governing EC coupling, is the bridge of the communication between cardiac electrical activity and excitation-contraction coupling. In HF, Ca2+ leaks through the channel RyR2. Current research has identified several potential factors for Ca2+ leakage, including the conformational change of RyR2 induced by hyperphosphorylation [2+ signaling, and then give rise to ROS accumulation 2+ Uniporter (MCU) [2+ dynamics are widespread that deteriorate the cellular environment.Carylation ,59. In aer (MCU) ,62. Late2+ is a major factor affecting arrhythmia during heart failure. β3-AR agonists can reduce the incidence of ventricular arrhythmia in CHF rabbits, and inhibit INCX, Ca2+ transient, SR Ca2+ load and leakage Heart failure causes significant changes in hormone receptors, which typically manifests as the failing heart escaping from the supervision of adrenergic. And the downregulation of β1 receptor is the most significant In heart failure, the pathophysiological differences of different types of HF make the uptake and utilization of energy substrates present different metabolic profiles. A study collected the coronary blood samples of 15 patients with HFrEF. The results indicated that compared with the control group, the absorption of FFA and glucose in HFrEF patients had not increased, while the plasma β-hydroxybutyrate and acetylacetate levels were slightly elevated, and the concentration of multiple acylcarnitines increased. In terms of energy contribution, the relative contributions of FFA and glucose decreased, while the relative contributions of ketone bodies increased. And the contribution of beta-hydroxybutyric acid and acetoacetic acid on the ATP production increased from 6.8% in the control group to 24% in the HFrEF group + and lactic acid in the cytoplasm, in turn, avoids calcium overload and other adverse cardiac events 2/kg min) 2/kg min). Basic experiments showed that metoprolol alleviated cardiac dysfunction, reducing palmitate oxidation rate, stimulating glucose oxidation, and increasing tissue ATP levels in diabetic rats Whether the reduction in FAO during HF is either protective or unadapted determines administration of medication. Based on what boosted FAO is strongly correlated with the increase of ROS, inhibition of FAO may exert a great cardioprotective effect by inhibiting intracellular oxidative stress levels, preventing the accumulation of toxic lipid intermediates, and maintaining cellular environmental homeostasis Besides, reducing cardiac FAO through the modification of PPARs is a latent therapeutic approach. Fibrates, the PPARs modulators, decrease β-oxidation by reducing FFA levels and increasing the utilization by other tissues. Studies confirmed that pemafibrate induced the expression of Lipoprotein Lipase (LPL) in mice by activating PPARα to reduce TG and lessen the secretion of LDL-C. At the same time, based on the concept of easing the burden on kidneys, pemafibrate is mainly metabolized through the liver, and excretion through urine only accounts for 14.5%. Thus, pemafibrate can be safely applied in patients with chronic kidney disease Insulin signaling is an important metabolic pathway in the cellular environment. In heart failure, neurohumoral and cytokine imbalances and cellular oxidative stress induce insulin resistance, resulting in increased fatty acid flux into cardiomyocytes. Since glucose is a more efficient substrate, the switch in cardiometabolic metabolism from glucose utilization to fatty acid oxidation may reduce cardiac efficiency. In the severe stage of heart failure, ATP level declines sharply and the inhibition of FAO may further damage cardiac function. Thus, direct stimulation of glucose oxidation may be a better option. Metformin was verified to have a relation with reduced HF mortality as well as readmission in a meta-analysis, with a 22% lower all-cause mortality for patients taking metformin than for those not +/H+ Exchanger 1 (NHE1) in the cardiomyocytes 2021 ESC Guidelines clearly brought Sodium-Glucose cotransporter-2 inhibitors (SGLT2i) as the primary drug for the treatment of heart failure with reduced ejection fraction (HFrEF). Its latent beneficial effects on the heart are as follows: Firstly, SGLT2i, acting like osmotic diuretics, improves ventricular load conditions, optimizes volume management, and reduces cardiac energy consumption by boosting sodium and glucose excretion +/NADH, at a relatively low-level amount patients with HF, is another prospective approach to restore the metabolic balance in the failing myocardium +, the electron donor, participates in glycolysis, TCA cycle and oxidative phosphorylation while acting as a signal transduction molecule to regulate acetylation of mitochondrial proteins + repletion has been demonstrated to improve the mitochondrial function as well as exercise capacity and ameliorate the HFpEF phenotype + levels in the failing heart, but the efficacy and safety of exogenous NAD+ supplement therapy for HF need to be verified in clinical trials  supplementation has turned into a safe and effective option l trials .Table 3PInjured cardiomyocytes enter the new cell cycle, and some endogenous cardiac stem cells may participate in the process of regeneration and repairment by regulating the secretion of cytokines. In the last decade, several types of stem cells have been applied to repair damaged cardiomyocytes in pre-clinical and clinical trials. For now, the hypotheses about latent mechanisms include promoting cardiomyocyte regeneration, angiopoiesis, reducing apoptosis, and intervening in ventricular remodeling by paracrine ,108. Stu+ cells failed to increase, suggesting that abnormal glucose metabolism and diabetes might impair the responsiveness of stem cells + stem cells failed to take effect among patients with diabetes, while putting up great responsiveness in patients with insulin resistance, implying that the effect of stem cell therapy might be regulated by glucose metabolism Whereas, dysregulation of glucose metabolism may interfere with the positive effect of stem cells on heart failure. Among diabetic patients, CD26/DPP-4 on CD34A sound heart can make adaptive adjustments to cope with environmental changes according to the availability of the substrate. In heart failure, transcription of key enzymes involved in cardiac metabolism, REDOX, and changes in signal transduction give rise to cardiac disturbance of energy metabolism and impairment of metabolic flexibility. And put up with a harder challenge for survival. Hence, A better and more thorough understanding of the role and regulatory mechanisms of cardiac metabolism remodeling in HF may pave the way for the resolution of HF. Existing studies have proved that regulation of substrate utilization, oxidative phosphorylation, mitochondrial function, and cytothesis improved cardiac function and the quality of patients’ life. Taking all these factors into account, shifting the emphasis of pharmacotherapy from neurohormonal therapy to metabolic regulation is expected to be a key point in reducing the high readmission rate of HF. For decades to come, research ought to focus on clarifying the efficacy and safety of improving the cellular metabolic environment and seek systematic and feasible therapeutic regimens.Lu Fan wrote the paper draft. ChenChen Meng and Xiaoming Wang drew the figures. Yunjiao Wang and Yanyang Li drew the tables. Shichao Lv and Junping Zhang corrected the draft. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. The authors declare that all data were generated in-house and that no paper mill was used.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Ewing sarcoma is a rare neoplasm that mostly grows in bones or soft tissues around bones. Most cases of Ewing sarcoma occur in intra-skeletal locations  and rarely in extra-skeletal regions such as paravertebral or epidural space, whereas a primary intradural extramedullary Ewing sarcoma (IEES) is extremely rare. Fifty cases of primary IEES including our case were identified in the literature, so far, of which two-thirds are in the lumbosacral region, while only 12 reports described a cervical location of the tumor. Herein, we are presenting a case of C7-T1 primary IEES for a 24-year-old male, followed by a review of updated literature about the primary IEES in the cervical spine. Ewing sarcoma is a malignant tumor, characterized by a poor prognosis and short survival rate, with the five-year survival rate being 60% . Most caDue to the rarity of this tumor, there is no consensus about its treatment guidelines. Surgery is currently the mainstay treatment in order to obtain a histological diagnosis and relieve compression of the cervical spinal cord due to the mass effect of the tumor. It is believed by some authors in the literature that patients who do not receive adjuvant treatment will have a poor prognosis in comparison with those who receive a combination of radiation and/or chemotherapy after surgery .A previously healthy 24-year-old male with a history of traumatic thoracic vertebral fracture four years prior to this presentation, came with progressive symptoms of upper back pain, right upper and lower limbs weakness to the extent of inability to walk and decreased sensations from nipples down over the last two weeks before presentation. In addition, he had difficulty passing urine for two days.Physical examination revealed a significant motor weakness of the right upper and lower limbs; the power about three out of five, spastic lower limbs bilaterally, the sensory level at T4, increased patellar and Achilles tendon reflexes bilaterally, and the reflexes grade 3. Anal tone and perianal sensation were intact, and clonus and Hoffmann signs were not present.Magnetic resonance imaging (MRI) of the cervical spine with contrast showed a well-defined intradural, extramedullary homogenously enhanced lesion at the level of C7-T1 compressing the spinal cord significantly toward the left side of the spinal canal and the lesion is extending toward and outside the right C7-T1 neural foramen with dumbbell-shaped appearance, it measures approximately 20 x 48 x 42 mm in AP, transverse and craniocaudal dimensions and there is widening of right C7-T1 neural foramen  of the chest, abdomen, and pelvis, and whole‑body positron emission tomography (PET) were negative for any tumors.Microscopic examination revealed a malignant small round blue cell tumor that was characterized by nests, trabeculae, and clusters figure  of atypiA panel of immunohistochemical antibodies was performed and showed the tumor cells to be positive with CD99 figure , BCL-2, We discussed his condition in our multidisciplinary teams meeting regarding adjuvant treatment and decided to offer him adjuvant chemotherapy and local radiotherapy at the region of the tumor. Indeed, the patient was started on VAC/IE chemotherapy protocol . The patient in general tolerated chemotherapy with no complications apart from a short duration of significant drop of the absolute neutrophil count (ANC) discovered on routine labs, following which filgrastim was given. For a few months after the surgery, patient continued to improve clinically. Unfortunately, after 6 months of treatment, he decided not to complete his medical course of chemoradiotherapy and wanted to go back to his home country. One month later he presented with acute hydrocephalus, ventriculoperitoneal (VP) shunt was inserted, two weeks later (7 months since time of diagnosis) he passed away.We had done a thorough literature review of the cases of Ewing sarcoma involving the cervical spine. A total of 11 confirmed cases of primary intradural extramedullary Ewing sarcoma/peripheral primitive neuroectodermal tumor in the cervical spine were identified in the literature. Our reported patient will be case number 12. The collective and individual data presentations of the literature review are detailed in Table Typically, young adults are affected . Of these 12 cases, 7 were females (58%) and 5 were males (42%). Clinical presentation is commonly characterized by paraesthesia 11 cases), motor weakness (10 cases), neck/back pain (6 cases), and urinary dysfunction (4 cases). The duration of symptoms before diagnosis was over a short period of time, which ranges from 2 to 12 weeks and a median duration of 6 weeks. All 12 patients underwent urgent surgical decompression and resection of the lesion. Gross total resection was achieved in only 4 cases (36%) and this is largely because the tumor is usually adherent to the surrounding nerve roots/spinal cord or sometimes engulfs or even invades them in addition to the finding of moderate to high vascularity, which was reported in 5 cases; all of these findings are in accordance with our intra-operative findings as well as Tan et al. and Lu et al. in their systemic reviews  cases, m. All repThis pooled review of reported cases showed a very poor prognosis for this tumor which is in accordance with the other reviews done previously. Progression-free survival was ranging from 10 days to 36 months. Overall survival ranges from 1 month to 48 months.Ewing sarcoma is a high-grade small round cell tumor of neuroectodermal origin and it shares the presence of a common chromosomal translocation with other tumors like peripheral primitive neuroectodermal tumor (pPNET), atypical Ewing sarcoma, and Askin tumor . Most caSurgery is currently the mainstay treatment for IEES because it is often difficult to differentiate Ewing sarcoma or pPNET from other diseases in the spinal canal, including schwannoma and meningioma. Therefore, a pathological examination after surgery is essential for making the initial diagnosis. Secondly, reducing the tumor volume by surgical resection is the basis for other adjuvant treatments, including radiotherapy and chemotherapy. According to the previously reported cases, patients who do not receive adjuvant treatment show a poor prognosis , whereasIn summary, we added a rare case of cervical intradural extramedullary Ewing sarcoma with a comprehensive review of the previously reported cases of IEES in the cervical spine. This review highlights the importance of considering Ewing sarcoma as one of the differential diagnoses of intradural extramedullary lesions of the spinal cord. Early surgical decompression is necessary to improve neurological function, although gross total tumor resection is mostly difficult to achieve; it should be followed by adjuvant therapy.