Coronary artery disease (CAD) remains one of the most important causes of morbidity and mortality worldwide, and revascularization through percutaneous coronary interventions (PCI) significantly improves survival. In this setting, poor glycaemic control, regardless of diabetes, has been associated with increased incidence of peri-procedural and long-term complications and worse prognosis. Novel antidiabetic agents have represented a paradigm shift in managing patients with diabetes and cardiovascular diseases. However, limited data are reported so far in patients undergoing coronary stenting. This review intends to provide an overview of the biological mechanisms underlying hyperglycaemia-induced vascular damage and the contrasting actions of new antidiabetic drugs. We summarize existing evidence on the effects of these drugs in the setting of PCI, addressing pre-clinical and clinical studies and drug-drug interactions with antiplatelet agents, thus highlighting new opportunities for optimal long-term management of these patients. Coronary artery disease (CAD) still represents a leading cause of cardiovascular death, alongside cerebrovascular diseases. Since Andreas Grüntzig performed the first percutaneous coronary angioplasty in 1977, percutaneous coronary intervention (PCI) has taken off, becoming a milestone in the treatment of CAD. Moreover, in the last 40 years, revolutionary changes in technologies such as new-generation drug-eluting stents (DES), intracoronary imaging modalities, and debulking strategies have resulted in a significant widening in PCI indications and improved clinical success, such that most revascularization procedures are currently performed percutaneously . Despite these significant improvements, patients with diabetes mellitus (DM) still have worse clinical outcomes following PCI than non-diabetics ,4. DM reNowadays, a new paradigm shift in diabetes treatment occurs since clear evidence of cardiovascular benefits associated with novel antidiabetic drugs has been reported in several randomized trials ,9,10. ThThis review aims to provide an overview of the pathophysiological mechanisms linking hyperglycaemia with short- and long-term PCI complications. We also sought to investigate the potential contrasting actions of new antidiabetic drugs, summarizing existing pre-clinical and clinical evidence on the effects of these drugs in the setting of PCI.In patients undergoing PCI, hyperglycaemia and GV, irrespective of the presence of known DM, have been demonstrated to induce an increase in reactive oxygen species (ROS) production, inflammation, endothelial dysfunction, and platelet reactivity, thus favouring the progression of atherosclerotic plaque and increasing the risk of in-stent complications such as thrombosis and restenosis  Figure .2−), lipid radicals (ROO−), hydroxyl radical (HO−), and not free radicals such as hydrogen peroxide (H2O2), hypochlorous acid (HClO) and peroxynitrite (ONOO−) .,61.60,61No-reflow is a rare PCI complication associated with increased mortality . It is dSeveral mechanisms could explain the increased risk of no-reflow in hyperglycaemic conditions. Firstly, hyperglycaemia induces enhanced expression of P-selectin and other pro-thrombotic adhesion molecules on the coronary capillaries, which favours the trapping of leukocytes . SecondlSeveral investigations demonstrated the predictive role of pre-PCI glucose levels on periprocedural myocardial injury. Notably, this complication has been associated with increased rates of cardiac adverse events on long-term follow-up . TherefoIn addition, the risk of periprocedural myocardial damage is also increased by hypoglycaemia before or during PCI. Indeed, hypoglycaemia was demonstrated to be an independent predictor of periprocedural injury  and long-term major adverse cardiovascular events (MACEs) in a cohort of patients undergoing elective PCI . ConcordPlatelet hyperreactivity, endothelial dysfunction, increased inflammation and oxidative stress are assumed to be the underlying pathophysiological mechanisms of periprocedural myocardial damage, inducing coronary vasoconstriction and microembolization. Since hyperglycaemia, increased GV, and hypoglycaemia might amplify all of these mechanisms, the link between abnormal glucose levels and procedural myocardial damage seems plausible.ST is an insidious complication after stent implantation, with most patients presenting with AMI . Several clinical investigations reported the direct association between ST and an abnormal glycaemic status. In a large registry, ST occurred more frequently in diabetic patients undergoing PCI than in those without diabetes . In anotPoor glycaemic control can also lead to the recurrence of ST. A retrospective study examined the clinical outcomes among 243 consecutive patients who suffered from ST after the index PCI. The endpoint was 1-year cardiac death or recurrent ST after the initial ST. According to this study, patients with recurrent events had higher blood glucose and HbA1c levels than those without, indicating that glycaemic control is a key factor in cardiovascular risk .Hyperglycaemia by itself has also been correlated with ST, regardless of diabetes. In a study conducted by Zhang et al., patients without DM who suffered from ST-elevation myocardial infarction (STEMI) with stress hyperglycaemia, defined as blood glucose >180 mg/dl measured on admission, experienced a significantly higher incidence of ST than patients with normal glycaemic values .Type 2 DM is an independent predictor of ISR after coronary angioplasty, even doubling its risk. Moreover, long-term type 1 DM enhances ISR development after endovascular stenting in pre-clinical studies .Pre-procedural abnormal glycaemic values have been associated with increased incidence of ISR and target vessel revascularization in diabetic and not diabetic patients undergoing elective PCI . In anotSome key processes might contribute to ISR and neoatherosclerosis development in diabetic patients, such as increased VSMC proliferation, enhanced macrophage infiltration, and greater vasa vasorum neovascularization leading to intraplaque haemorrhage . MoreoveGiven the solid pathophysiological basis, efforts have been concentrated over the years to investigate the benefit of optimal glycaemic control during and after percutaneous coronary revascularization. In this regard, Marfella et al.  showed tWhile periprocedural hyperglycaemia is undoubtedly related to adverse short- and long-term outcomes after PCI, the best strategy to treat hyperglycaemia during the hospital stay is still uncertain. Some clinical trials investigated the effects of glucose-insulin-potassium (GIK) therapy, which was proposed for the first time in the 1960s. The assumption was that GIK infusion might have an antiarrhythmic effect through potassium uptake and a beneficial effect on ischemic myocardium by reducing free fatty acid levels and enhancing myocardial efficiency by providing high-dose glucose ,92. HoweInsulin-based strategies for periprocedural glycaemic control have been largely evaluated with conflicting results. The DIGAMI 1 trial showed a mortality rate decreasing about 30% after one year and an absolute reduction in mortality of 11% in the long-term follow-up (3–5 years) in patients with AMI treated with insulin-glucose infusion followed by subcutaneous insulin . The DIGTherefore, optimal glycaemic control in the setting of percutaneous coronary revascularization should take into account three different domains: hyperglycaemia, hypoglycaemia, and GV. Insulin is the only treatment studied in depth; advantages of insulin treatment include the rapid corrections of blood glucose and its use in critically ill patients who cannot eat regular meals . HoweverThiazolidinediones showed anti-atherosclerotic effects but exacerbated HF by inducing fluid retention ,102. TheIn this background, novel anti-diabetic agents, like dipeptidyl peptidase-4 inhibitors (DPP-4i), glucagon-like peptide-1 receptor agonists (GLP-1 RAs) and sodium–glucose cotransporter-2 inhibitors (SGLT-2i) seem to be particularly attractive due to the low risk of hypoglycaemia and the potential beneficial effects on cardiovascular outcomes reported in large, randomized trials on patients with CAD regardless of diabetes .GLP-1 is a gut-derived peptide which increases glucose-stimulated insulin secretion in response to nutrient intake . Since tAlbeit the mechanisms responsible for the cardiovascular effects of these agents are still unclear, a direct cardiac and vascular action has been reported beyond the improvement of glycaemic control . GLP-1 RGLP-1 causes vasodilation and improves endothelial function . Indeed,In addition, several studies also demonstrated the antiatherogenic activity of GLP-1 RAs independently of their effect on glycaemic stabilization . SpecifiFurthermore, GLP-1 RAs provide a direct cardioprotective effect, increasing the β-catenin signalling and canceling apoptosis . GLP-1 Rp < 0.0001) and it was associated with a significant reduction of all-cause mortality, hospital admission for HF, and a composite kidney outcome  . T. T137]. Concerning their cardiovascular effects, previous studies showed that DPP-4i reduce the expression of several adhesion molecules on endothelial cells , preventing atherosclerotic plaque progression and potentially stent-related complications such as ST and ISR ,140. In Furthermore, several studies suggest anti-inflammatory properties for DPP-4i . SitagliSeveral randomized clinical trials have assessed the cardiovascular effects of DPP-4i in diabetic patients. Although the experimental evidence of anti-inflammatory and anti-atherogenic effects of DPP-4i, none of the trials showed a significant benefit on cardiovascular outcomes ,152,153.Few studies focused on the potential role of DPP-4i in the setting of coronary revascularization. Some experimental analyses evaluated the effects of DPP-4i on the processes of post-angioplasty reendothelization, considering that the regenerated endothelium is usually dysfunctional in diabetic patients and strictly correlated to ISR, neo-atherosclerosis and late ST development . In a miIn humans, there is limited evidence about the effects of DPP-4i on PCI related outcomes. A retrospective analysis, including data from the Acute Coronary Syndrome Israeli Survey (ACSIS) 2010, showed that chronic treatment with sitagliptin was associated with a lower risk of in-hospital complications and 30-day MACEs among diabetic patients presenting with ACS . In-hospSGLT-2i inactivate sodium-glucose cotransporters located in proximal renal tubules, thus inducing sodium and glucose excretion to improve glycaemic control . HoweverDapagliflozin also decreases cardiac fibrosis by activating anti-inflammatory cells and attenuating myofibroblast activation after myocardial injury in mice . SimilarDespite these beneficial pleiotropic properties, some critical side effects should be mentioned for this class of agents. Further to the most common genitourinary tract infections, volume depletion and haemoconcentration induced by these drugs raised the question of a possible ischemic and pro-thrombotic effect. Hence, these mechanisms have been called into question to explain the increased risk of peripheral ischaemia with limb amputation and acute kidney injury observed in patients receiving SGLT-2i ,168; conFour large clinical trials have investigated the effects of SGLT-2i in patients with type 2 diabetes and high cardiovascular risk: EMPA-REG OUTCOME for empagliflozin, CANVAS for canagliflozin, DECLARE-TIMI 58 for dapagliflozin, VERTIS-CV for ertugliflozin ,170,171.Furthermore, the anti-inflammatory, antioxidant and antithrombotic properties of this class of agents, along with favourable alterations in lipid metabolism, may support the role of SGLT-2i in preventing stent-related complications. However, there is limited evidence about how SGLT-2i influence outcomes in patients receiving PCI.Some pre-clinical and clinical studies have suggested that SGLT-2i could affect vascular remodelling and attenuate NIH after angioplasty. For instance, an experimental study conducted by Mori et al. showed that luseogliflozin attenuated NIH of the femoral artery after wire injury in high-fat diet-fed mice by suppressing the macrophage PDGF-B . SimilarAs GLP-1 RAs, even SGLT-2i were found to reduce infarct size in experimental ischemia/reperfusion injury models. Specifically, long-term treatment with empagliflozin reduced myocardial infarction size and improved myocardial function in mice with type 2 diabetes subjected to 30 min of ischemia and 2 h of reperfusion . An addiAmong the new glucose-lowering agents, SGLT-2i have been proved to have potential nephroprotective effects. Since contrast-induced acute kidney injury (CI-AKI) causes clinically acquired nephropathy and worsens pre-existing nephropathy in PCI patients, it is reasonable to ask if SGLT-2i could prevent periprocedural renal complications. In this regard, an experimental study on rats demonstrated that dapagliflozin attenuates CI-AKI by suppression of hypoxia-inducible factor 1-alpha (HIF-1α)/human epididymis protein-4 (HE4)/NF-κB signalling, which is related to hypoxic injury .Patients undergoing PCI require pharmacological therapy including antiplatelet drugs, beta-blockers, statins, and ACE inhibitors. Therefore, possible interactions with this standard therapy should be considered when antidiabetic drugs are administered.The presence of diabetes mellitus, such as hyperglycaemia and high glycaemic variability, has been associated with high platelet reactivity in patients undergoing coronary stenting, with current antiplatelet agents proven to be less effective in these patients . Of noteIn this context, some glucose-lowering agents have been reported to reinforce the antithrombotic effects of antiplatelet agents, and others have been shown to counteract the effects of these drugs. Indeed, metformin furtherly decreases platelet activation among diabetic patients on aspirin therapy . ConversNotwithstanding the well-documented antiplatelet effects of the novel antidiabetic agents, no studies specifically designed to find interactions between GLP-1 RAs, SGLT-2i and DPP-4i with antiplatelet drugs are available to date. Whether on one side, this could indeed represent an interesting topic for further studies; on the other, these drugs have been reported to have critical interactions with other cardiovascular drugs. The DPP-4i vildagliptin was reported to increase the bioavailability of ACE inhibitors, thus potentially increasing the risk of angioedema . GLP-1RAIn this review, we focused on patients undergoing percutaneous coronary revascularization summarizing the existing evidence on the effects of new antidiabetic drugs in the setting of PCI. We have not considered the effects of these drugs on patients undergoing surgical revascularization through coronary artery bypass graft (CABG). However, it is well known that CABG is a relevant treatment option in diabetic patients with CAD, being superior to PCI with DES in reducing the rate of MACE in patients with DM and multivessel disease in several randomized trials . Indeed,Additionally, we included all of the evidence in the PCI setting without differentiating the acute from the elective revascularization. Patients with ACS are more likely to experience stress hyperglycaemia, regardless of a previous history of known diabetes. Notably, stress hyperglycaemia is associated with worse short- and long-term prognosis post PCI . TherefoFinally, notwithstanding all our efforts to summarize all available data on this topic carefully and thoroughly, as all review articles, also our manuscript might be subject to potential bias, such as gaps in literature searching, and mistranslation or misinterpretation of data from the primary literature.The strict association between abnormal glycaemic values and the progression of cardiovascular disease is well-known and widely investigated so far. An abnormal glycaemic status, regardless of the presence of DM, has also been related to poor outcomes during and after PCI. Pathophysiologic mechanisms by which glucose abnormalities may be a causal factor for procedural and long-term complications in patients undergoing coronary stenting include endothelial dysfunction, oxidative stress, inflammation, and platelet activation. Given the negative role of glucose abnormalities in patients receiving stent implantation, considerable attention has been focused on determining whether optimal glycaemic control may lead to improved cardiovascular outcomes in these patients. Novel antidiabetic agents such as GLP-1 RAs, DPP-4i, and SGLT-2i significantly improve glycaemic parameters and exert direct protective effects on endothelial and vascular smooth muscle cells through anti-oxidative effects, anti-inflammatory and anti-thrombotic properties, potentially attenuating PMI, ST, and ISR. Pre-clinical studies have provided exciting evidence in this direction, whereas few clinical data are available until now. However, given the increasing use of percutaneous techniques for coronary atherosclerosis treatment in the last years, further studies aiming to examine the role of these new agents in this setting are strongly demanded in order to identify new effective strategies for secondary prevention of patients undergoing PCI.


Traumatic brain injury (TBI) is a complex, life-threatening condition that causes mortality and disability worldwide. No effective treatment has been clinically verified to date. Achieving effective drug delivery across the blood–brain barrier (BBB) presents a major challenge to therapeutic drug development for TBI. Furthermore, the field of TBI biomarkers is rapidly developing to cope with the many aspects of TBI pathology and enhance clinical management of TBI. Exosomes (Exos) are endogenous extracellular vesicles (EVs) containing various biological materials, including lipids, proteins, microRNAs, and other nucleic acids. Compelling evidence exists that Exos, such as stem cell-derived Exos and even neuron or glial cell-derived Exos, are promising TBI treatment strategies because they pass through the BBB and have the potential to deliver molecules to target lesions. Meanwhile, Exos have decreased safety risks from intravenous injection or orthotopic transplantation of viable cells, such as microvascular occlusion or imbalanced growth of transplanted cells. These unique characteristics also create Exos contents, especially Exos-derived microRNAs, as appealing biomarkers in TBI. In this review, we explore the potential impact of cell-derived Exos and exosome-derived microRNAs on the diagnosis, therapy, and prognosis prediction of TBI. The associated challenges and opportunities are also discussed. Traumatic brain injury (TBI) is a common public health problem, and no effective clinical treatment strategies have been found; nearly half of the world’s population will experience one or more TBIs in their lifetime ,2. Despi2+ overload, and neuroinflammation, leading to further deterioration of neurological functions [TBI is a multi-phase pathology with complex interactions between the brain, periphery, and immune system . The disunctions . Despiteunctions , and theThe family of extracellular vesicles (EVs) comprises a heterogeneous mixture of exosomes , microvesicles (MVs) , 100–1000 nm diameter), and apoptotic bodies (1–5 nm diameter) . EVs mayMiRNAs are a class of small and non-coding endogenous RNA molecules that regulate the expression of various genes at the post-transcriptional level via binding to complementary seed sequences in the 3′-untranslated regions of target mRNAs, resulting in mRNA degradation or translational repression . NumerouThe action of Exos has been extensively studied among EVs, especially Exos-derived miRNAs as cargoes .To date,Microglia are resident mononuclear phagocytes that comprise 10–15% of all cells in the central nervous system (CNS) . NumerouFurthermore, studies have revealed that microglia aggregate onto the damage site within minutes and undergo time-dependent and injury-related transcriptional profile changes after TBI . In the Increasing studies have shown that MD-Exos can mediate microglia–neuron interactions after TBI. A recent study demonstrated that injured microglia partially suppress neurite outgrowth and synapse recovery by downregulating exosome-derived miR-5121 delivered to injured neurons after TBI. A severe stretch-induced injury increases Exos release in microglia, and neurons absorb these MD-Exos in vivo and in vitro. Stretch-injured MD-Exos decreased the neurite outgrowth and the dendritic complexity in stretch-injured neurons, reducing the neuron percentage and the apical dendritic spine density in the peri-contusion region after TBI. Meanwhile, the miR-5121 level decreased most significantly in stretch-injured MD-Exos. The overexpression of miR-5121 in stretch-injured MD-Exos partially reversed the inhibition of neurite outgrowth and neurons’ synaptic recovery by directly targeting RGMa after TBI. Moreover, the motor coordination in miR-5121 overexpressed MD-Exos treated mice was significantly improved after TBI . One intEVs also propagate neuroinflammation from the CNS to the circulatory system. A study was designed to determine the role of microglia-derived microvesicles (MD-MVs) in TBI, and researchers found that the number of MVs increased within 24 h after injury. These MVs originated from microglia, suggesting that MD-MVs were released from the brain after TBI and then reached the circulatory system. Moreover, MD-MVs isolated from TBI were injected into un-injured mice, demonstrating that these MD-MVs loaded with pro-inflammatory molecules such as miR-155, TNF-a, and IL-1β could transfer the post-traumatic neuroinflammatory phenotype into animals. This evidence suggests that MVs can propagate neuroinflammation in TBI .Therefore, miR-5121, miR-124-3p, and miR-155 from MD-Exos or MD-MVs can be novel therapeutic targets for interventions of neurodegeneration and neuronal inflammation after TBI. Furthermore, miRNA-manipulated MD-Exos may provide promising therapy for TBI .Astrocytes are the most abundant glial cells in the human brain, numbering five times that of neurons, and play a significant role in the brain in maintaining the BBB, regulating synaptic circuits, participating in neurotransmitter recycling, providing metabolic support for neural tissue, regulating regional blood flow, and repairing and scarring processes of the brain following various injuries ,37,38,39Astrocyte-derived Exos (AD-Exos) and astrocyte-derived EVs (AD-EVs) are enriched with a variety of biological molecules including miRNAs. It has been reported that Exos secreted by astrocytes may be channels for transferring the cargo of macromolecular substances to nearby and distant cells, resulting in an extensive range of functional changes in recipient cells . AD-EVs Firstly, astrocytes and neurons regulate each other through Exos and EVs. A few interesting studies conducted on experimental TBI models reported that AD-Exos could repair and protect injured neurons. Exos were absorbed by neurons and released GJA1-20 k protein after TBI in a rat model. These AD-Exos secreted GJA1-20 k proteins reduced the phosphorylation of Cx43, protected and restored mitochondrial function, and down-regulated the apoptosis rate, thereby promoting neuronal functional recovery . AD-ExosSecondly, astrocytes and microglia regulate each other through Exos in TBI. One study explored how miRNAs secreted by Exos derived from activated astrocytes play a significant role in the astrocyte–microglia interaction. AD-Exos enriched with miR-873a-5p could promote the M2 phenotype transformation of microglia in the early stage of TBI and inhibit microglia-mediated neuroinflammation, thus improving neurological deficits after TBI by inhibiting the Erk/NF-κB signaling pathway. Meanwhile, the anti-neuroinflammatory effect of miR-873a-5 can also reduce brain edema in a mice TBI model. In addition, the expression of miR-873a-5p in the brain tissue of TBI patients was detected in this study .Thirdly, apoptosis contributes to the pathogenesis of TBI , and AD-Neuroinflammation is one of the hallmarks of several neurodegenerative diseases and disorders, and AD-Exos have the potential to serve as neuro-inflammatory biomarkers in TBI. In an in vitro study, primary human astrocytes were activated by IL-1β stimulation, and AD-Exos-derived miRNA cargoes released in a neuroinflammatory stress model were examined. The results suggested that IL-1β-induced acute neuroinflammation and oxidative stress release a very distinct miRNA expression profile such as miR-141-3p or miR-30d through Exos, which may regulate the inflammatory response .Finally, we noted that the plasma AD-Exos levels of Aβ42, postsynaptic protein, and NRGN could discriminate between military service personnel with mild traumatic brain injury (mTBI) and those without TBI with moderate sensitivity and accuracy. Injury-induced aggregation of Aβ42 and P-396-tau activates the phagocytic response in astrocytes; this response may lead to higher protein levels within astrocytes, which are subsequently expelled and trafficked to the periphery via plasma AD-Exos in mTBI patients. In addition, plasma AD-Exos were not toxic to neuron-like cells. Plasma AD-Exos contained detectable levels of Aβ42 and p-tau, and these cargo proteins were still not toxic to neuron-like cells in vitro . Table Neuron-derived exosomes (ND-Exos) collect neuronal proteins of cellular membranes and cytosol through the endosomal pathways prior to being secreted into the extracellular fluid of the CNS and entering into other neuronal cells or passing through the BBB into the blood . ND-ExosIn the CNS, neurons and microglia influence each other through Exos in TBI. A report by Yin et al. showed that ND-Exos containing miR-21-5p were phagocytosed by microglia and induced microglial polarization, while the miR-21-5p expression increased in M1 microglia. The polarization of M1 microglia accelerated the release of neuroinflammatory factors, increased the accumulation of P-tau protein, inhibited the neurite outgrowth, and promoted the apoptosis of neurons, which formed a model of cyclic cumulative damage similar to the TBI model. Therefore, regulating the expression of miR-21-5p or the secretion of ND-Exos may be a novel strategy for treating neuroinflammation in TBI .Neurons improve neuronal autophagy through Exos in TBI. A study indicated Rab11a as a potential target gene of miR-21-5p, and miR-21-5p acts as cargo in the Exos playing a critical role in the inhibition of neuronal autophagy. The increased levels of miR-21-5p in ND-Exos can be translocated to damaged neurons to regulate excessive neuronal autophagy by inhibiting Rab11a, thereby resulting in protective effects against neuronal injury after TBI .There is growing evidence that plasma ND-Exos cargoes have diagnostic and predictive value as biomarkers in patients after TBI. It has been suggested that the slope of change in ND-Exos synaptopodin (SYNPO) between 8 h and 14 h after TBI is a promising biomarker for three clinical study outcomes . A cliniMesenchymal stem cells (MSCs) are multipotent stromal cells obtained from various sources, including bone marrow, the umbilical cord, and adipose tissue ,61. MSCsOur initial hypothesis was that MSCs could readily differentiate into neurons or other cells in the injured brain after transplantation. However, only a limited number of MSCs undergo such differentiation. The paracrine factors secreted by MSCs are now suggested to exert therapeutic effects rather than differentiating MSCs . AbundanFirstly, MSCs-Exos may play a vital role in functional improvement after TBI by promoting neuronal recovery function. In an interesting study involving a significantly high expression of miR-216a-5p, BDNF-induced bone-marrow-derived MSCs-Exos (BM-MSCs-Exos) improved neuronal regeneration and cell migration and inhibited inflammation and apoptosis in vivo and in vitro, thus promoting the recovery of spatial learning abilities and sensorimotor functions . Then, MSCs-Exos may exert a positive neuroprotective effect by interfering with astrocytes after TBI. A study observed that human umbilical cord derived MSCs-Exos (UC-MSCs-Exos) could incorporate into hippocampal astrocytes and reduce reactive astrogliosis and inflammation in vitro and in vivo after TBI. UC-MSCs-Exos could also ameliorate LPS-induced calcium signaling dysregulation, mitochondrial dysfunction, and SE-induced learning and memory impairments in mice. The Nrf2-NF-κB signaling pathway was also involved in inhibiting astrocytic activation by UC-MSCs-Exos . MSCs coMoreover, a few studies validated that MSCs-Exos ameliorate BBB integrity after TBI. In a series of studies on the TBI swine model by Williams et al., early treatment with a single dose of BM-MSC-Exos significantly attenuated cerebral swelling and lesion size with a corresponding decrease in intracerebral pressure; decreased levels of blood-based cerebral biomarkers including GFAP, laminin, and claudin-5; decreased albumin extravasation; and improved BBB integrity . SubsequLastly, MSCs-Exos have shown promise in treating brain disorders, with some results highlighting neuroprotective effects through anti-neuroinflammation, pro-neurogenesis, and pro-angiogenesis after TBI. A certain research group developed an in vivo assay for BM-MSCs-EVs efficacy in suppressing neuroinflammation after TBI in mice and found that intravenous infusion of those isolated BM-MSCs-EVs shortly after inducing TBI improved spatial learning and pattern separation deficits 1 month later . AnotherIn conclusion, the protein components and miRNA of MSCs-Exos are involved in stimulating various biological processes in the target cells through neurite regrowth and neuron survival, microglia polarization, exosome biogenesis, cellular motility, BBB integrity, immunomodulation, inflammation regulation, neurogenesis, angiogenesis, extracellular matrix modification, antioxidation, self-renewal, or differentiation .Despite various MSC-related studies, some scientists still turn their attention to other stem cells Exos (SC-Exos) or stem cell EVs (SC-EVs) for the treatment of TBI .Adipose-derived stem cells (ADSC-Exos) might have beneficial biological functions after TBI. On the one hand, Exos derived from human ADSC-Exos reduce motor and cognitive impairments following TBI. ADSC-Exos containing MALAT1 may reduce cortical damage via modulating the NRTK3 (TrkC)/MAPK pathway, and the therapeutic window would span at least 48 h . On the Similar to MSCs, SC-Exos reverse inflammation following TBI via regulating the key resident immune cells and neurogenesis in the CNS. SC-Exos derived from human exfoliated deciduous teeth (hEDSC-Exos) may promote functional motor recovery and decelerate neuroinflammation by shifting microglia M1/M2 polarization in the TBI rats model . Human eAlthough the cause of TBI is largely unavoidable, the consequently impaired responses including inflammatory response to injury may require therapeutic intervention . HoweverSince peripheral blood is easier to collect than damaged brain tissue, a large number of HCD-Exos-related clinical studies have been carried out. The discovery of possible trauma-specific biomarkers in peripheral blood samples could serve as a de-facto “liquid biopsy” for TBI and could greatly aid clinicians in making the correct differential diagnosis and assessment of TBI. A multicenter study of 195 veterans involved in the chronic effects of neurotrauma consortium suggested that repetitive chronic TBI may contribute to chronic neuropsychological symptoms including loss of consciousness or posttraumatic amnesia. Outcomes in TBI patients were associated with elevated p-tau and tau from plasma cell-derived exosomes (PCD-Exos) that may provide peripheral sources of central and informative biomarkers in remote TBI . SimilarThe levels of miRNAs in Exos and EVs of peripherally circulating cells have been assessed in several clinical TBI-related studies in search of promising biomarkers. Vorn et al. profiled peripheral blood cell-derived Exos (PBCD-Exos) and their microRNAs from young adults with or without chronic mild TBI and identified 25 significantly dysregulated microRNAs associated with pathways of neurological disease, psychological disease and organismal injury and abnormalities, thereby resulting in the possibility of long-lasting post-injury symptoms. Moreover, pathway analysis revealed that 14 PBCD-Exos-miRNAs were associated with neurological disorders, 13 with psychological disorders and 23 with organismal damage and abnormalities . Devoto Similar to AD-Exos, ND-Exos, and MSCs-Exos, recently published studies revealed that endothelial cell-derived Exos/EVs/MVs may also promote angiogenesis and BBB continuity after TBI. It is well known that brain microvascular endothelial cells (BMECs) are one of the major components of BBB that maintain brain homeostasis. Endothelial progenitor cell-derived microvesicles have been reported to promote angiogenesis in rat BMECs in vitro . In an e181 in CSFCD-EVs were positively correlated with the levels of t-tau and p-tau181 in total CSF in those participants, respectively. MAPT (tau) may be a potential upstream regulator for predicting the risk of chronic traumatic encephalopathy in TBI players [Furthermore, although CSF collection is more difficult than peripheral blood, few investigators have recently designed CSF cell-derived Exos/EVs/MVs (CSFCD- Exos/EVs/MVs) related studies in TBI patients. Manek et al. have found elevated levels of GFAP, UCH-L1, presynaptic terminal protein synaptophysin and αII-spectrin breakdown products (BDPs) in CSFCD-MVs/Exos from severe TBI patients. These CSFCD-MVs/Exos were likely derived from various brain cell types after TBI—neurons and astroglia, for example . The res players . It shou players .Purely humoral pathways were believed to be responsible for initiating the acute phase response after TBI; however, new studies have raised doubts about these views. Studies have revealed that vagotomized animals continue to present a peripheral response to CNS injury . AlthougSaliva, intriguingly, also secretes EVs ,118. A cFurthermore, in an orthopedic-related study, investigators observed that miRNA-1224 was upregulated in bone marrow-derived extracellular vesicle (BM-EVs) cargoes of TBI mice. These BM-EVs activated NF-κB signaling genes and enhanced their colony-forming ability and osteoclast differentiation efficacy, leading to the reversal of bone loss and reduction in fracture rate  Table 7Table 7.Despite the attraction of specific cerebral cell-derived Exos, few studies have focused on purely brain-derived Exos, EVs, or MVs (BD-Exos/EVs/MVs), regardless of the cell types . TBI incSurprisingly, a research team working on coagulopathy after TBI revealed that lactadherin might prevent coagulopathy and improve the survival of severe TBI mice via promoting BD-MVs clearance . MoreoveOne of the major problems with the clinical application of cell-derived Exos or EVs treatment is the large variability in cell quality, due to the usage of different donors and their tissues, known as donor heterogeneity ,131,132.Despite recent efforts to understand the role of various cell-derived EVs and Exos in CNS diseases, future research is required to determine the role of cell-derived Exos, especially their associated biomolecules, their presence in both healthy and abnormal cells, and their functional effect. Identifying the effect of cell-derived exosomes on both shorter and longer distance targets and their survival strategies may lead to the discovery and development of new diagnostic and therapeutic approaches. To further elucidate the roles of Exos/EVs in the pathophysiology of various CNS diseases, further narrative studies on various biomolecules in cell-derived Exos or cell-derived EVs are required.Exos-based cell-free therapy delivers targeted regulatory genes such as miRNAs to enhance multifaceted aspects of neuroplasticity, reduce neuroinflammation, and enhance neurological recovery for effective and reparative therapies for several nerve injuries following TBI. In addition to the role of the miRNAs, further investigations of Exo proteins could fully investigate the active trophic mechanisms underlying Exos-induced therapeutic effects in TBI. Although Exos provide promising therapeutic effects in rodent models of TBI and some clinical patients, further investigations are required for clinical translation. Meanwhile, despite the critical and prominent role of Exos/EVs-derived miRNA in ameliorating TBI outcomes and modulating brain recovery using experimental models and clinical studies, further clinical studies are required to investigate human brain efficacy, limit side effects, and improve targeted mechanisms in the human body.Overall, we show that some neural cells, especially neural stem cells, astrocytes and microglia, and their miRNAs such as miR-124, miR-141, miR-32, etc., may become promising strategies for targeted drug delivery in the treatment of TBI. However, in this review, we cover the broad topic of Exos and their miRNAs with TBI, and therefore it appears narrow to discuss Exos and their miRNA-related signaling pathways and molecular mechanisms in TBI. A more detailed summary is required in the future.


The term ferroptosis was put forward in 2012 and has been researched exponentially over the past few years. Ferroptosis is an unconventional pattern of iron-dependent programmed cell death, which belongs to a type of necrosis and is distinguished from apoptosis and autophagy. Actuated by iron-dependent phospholipid peroxidation, ferroptosis is modulated by various cellular metabolic and signaling pathways, including amino acid, lipid, iron, and mitochondrial metabolism. Notably, ferroptosis is associated with numerous diseases and plays a double-edged sword role. Particularly, metastasis-prone or highly-mutated tumor cells are sensitive to ferroptosis. Hence, inducing or prohibiting ferroptosis in tumor cells has vastly promising potential in treating drug-resistant cancers. Immunotolerant cancer cells are not sensitive to the traditional cell death pathway such as apoptosis and necroptosis, while ferroptosis plays a crucial role in mediating tumor and immune cells to antagonize immune tolerance, which has broad prospects in the clinical setting. Herein, we summarized the mechanisms and delineated the regulatory network of ferroptosis, emphasized its dual role in mediating immune tolerance, proposed its significant clinical benefits in the tumor immune microenvironment, and ultimately presented some provocative doubts. This review aims to provide practical guidelines and research directions for the clinical practice of ferroptosis in treating immune-resistant tumors. Ferroptosis is a programmed cell death induced by iron-dependent lipid peroxidation, which is modulated by various cellular metabolic and signaling pathways.Ferroptosis has been linked to various diseases and acts as a double-edged sword while mediating immune tolerance.Drug-resistant cancer cells, especially those in a mesenchymal state and prone to metastasis, are remarkably sensitive to ferroptosis, which could bring a promising strategy for drug-resistant cancer treatment.The mechanisms and vulnerability of ferroptosis in varied tumor cells demand further investigations to target precisely and maximize clinical therapeutic benefits.How to modulate immune tolerance by inducing or suppressing cellular ferroptosis in an artificially regulated manner still should be explored.Discovering a method to balance the dual role of ferroptosis in tumor cells, anti-tumor immune cells, and immunosuppressive cells is especially crucial to broaden the application of ferroptosis in anti-cancer treatment.Currently whether ferroptosis is immunogenic and whether mitochondria play an essential role in all forms of ferroptosis or only GPX4 inhibition-induced ferroptosis are controversial and still require experimental demonstrations in the future.Cells serve as the fundamental organizational unit of life. Their multiple activities, including proliferation, differentiation, and ultimately cell death, are essential for ontogeny, homeostasis, and disease progression . Among tDuring the neoplastic process, tumor cells obtain immune tolerance to elude immunity, thereby contributing to the defeat of targeted therapy . Immune Ferroptosis could be observed throughout the process of peripheral immunotolerance –42. As aDolma et al. began high-throughput screening in the early 20th century in search of new small-molecule anti-cancer therapies, and in 2003 published a series of compounds that can induce a different mode of cell death from necrosis and apoptosis  Table 11. In subTwo pivotal molecules in the amino acid metabolism of ferroptosis are GPX4 and system xc- Fig. . GPX4 beIn the 1950s, vitamin E and cysteine, as well as the trace element selenium, were certified as the bond that restrained lipid peroxidation –52. LipiFrom the term “ferroptosis”, iron metabolism serves a vital function in the death of ferroptotic cells, including iron uptake, utilization, storage, and efflux . Cells gFor lipid peroxidation and ferroptosis, the generation of mitochondrial reactive oxygen species (ROS) is crucial. As an essential supplier of ROS, mitochondria could generate hydroxyl radicals via the Fenton reaction, and then drives the PUFA-FL peroxidation . The mitThe hippo-YAP pathway in ferroptosis, which could be involved in multiple biological functions such as cell proliferation, was identified by the heightened resistance of densely grown cells to cysteine deficiency- and GPX4 degradation-induced ferroptosis , 77. MecEnergetic loss induced by the energy and metabolic disorder could bring about a serial breakdown of systems required to retain homeostasis, consequently contributing to cell death , 79. StrHypoxia-inducible factor (HIF) is composed of an oxygen-depleted α-subunit consisting of HIF1α, EPAS1 (HIF2α), and HIF3α, and a constitutively expressed β-subunit (ARNT) . In specHIF possesses a double capacity in modulating ferroptosis . HypoxiaThe epithelial-mesenchymal transition (EMT) is a cellular process whereby epithelial cells repress characteristics, such as the polarity and intercellular adhesion, and progressively attain migratory and invasive faculties relevant to the mesenchymal phenotype .In clinical practice, EMT is considered a cellular process to yield stem cells causing tumor metastasis and treatment resistance, for instance, SNAI1, TWIST1, and ZEB1 . EquivalFerroptosis is a critical anti-tumor mechanism. Tremendous evidence suggests that the tumor has evolved at least three mechanisms to avoid ferroptosis, such as downregulating PUFA-PL levels and lipid peroxidation, astricting the storage of iron in LIP, and uprising defense systems against ferroptosis.The antioxidant enzyme GPX4 is the only GPX member that straightway converts phospholipid hydroperoxide to phospholipid hydroxyphospholipid Fig. , servingFerroptosis suppressor protein 1 (FSP1), with its plasma membrane localization being a prerequisite for its function, operates as a NAD(P)H-dependent oxidoreductase capable of converting CoQ to CoQH2 , 15, 98.A recent study unveiled a dihydroorotate dehydrogenase (DHODH)-mediated ferroptosis defense system in mitochondria that could compensate for GPX4 deficiencies to antitoxify mitochondrial lipid peroxidation . RenderiGTP cyclohydrolase 1 (GCH1) is also identified as a conditioner of ferroptosis , 17. BH4The finding of the antioxidant effect of vitamin K predates the term ferroptosis, which has recently been demonstrated to be essential in anti-ferroptotic activity , 101. AlIn certain cancer cells, not only the specific cellular states but also mutations in some genes will exhibit unexpected sensitivity to ferroptosis. As for the metabolic features, therapy-refractory cancer cells in certain statuses could perform surprisingly sensitive to ferroptosis , 105. CaAdditionally, genetic mutations, for instance, the inactivation of tumor suppressor genes in certain cases, also increase susceptibility to ferroptosis. Incapacitation of any element of the E-cadherin-NF2-Hippo pathway contributes to the expression or activity of its downstream molecule YAP, leaving cancer cells or mutant tumors like NF2- mutant mesotheliomas especially vulnerable to FINs , 109.Ferroptosis can be suppressed by GPX4-dependent and GPX-independent methods. Once one method is inactivated or exhausted, cancer cells become extremely reliant on the other method to fight against ferroptosis and are supremely sensitive to ferroptosis induced by the other defense system.Inducing and maintaining robust immune tolerance have been the holy grail of immunology for decades. How to effectively restrain tumor immune tolerance still requires long-term investigation.Peripheral immune tolerance can be induced by the co-stimulatory or co-inhibitory signaling pathway, equally vital as which, additional controls on the surface of activated T cells, acknowledged as immune checkpoints, can also generate active immune tolerance. Another key mechanism is the presence of specialized cell populations designed to suppress pathogenic immune responses that inadvertently target self-tissue . FerroptImmune cell functions are regulated by co-inhibitory and co-stimulatory receptors. Effective activation of these receptors may have significant therapeutic benefits and prospects in anti-cancer immunity –114. FerEffective T cell signaling requires both participation of primary antigen-specific receptors and a second costimulatory signal to generate proliferation, differentiation, and survival . As a coCTLA-4 blockade contributed to the CD28-mediated proliferation of tumor-associated Tregs, inducing tumor immune tolerance ultimately. This procedure allows polyclonal Treg repertoire to be pre-enriched for recognizing antigens submitted by tumor-associated conventional dendritic cells (CDCs). Unsteady CDC contacts ensured Treg capability, while CTLA-4-mediated downregulation of CDC co-stimulatory B7 proteins, via Tregs, resulted in contact instability. CTLA-4-blockade sparked CD28-dependent Tregs hyper-proliferation in the TME. Therefore, the blockade of CTLA-4 may lessen therapeutic benefits in cancer patients .Studies revealed that CTLA-4 expression was higher in high ferroptosis scores groups than in the lower ones. In bladder cancer, low ferropscores indicated a better immunotherapy response, while in clear cell renal cell carcinoma, the high ferropscores were characterized by a higher immune and enhanced response to anti-CTLA-4 immunotherapy , 118. AcCD40 is involved in humoral immunity and inflammatory response. Belonging to the tumor necrosis factor (TNF) receptor superfamily, CD40 is expressed in a variety of cells, including antigen-presenting cells (APCs) and fibroblasts –124. CD1As the most well-characterized damage-associated molecular patterns (DAMPs) implicated in immunogenic cell death, ATP and high mobility group box chromosomal protein 1 (HMGB1) have been elucidated to be automatically emitted following the timeline of ferroptosis, which also serve as immune signals correlated with the immunogenicity of early ferroptotic cancer cells. Further, ATP and HMGB1 may be essential in accelerating ferroptotic immunogenic cell death (ICD) . TherefoCD86 could prohibit immune tolerance . As reseImmunotherapy is a promising strategy to treat malignant tumors by harnessing the cytotoxic potential of the immune system, especially tumor-specific cytotoxic T cells. ICIs have an extensive impact and early success in the clinic. Such as PD-1 ligand (PD-L1) within TME, the expression of immune checkpoint molecules has been shown to predict response to ICIs in some but not all cases , 150. SoPD-1 and PD-L1 are significant proteins for immune regulation and have a dual impact on cancer prognosis . PD-L1 eIn prostate cancer, PD-L1 was positively related to heterogeneous nuclear ribonucleoprotein L (HnRNPL) , which o3O4-siPD-L1@M-BV2, was constructed to facilitate ferroptosis for immunotherapy of drug-resistant GBM. A cascade of amplification between ferroptosis and immune activation was formed via the system, ultimately prohibiting tumor growth [Glioblastoma (GBM) is an invasive intracranial malignant tumor . Frustrar growth .Tumor-derived exosomes could restrain DC maturation, down-regulate the expression of surface markers like CD80, CD86, and MHC-II, and up-regulate the expression of CD11b and PD-L1, which ultimately obstructed the anti-tumor activity of Teff and reinforced immune evasion –187. XieAlthough immunotherapy with ICIs has generated significant positive applications in a subset of cancer patients, there are still numerous mechanisms of tumors to motivate drug resistance. It was revealed that TYRO3 prohibited the ferroptosis of tumor cells induced by anti-PD-1/PD-L1 via the AKT/NRF2 axis and amplified a pro-tumor microenvironment by downgrading the ratio of M1/M2 macrophages, consequently contributing to anti-PD-1/PD-L1 treatment . Thus, TSimultaneous induction of cancer-target cytotoxicity and anti-tumor immunity could be a prospective method for treating drug-resistant tumors. An investigation highlighted the potential of zero-valent-iron nanoparticles (ZVI-NP) as an innovative integrative cancer-fighting strategy. Notably, ZVI-NP enhanced anti-tumor immunity via transforming pro-tumor M2 macrophages into anti-tumor M1, reducing Tregs and down-regulating CTLA-4 and PD-1 in CD8 + T cells to provoke their cytolytic activity against cancer cells. Therefore, the dual mechanism of anti-cancer activity of ZVI-NPs insightfully exploited the prospect of novel anti-cancer therapies while reducing adverse effects and improving prognosis . To concNevertheless, ferroptosis in glioma might generate an immunosuppressive microenvironment, eventually contributing to immunotherapy resistance. Enhanced ferroptosis revealed the induction of immune cell activation and infiltration, but attenuated anti-tumor cytotoxic killing, among which tumor-associated macrophages (TAMs) were involved in ferroptosis-mediated immunosuppression. TAM was the most abundant immune cell in GBM tissue and initiated pro-inflammatory (M1) or immunosuppressant (M2) responses according to its polarization state. Patients with enhanced ferroptosis were characterized by recruitment of TAM and M2 polarization. The combination of ferroptosis obstacle with PD-1/L1 blockade triggered synergistic therapeutic results in GBM mouse models . Hence, Specialized cell populations include naturally occurring Treg and in vitro- induced Tregs (iTregs), as well as type 1 regulatory T (Tr1) cells and TGF-β producing type 3 helper T (Th3) cells. Of these subpopulations, the most extensively studied are Tregs . By acquTh3 regulatory cells are a unique T cell subset that secretes TGF-β and assists IgA . TGF-β fApart from cancer cells, TME also comprises immune cells, including T cells, macrophages, myeloid-derived suppressor cells (MDSCs), etc. , which aAs a portion of CD4 + T cells, Tregs can hamper anti-tumor immunity. Tregs rapidly induce GPX4 expression after TCR/CD28 co-stimulation activation to avert ferroptosis . FerroptMDSCs in TME have potent immunosuppressive capacity exhibiting resistance to ferroptosis . FerroptIt was widely validated that natural killer (NK) cells were also affected by the ferroptosis, resulting in dysfunction , and NRFIn summary, ferroptosis, a non-traditional pattern of cell death, operates as a double-edged sword in mediating tumor immune tolerance. The mechanisms of ferroptosis in diverse tumor cells require further exploration to maximize clinical therapeutic benefits. Meanwhile, the susceptibility of tumor cells to ferroptosis, to some extent, implies that regulating ferroptosis could be a novel treatment for drug-resistant tumors.Nevertheless, to fully recognize the potential of ferroptosis-inducing strategies in cancer therapy, there are still vast thought-provoking uncertainties to be clarified in future investigations. Firstly, how to target tumor types or patients more accurately that are considered suitable for proferroptotic therapy? How to detect the iron level of tumor cells? It was confirmed that appropriateness could be assessed by iron level, gene, and mutation levels in tumor cells , 223. IrTo sum up, ferroptosis will be an extremely promising research direction and can provide a new method for drug-resistant cancer treatment. It will generate massive clinical benefits only if balancing the double-sided effect of ferroptosis in the tumor immune microenvironment.


Flaviviruses cause a spectrum of potentially severe diseases. Most flaviviruses are transmitted by mosquitoes or ticks and are widely distributed all over the world. Among them, several mosquito-borne flaviviruses are co-epidemic, and the similarity of their antigenicity creates abundant cross-reactive immune responses which complicate their prevention and control. At present, only effective vaccines against yellow fever and Japanese encephalitis have been used clinically, while the optimal vaccines against other flavivirus diseases are still under development. The antibody-dependent enhancement generated by cross-reactive immune responses against different serotypes of dengue virus makes the development of the dengue fever vaccine a bottleneck. It has been proposed that the cross-reactive immunity elicited by prior infection of mosquito-borne flavivirus could also affect the outcome of the subsequent infection of heterologous flavivirus. In this review, we focused on five medically important flaviviruses, and rearranged and recapitulated their cross-reactive immunity in detail from the perspectives of serological experiments in vitro, animal experiments in vivo, and human cohort studies. We look forward to providing references and new insights for the research of flavivirus vaccines and specific prevention. Flaviviruses are mainly transmitted between humans and animals through blood-sucking arthropods including mosquitoes and ticks. At present, mosquito-borne flaviviruses are extremely widespread, of which dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are responsible for significant human morbidity and mortality. Particularly, ZIKV has been transmitted rapidly throughout the Americas in recent years and is one of the causes of congenital brain abnormalities . FollowiFlaviviruses cause a spectrum of potentially severe diseases. Clinical studies  demonstrAedes mosquitoes  or Culex mosquitoes (such as JEV and WNV). The insect vectors tremendously affect viral distribution, so epidemic regions of different flaviviruses overlap widely [Akin to DENV, common mosquito-borne flaviviruses also elicit abundant cross-reactive immune responses and affect each other ,5,6. Mosp widely ,8  live attenuated vaccine (LAV) (YF-17D), Japanese encephalitis (JE) LAV (SA 14-14-2), JE inactivated vaccine (INV)  , and tetFlavivirus of the family Flaviviridae are enveloped viruses with a positive-sense single-stranded RNA genome, encoding 10 proteins—capsid (C), premembrane (prM), envelop (E), and nonstructural proteins 1–5 . The flaviviruses are antigenically related by sharing similar antigenic determinants on their C and E proteins [Viruses in the genus proteins ,15. Accoproteins .We used available published proteomic data to align the sequences of both complete polyprotein and E protein among the five flaviviruses mentioned above . The aliThe structural similarity of flaviviruses provides a molecular basis for cross-reactive immunity. Structural approaches such as cryoelectron microscopy and X-ray crystallography figure out the epitopes of monoclonal antibodies toward flaviviruses . Epitope(1)In vitro serological experimentDENV infection induced few cross-reactive neutralizing antibodies (NAbs) against ZIKV. Swanstrom, et al. revealed that 73% of dengue fever patients’ sera failed to cross-neutralize ZIKV, 18% cross-neutralized ZIKV slightly, and only 9% strongly . Among tInstead, sera or monoclonal antibodies of FL epitope derived from dengue fever convalescent patients could induce ADE of ZIKV in U937 cells . In cyno(2)The in vivo mouse model−/−ifnar Sv/129 mouse model used by Watanabe, et al., all DENV2 inoculated mice survived when challenged with ZIKV, while the passive transfer of DENV2-immune human or mouse sera to −/−ifngr−/−ifnar mice did not affect mortality and viremia levels when challenged with ZIKV [−/−Stat2 mice, which were passively transferred with the DENV-immune human sera [The DENV-immune background could cross-protect mice against ZIKV infection in a way other than cross-reactive antibodies. In the ith ZIKV . Insteadman sera , indicatifnar−/−1 mouse model, depletion of CD8+ T cells enhanced ZIKV infection in DENV2-immune mice, and passive transfer of DENV2-immune CD8+ T cells protected the naïve mice against ZIKV infection [+ T cell epitopes of both DENV and ZIKV significantly reduced ZIKV infection, indicating the protective role of CD4+ T cells in the cross-reactivity [As for the cellular immune response, multiple studies proved that T cell immunity of DENV and ZIKV are cross-reactive ,24,25,26ifnar−/−1 mouse model and the ifnar1 transient blockaded mouse model, confirming a protective role of prior DENV immunity to the fetuses [+ T cell depletion and CD8+ T cell passive transfer experiments proved that the cross-reactive CD8+ T cells elicited by DENV infection were activated by ZIKV, and aggregated on the placenta, thereby acting as a protective factor. In addition, CD4+ T cells also played a supporting role in it [Prior DENV immunity could protect the fetal mice against ZIKV infection. Fetuses of DENV-immune C57BL/6 mice could maintain normal size when the female mice were challenged with ZIKV during pregnancy, with reduced viral loads in the placenta and fetal mice, while the fetuses of non-immune mice showed atrophy and weight loss . The res fetuses . Subsequle in it .Therefore, cellular immunity might play a more protective role than humoral immunity in sequential infection with DENV and ZIKV.(3)In vivo non-human primate animal model+ T cells, and CD8+ T cells when compared to the naïve control [In non-human primate animal models, ZIKV infection did not cause obvious changes in their body temperature, body weight, or hematological parameters. Other indicators such as RNA loads and immune responses could be used to decipher the cross-reactivity ,28,29. W(4)Study in humansCohort studies revealed that neonatal microcephaly caused by ZIKV can occur in people with DENV-immune backgrounds . An ecol(1)In vitro serological experimentLow titers of cross-reactive NAbs to DENV could be detected in sera of C57BL/6 mice and rhesus monkeys with prior exposure to ZIKV, but when the mice were infected twice, no cross-reactive NAbs were detected ,34. Inci(2)In vivo mouse model−/−ifnar Sv/129 mouse model, Watanabe, et al. corroborated that when challenged with DENV2, mice primoinfected with ZIKV displayed lower mortality and viral load than the control group [−/− ifngr−/−ifnar mouse model, both passive transfer of murine ZIKV-immune sera and immunization of inactivated ZIKV elevated the mortality when challenged with DENV2 [In the ol group , suggestth DENV2 , reveali(1)In vitro serological experimentIn a study to investigate experimentally how cross-reactive immunity among flavivirus modifies the course of sequential infection, Saron, et al. used YFV-17D to vaccinate immunocompetent mice and found that the immune sera generated high-titer cross-reactive antibodies against DENV1, but failed to cross-neutralize DENV1 .(2)In vivo mouse modelIn the mouse model, Saron, et al. confirmed that either the immune history of YFV-17D or passive transfer of YFV-17D immune sera or spleen cells did not affect the DENV1 viral load in the mice’s lymph nodes . Consist+ and CD8+ effector T cells significantly increased after sequential infection of DENV1 [However, DENV1 antigens could stimulate the proliferation and activation of splenocytes harvested from the YFV-immune mice, and the numbers of activated CD4of DENV1 , demonst(3)Study in humans+ and CD8+ T cell immunity against other flaviviruses including DENV [In a retrospective study in Brazil, Luppe, et al. evaluated the association between YF vaccination and dengue severity in 11448 lab-confirmed dengue cases. They concluded that YF vaccination did not affect the clinical symptoms of dengue patients . Anothering DENV .The finding of Saron’s study displayed that DENV1-immune mouse sera embodied no cross-reactive antibodies or NAbs against YFV, and the proliferation of splenocytes could not be stimulated by YFV-17D antigens .During the outbreak of YF in a jungle garrison and its detachment, Izurieta, et al. collected the sera of 338 survivors (97% of the whole team). They found no relationship between the level of DENV reactive antibodies and the risk for YFV infection but supposed that prior DENV immunity significantly alleviated the severity of the YF disease . However(1)In vitro serological experimentELISA testing proved that sera of JEV-immune mice or humans contained ZIKV cross-reactive antibodies, and He, et al. demonstrated that the titers of JEV antibodies were positively correlated with the titers of ZIKV cross-reactive antibodies in ZIKV non-endemic areas ,42. Howe(2)In vivo mouse model−/−ifnar mouse model, Zhang, et al. reported that the immune history of JE LAV could protect the mice against ZIKV infection without the mediation of cross-reactive antibodies [ifnar1 transiently blockaded mouse model [By sequential infection of JEV–ZIKV and passive administration of JEV antisera in the tibodies . Passivese model ,43. Addi−/−ifnar adult mouse model, depletion of CD8+ T cells increased mortality, weight loss, and severity of clinical symptoms during ZIKV infection, indicating that CD8+ T cells were indispensable for the protective effect in the sequential infection of JEV–ZIKV [+ T cells conferred less efficient heterotypic protection against ZIKV infection [+ T cells ameliorated the behaviors of one-day-old suckling mice during episodes of ZIKV infection and abrogated the ADE of ZIKV elicited by the antisera of JEV [Cellular immunity played an important role in the cross-protective effect of prior JEV immunity on the sequential ZIKV infection ,45. In tJEV–ZIKV . Howevernfection . In conta of JEV . In a wo(3)Study in humans+ T cell responses, with more obviousness in conserved epitopes among flaviviruses, suggesting that prior JEV immunity altered the immunogenicity of subsequent ZIKV vaccine [In a phase 1 clinical trial of a ZIKV vaccine, participants preimmunized with the JEV vaccine maintained more durable ZIKV-specific CD4 vaccine .ZIKV-immune murine sera demonstrated prominent JEV cross-reactive antibody responses. Adoptive transfer of ZIKV-immune sera led to the earlier death of one-day-old suckling mice challenged with JEV than the mock sera, indicating cross-reactive antibodies in anti-ZIKV sera induced ADE of JEV . However+ T cells displayed a cross-protective effect against JEV infection in the suckling mouse model [Passive transfer of ZIKV-immune CD8se model . This di(1)In vitro serological experimentJEV vaccination elicited high-titer cross-reactive antibodies against DENV1–4 in humans or mice ,47. Li, (2)In vivo mouse modelPrior JEV immunity was cross-protective against DENV in immune-competent mouse models ,48. Li, Cross-reactive NAbs and cytokines elicited by JE vaccination were boosted during the subsequent DENV challenge, suggesting that prior JEV immunity positively modulated both humoral and cellular immune responses against subsequent DENV infection ,49. Howe(3)Study in humansPrior anti-JEV immunity promoted the development of dengue symptoms. In a prospective cohort study conducted in Thailand, Anderson, et al. revealed that among dengue fever patients without anamnestic DENV serology, the positivity of anti-JEV NAbs elevated the probability of symptomatic infection . In addiTo ascertain the quality of cross-reactive antibodies elicited by various flaviviruses, Saron, et al. tested IgG and NAbs levels of sera of flavivirus-immune immunocompetent mice, and proved that DENV-immune sera were slightly cross-reactive against JEV while with no cross-neutralizing ability .In the human study, an analysis of clinical data of 182 JE cases revealed that patients who were dengue IgG-positive performed better in clinical outcomes and prognoses than dengue IgG-negative patients , suggest(1)In vitro experimentJE-vaccinated human sera exhibited cross-reactivity against WNV. Yamshchikov, et al. immunized seven volunteers with JE INV and YFV-17D, monitoring their seroconversion for four years . They deHowever, in the mouse model, other research demonstrated that JE INV induced low titers of WNV cross-reactive NAbs only in the presence of adjuvants . Similar(2)In vivo mouse modelExperiments in various mouse models revealed that prior JEV immunity promoted cross-protection against WNV infection ,55. In a+ T cells in the cross-protective effect [+ T cells to C57BL/6 mice significantly ameliorated their mortality and weight change during WNV infection [Cellular immunity was less important in the cross-protection. In sequential infection of JEV–WNV, Petrovsky, et al. observed identical cross-protection between β2-microglobulin-deficient mice and immune-competent mice, confirming the needlessness of CD8e effect . Howevernfection , demonstThe considerable sequence identity created antigenic similarities between JEV and WNV . NowadayFlaviviridae viruses spread worldwide and have a far-reaching effect on human life. Investigating the interactions among flaviviruses can provide theoretical clues for the prediction of flavivirus epidemics and the development of effective vaccines. We retrospected the research of cross-reactivity among several important mosquito-borne flaviviruses in detail. Prior flavivirus immunity mentioned above achieved cross-reactive effects on resisting subsequent infection of heterologous flaviviruses besides the YFV ( the YFV , which i the YFV .At present, there are still some limitations in the test of cross-reaction, especially in the evaluation of ADE. Although Fcγ receptor-bearing cells have been widely used to measure the enhancement of flavivirus infection ,37, reacThe cross-reaction depends on the balance between cross-reactive humoral immunity and cross-reactive cellular immunity, and the equilibrium point in the tug of war varies with the immunological conditions of the biosome. However, immune-competent mouse or non-human primate models are not susceptible to some flaviviruses, such as DENV and ZIKV. The relevant studies ,27,43,45In recent years, demographic characteristics demonstrated that fewer severe dengue cases in China were observed than that in other dengue hyperendemic countries . MeanwhiCo-circulation of different flaviviruses has made people living in the epidemic regions suffer a lot. It is of great significance to figure out the impact of preexisting cross-reactive immune responses upon subsequent flavivirus infections. These studies provoke cautions for the potential risk of enhancement in sequential infections, especially in ZIKV possible epidemic regions. Additionally, the development of diagnostic reagents for flavivirus infections should avoid cross-reactive epitopes to improve the specificity. For the invention of vaccines, sub-neutralizing antibodies that bind to poorly accessible epitopes need to be carefully eschewed ,68. It w


TREK-1/TRAAK channels are activated by membrane depolarization as well as various stimuli, such as temperature, pH, arachidonic acid, and mechanical membrane stretch. Although membrane mechanical stretch has been suggested to modulate action potential conduction, how membrane stretching modulates intrinsic electrophysiological properties at the node of Ranvier remains unclear. In the present study, we examined the effects of membrane stretch on neuronal membranes at the node of Ranvier in rat sciatic nerves. The single-channel conductance was approximately 90 pS at 80 mV. Membrane stretch increased the single-channel event numbers and open probability in a pressure-dependent manner. Consistent with single-channel activity, intra-pipette positive pressure increased outward leak currents and decreased membrane excitability in a whole-cell configuration. Furthermore, blockage of TREK-1/TRAAK channels by Ba2+ reversed the changes in the intrinsic electrophysiological properties induced by intra-pipette pressure. These results indicate that the activation of mechanosensitive TREK-1/TRAAK channels may suppress neuronal excitability following axonal stretch. Our findings suggest that TREK-1/TRAAK channels may play an important role in the prevention of ectopic action potential discharge at the axon by intense mechanical nerve stretch under physiological conditions.Saltatory conduction is the propagation of action potentials along myelinated nerves, which enables fast propagation through the node of Ranvier. Recently, we demonstrated that K2P channels, TWIK-related KThe online version contains supplementary material available at 10.1186/s13041-023-01000-6. Liu et al. recently developed an experimental technique to apply an axonal mechanical stretch to ex vivo sciatic nerve preparation. Using these methods, they demonstrated that mechanical nerve stretch instantaneously mediates conduction delay in A-fibers [The axon ensures stable action potential (AP) propagation from the axon initial segment to the axonal terminus . While ativation , 5. Intetivation . The suptivation , 9. SincA-fibers . AlthougTo determine the effect of membrane stretch at NRs of myelinated sensory nerves, we performed a pressure-clamped patch-clamp on intact NRs of rat sciatic nerve  Fig. 1AA1A. We fleak) currents following depolarizing voltage steps mainly mediated by activation of TREK-1/TRAAK channels [leak currents by this activation at the NRs, we applied positive pressure into cells through a recording pipette under the whole-cell configuration (Fig. leak conductance compared to that at 0 mmHg (Fig. 2+. The bath application of Ba2+ significantly suppressed pressure-enhanced outward currents and IKleak conductance (Fig. At NRs, strong leak potassium outward (lKchannels . To dete2+ reversed the changes in the intrinsic electrophysiological properties induced by intra-pipette pressure (Fig. We further confirmed whether membrane stretch modulates nodal membrane excitability in NRs in the current-clamp mode. The input resistance was significantly decreased by 10 mmHg intra-pipette positive pressure (Fig. + channels [leak currents and suppressed membrane excitability. Furthermore, pharmacological blockage of TREK-1/TRAAK channels reversed the changes in intrinsic electrophysiological properties induced by intra-pipette pressure. These results indicate the importance of TREK-1/TRAAK channels in the prevention of ectopic AP discharge at the axon by intense mechanical nerve stretch under physiological conditions.In conclusion, our study demonstrates that mechanosensitive TREK-1/TRAAK channels may be activated by axonal stretch. The activation could suppress neuronal excitability. We previously found that TREK-1 and TRAAK are highly expressed in the NRs of mammalian sensory nerves, which leads to AP repolarization instead of voltage-gated Kchannels . In the As a limitation of this study, we used intra-pipette pressure to study ion channel function at the NRs instead of axonal stretch, because the membrane seal for patch-clamp recording is easily disrupted by membrane displacement. Thus, the application of mechanical pressure with a high-speed pressure-clamp device may not fully represent axonal stretch.In conclusion, our study demonstrates the effect of membrane stretching on the intrinsic electrophysiological properties of NR. The findings provide important insights into the pathology of diseases, such as demyelination.Additional file 1. Materials and methods.


PD-L1 expression is associated with immune suppression, whereas expression of Hyal2 is associated with inflammation, because Hyal2+ myeloid cells can degrade the extracellular hyaluronan (HA), leading to the accumulation of pro-inflammatory HA fragments with low molecular weight. These findings implicate the potential involvement of monocytic MDSCs in both tumor-associated immune suppression and cancer-related inflammation. Analysis of organotypic tumor-tissue slice cultures prepared from cancer tissue of the same patient revealed the significant presence of PD-L1+ HLA-DR+ macrophage-like or dendritic cell-like antigen-presenting cells in tumor stroma. Interestingly, stroma-associated PD-L1+ cells frequently have intracellular hyaluronan. Collectively, data presented in this study suggest that the interplay between tumor-recruited myeloid cells and stromal HA may contribute to the inflammation and immune tolerance in kidney cancer.Renal cell carcinoma (RCC) patients frequently have increased number of immunosuppressive myeloid cells in circulation. High number of myeloid-derived suppressor cells (MDSCs) in the blood are associated with immune suppression as well as with cancer-related inflammation which drives the mobilization of myeloid cells to tumor tissue. Here, we show that peripheral blood from a previously untreated RCC patient has increased the number of monocytic CD33 More recently, it was demonstrated that monocytic MDSCs in patients with bladder cancer, in contrast to healthy donors, frequently co-express membrane-bound enzyme hyaluronidase 2 . Here, we show an increased presence of PD-L1+ myeloid cells in both peripheral blood and tumor tissue from a previously untreated patient with clear cell RCC. Furthermore, tumor-infiltrating PD-L1+ myeloid cells express a marker of antigen-presenting cells HLA-DR and show a significant amount of internalized hyaluronan, indicating the possible contribution of stroma and tumor-associated hyaluronan (HA) in the modulation of immune function of myeloid cells including antigen-presentation.Immune checkpoint inhibitors have improved the treatment of a broad spectrum of cancers including renal cell carcinoma (RCC), metastatic melanoma, and non-small lung cancer. These humanized monoclonal antibodies target inhibitory receptors  and ligands (PD-L1) expressed on T lymphocytes, antigen-presenting cells, and tumor cells and elicit an anti-tumor response by stimulating the immune system . HoweverThe 67-year-old man diagnosed with non-metastatic cell clear RCC with no prior cancer treatment history underwent radical nephrectomy. Clinical samples including peripheral blood and freshly excised kidney tumor tissue were transferred to the research laboratory for analysis. Clinical samples were collected from the patient after obtaining written informed consent. All samples were obtained according to federal guidelines and as approved by the University of Florida institutional review board (IRB). Final pathology demonstrated pT3a N0 clear cell RCC with ISUP grade 3, negative surgical margins, and no presence of adverse markers such as sarcomatoid, rhabdoid, or necrosis.Peripheral blood mononuclear cells (PBMCs) were isolated by gradient density centrifugation using Lymphoprep . CD11b myeloid cells were purified from PBMCs by positive selection using the anti-CD11b microbeads and columns . Immunofluorescent staining and analysis were performed according to the previously described protocol .+ myeloid cells from the peripheral blood of a previously untreated patient with RCC. First, we looked at the presence of CD33+ monocytic and CD15+ granulocytic MDSCs. Data presented in + in a patient with RCC, also co-expressed membrane-bound enzyme Hyal2 , we have isolated CD11bnd PD-L1  and D.2 incubator. The organotypic tumor tissue slice technique, which has been significantly used during recent years, provides a novel approach for studying the stroma-immune interactions and may offer a novel avenue to reduce the translational gap. Thus, cultures of precision-cut tissue slices prepared from freshly excised tissue, create nearly ideal conditions to explore the interaction between tumor stroma and immune cells. Once tumor slices are placed in a culture flask or plate, they start the formation of an adherent stroma which includes an extracellular matrix with attached fibroblasts, macrophages, and other immune cells.In addition to peripheral blood, the Hyal2-expressing myeloid cells can also be found in tumor tissue . To exam+ cells belong to the antigen-presenting cells. Also, staining of RCC stroma for HA revealed the PD-L1+ cells have a marked presence of intracellular HA  (20 kDa). Accumulation of LMW-HA fragments in tumor tissue has been associated with enhanced production of multiple inflammatory and pro-angiogenic factors. Since tumor stroma is rich in HA, it is plausible that tumor stroma is involved in the regulation of an anti-tumor immune response. However, the exact mechanisms of stroma-immune interactions in cancers including RCC remain largely unknown. Our data demonstrate the frequent co-localization of HA and fibroblast-like cells in RCC stroma.The enhanced HA metabolism has previously been described in several major subtypes of RCC such as clear cell, papillary, and chromophobe renal carcinomas . Thus, tal blood . Upon re+ MDSCs. These myeloid cells co-express immunosuppressive ligand PD-L1 as well as membrane-bound enzyme Hyal2. High levels of PD-L1 are associated with immune suppression (+ cells in RCC stroma frequently express immunosuppressive PD-L1 ligand and also show significant amounts of internalized tumor-associated HA. Both tumor epithelial cells and CAFs were recently identified as the main sources of HA in the tumor microenvironment (Detectable cellular HA in the RCC tumor microenvironment was associated with higher tumor grades in patients and has been identified as a prognostically unfavorable subgroup among low-grade carcinomas . Here, wpression –19, wherpression , 20–22. pression , 23. Mulironment . Taken t


Chronic graft-versus-host disease (cGVHD) is a serious immune-mediated complication after allogeneic haematopoietic stem cell transplantation (HSCT), but in patients with malignancy, cGVHD development is associated with superior survival. Lack of reliable biomarkers and clinical underreporting means there is insufficient understanding of cGVHD clinical outcomes and balance between cGVHD treatment and maintaining beneficial graft-versus-tumour effects.We performed a Swedish population-wide registry study following patients who underwent allogeneic HSCT 2006–2015. cGVHD status was retrospectively classified using a real-world method based on the timing and extent of systemic immunosuppressive treatment.cGVHD incidence among patients surviving ≥6 months post-HSCT (n = 1246) was 71.9%, significantly higher than previously reported. 5-year overall survival in patients surviving ≥6 months post-HSCT was 67.7%, 63.3%, and 65.3%, in non-, mild, and moderate-severe cGVHD, respectively. Non-cGVHD patients had a mortality risk almost five-fold higher compared to moderate-severe cGVHD patients 12-months post-HSCT. Moderate-severe cGVHD patients had greater healthcare utilization compared with mild and non cGVHD patients.cGVHD incidence was high among HSCT survivors. Non-cGVHD patients had higher mortality during the first 6 months of follow-up; however, moderate-severe cGVHD patients had more comorbidities and healthcare utilization. This study highlights the urgent need for new treatments and real-time methods to monitor effective immunosuppression after HSCT. Successful allogeneic haematopoietic stem cell transplantation (HSCT) relies on optimizing the balance between graft-versus-host disease and the graft-versus-leukaemia effect to mitigate the risk of disease relapse. Chronic graft-versus-host disease (cGVHD) is a severe complication of HSCT and a leading cause of morbidities following HSCT , 2. The Currently, there are significant clinical limitations to fine-tuning this immunological balance. First, effective therapeutic options are scarce  and relaThe 2005 National Institutes of Health (NIH) Consensus Conference established a framework to characterize cGVHD by standardising diagnostic criteria . New guiThere is limited data on the burden of cGVHD from real-world studies; there are no central databases of patients with cGVHD diagnosed by NIH criteria, and there is a lack of complete data. As an integral part of Swedish healthcare, national registers contain comprehensive details of treatment and drug dispensation, along with inpatient and outpatient visits . SimilarSweden has a universal, tax-funded healthcare system with equal access to all eligible residents. Eligible residents receive a unique personal identification number which enables longitudinal follow-up of individuals from birth to death. For the present study, four national population-based registers held by the National Board of Health and Welfare were linked: the Patient Register, the Cancer Register, the Cause of Death Register, and the Prescribed Drug Register. The diagnostic accuracy and completeness of the Swedish registers are high due to legally mandated reporting .All patients with a record of an allogeneic HSCT between 2006 and 2015 were identified in the Patient Register n = 2147). This cohort and methods used for identification of cGVHD have been described previously 7. This c, 17. ForPatients were classified as non-cGVHD if, after taper of post-HSCT GvHD prophylaxis immunosuppression, they received neither systemic corticosteroids nor systemic immunosuppressive treatment  during the entire observation period. In Sweden, GvHD prophylaxis post-HSCT is discontinued by 3 months for matched sibling donors, and by 5 months for matched unrelated donors.Patients with low-level cGVHD require less intensive immunosuppressive treatment. Based on this rationale, mild cGVHD was defined as patients receiving either corticosteroids or immunosuppressants alone. The following four mild cGVHD groups were defined: 1) patients who received systemic corticosteroid treatment >3 months alone, 2) patients whose last date of systemic corticosteroid treatment ended <3 months before censoring, 3) patients whose last date of systemic corticosteroid treatment ended <6 months before death, and 4) patients who received immunosuppressive treatment only .Treatment modalities are similar for patients with moderate and severe cGVHD, which meant that it was not possible to separate these two groups. Patients with moderate-severe cGVHD require more intensive treatment than those with mild cGVHD. Based on this rationale, the following three moderate-severe cGVHD groups were defined: 1) patients who received corticosteroid treatment (irrespective of duration) and immunosuppressive treatment, 2) patients who received corticosteroid treatment (irrespective of duration) and extracorporeal photopheresis (ECP), and 3) patients who only received ECP.Patients were assigned retrospectively to respective groups based on treatment (or not) received.All analyzes were performed based on patients who survived ≥6 months post-HSCT (index date). This was selected as it was more unlikely to include acute GVHD patients, however it may exclude some patients with true cGVHD who died early. Additionally, it may have included some patients who received donor lymphocyte infusion ≥ 6 months post-HSCT and developed acute GVHD. During the first 6 months post-HSCT, 193 patients died and were excluded from further analysis.Chi-square and Kruskal–Wallis tests were used to compare the demographic and baseline differences between the three cGVHD groups. Overall survival (OS) rates were estimated for each group using the Kaplan-Meier method and the difference among the groups was analyzed by log-rank/Mantel–Haenszel test. The time-dependent hazard ratios (HRs) were estimated using a multivariate Cox-regression model to assess the relative effect of cGVHD on death rates, adjusting for age, sex, calendar year of HSCT, donor and stem cell source. Relapse-related mortality (RRM) and transplant-related mortality (TRM) were determined using data from the Cause of Death Register. When a haematological malignancy was stated as the main cause of a patient’s death, this case was considered for RRM. All other causes of death were considered for TRM.Morbidities were classified using the International Statistical Classification of Diseases and Related Health Problems, 10th revision (ICD-10) codes. Morbidity crude incidence rates with 95% confidence intervals (CIs) were derived from a generalized linear Poisson univariate model for each complication. The adjusted incidence rate ratios (IRRs) and 95% CIs were obtained from a multivariate negative binomial model, adjusting for age, sex, and follow-up year with person-time at risk as offset.Rate of healthcare resource utilization for each follow-up year was calculated as the total number of days in inpatient/outpatient care reported in the Patient Register for all causes, divided by the total days contributed to follow-up time for that follow-up year.survival.Data cleaning and statistical analyzes were performed using the statistical software R version 3.5.1 with the aid of R statistical package Study approval was obtained from the Stockholm Ethical Review Board (dnr 2017-1716-31-1). The study has been granted an exemption from requiring written informed consent by the same ethical review board.We included 1246 patients in the final analysis who underwent HSCT from 2006 to 2015 and met our inclusion criteria. Of these patients, 28.1% were classified as non-cGVHD and 71.9% as cGVHD  . Age, seThe median follow-up time for patients surviving ≥6 months post-HSCT was 3.5 years. The best OS at 1-year post-HSCT was in patients with moderate-severe cGVHD, which decreased with decreasing cGVHD severity status. Moderate-severe cGVHD patients had a 1-year OS rate of 87.5%, compared to 74.3% for non-cGVHD patients and 82.0% for mild cGVHD patients (P < 0.0001) . The nonIn patients who survived the first 6 months post-HSCT, multivariate analysis was adjusted for sex, age, calendar period, donor source, and donor relationship. Multivariate and univariate analysis revealed that during the 0- to 6-month follow-up period (6–12 months post-HSCT), the risk of death was significantly higher in non- and mild cGVHD than in moderate-severe cGVHD patients . There wAfter adjusting for age, sex, and follow-up year, the IRRs for morbidities were assessed across all disease categories in the ICD system . The incWe determined the clinical burden associated with cGVHD in terms of healthcare utilization in patients who survived ≥6 months post-HSCT. Those patients who did not survive to ≥ 6 months post-HSCT (n = 193) were excluded. The number of transplants more than doubled during the 10-year study period, while the proportion of patients with cGVHD stayed approximately constant . More paAllogeneic HSCT is a potential cure for patients with haematological malignancies . While tThe NIH recommendations are the standard criteria used to assess cGVHD although there is a limited uptake in clinical practice (lack of adherence has been reported to be as high as 60%) . A recenIn the current study we have shown that 6–12 months post-HSCT, death rate was higher in the non- and mild cGVHD group compared with the moderate-severe cGVHD group. Strikingly, the non-cGVHD patients who survived ≥6 months post-HSCT had a 4.72 times higher death risk at 6 months of follow-up, compared with those with moderate-severe cGVHD. This suggests a negative clinical impact of over-immunosuppression through GvHD prophylaxis , and that a more personalized approach is required, balancing disease relapse and cGvHD risks. Furthermore, the 1-year overall survival rate improved with increasing cGVHD severity. This improved survival is supported by a systematic review which also showed that patients with cGVHD had higher overall survival compared with those patients with malignancy without cGVHD . PatientTo avoid including patients with acute GVHD, a cut-off 6 months post-HSCT was used. This is the most accepted time frame for the resolution of acute GVHD and the onset of cGVHD. In support of this, others have shown that some clinicians utilizing consensus criteria have historically considered any GvHD (including acute) to be cGvHD if it occurred after 100 days . HoweverHigher rates of morbidities translated into greater healthcare utilization for moderate-severe cGVHD patients. This is evident from the increased proportion of time that moderate-severe cGVHD patients spent in inpatient and outpatient healthcare compared with non- and mild cGVHD patients, highlighting the impact of cGVHD on healthcare resources as well as patient quality of life. We have previously shown that the cumulative total costs during the first 3 years of follow-up post HSCT were EUR 14,887,599, EUR 20,544,056, and EUR 47,811,835 for non-, mild, and moderate–severe groups cGVHD, respectively [Although our method to classify cGVHD, as opposed to the established NIH criteria is novel, it highlights an urgent need for standardized methods to monitor immune function post-HSCT in clinical practice. The necessary tools required to prospectively identify specific patients at risk are currently lacking. To date most research has focused on early identification of patients at risk of developing severe cGVHD with little attention directed at identifying patients who may be over-immunosuppressed. More research should now be directed at this non-cGVHD group who have a significantly increased risk of death during the first year after allogeneic HSCT. Efficient methods are therefore needed to monitor immune function that will enable data-driven treatment decisions to be made in real time. Once these have been identified and implemented, prospective trials can be initiated to validate the observations. Until then, clinical outcomes for patients with non- and moderate-severe cGVHD will remain inferior to patients with mild cGVHD, posing a considerable strain on healthcare delivery.Our registry-based method for identifying patients with cGVHD may not be fully concordant with the NIH cGVHD classification. However, the Prescribed Drug Register in Sweden has previously been used to define disease severity , 30. ImpIn conclusion, our population-based registry study indicates that cGVHD is underreported in Sweden. Patients without cGVHD had significantly increased death rates in the first year post-trantsplant, possibly due to the intensity or duration of GvHD prophylaxis, and suggesting that a more personalized approach balancing relapse risk and cGvHD risk is required. Patients with moderate-severe cGVHD had higher rates of morbidities and healthcare resource utilization versus patients with non- and mild cGVHD. We conclude that to improve patient outcomes, validated clinically approved and standardized assays to measure immune function are needed, as well as novel treatments that better balance the competing requirements of immune suppression and optimisation of graft-versus-leukaemia effects.S1 Fig(TIF)Click here for additional data file.S2 FigNon-cGVHD (A), mild cGVHD (B) and moderate-severe cGVHD patients (C), who survived ≥3 months post-HSCT. Non-cGVHD (D), mild cGVHD (E) and moderate-severe cGVHD (F) who survived ≥6 months post-HSCT.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


H17) cell differentiation, and interleukin-22 production. In addition, microbiota-derived Trp metabolites have direct effects on the vascular endothelium, thus influencing the development of vascular inflammatory phenotypes. Indoxyl sulfate was demonstrated to promote vascular inflammation, whereas indole-3-propionic acid and indole-3-aldehyde had protective roles. Furthermore, there is increasing evidence for a contributory role of microbiota-derived indole-derivatives in blood pressure regulation and hypertension. Interestingly, there are indications for a role of the kynurenine pathway in atherosclerotic lesion development. Here, we provide an overview on the emerging role of gut commensals in the modulation of Trp metabolism and its influence in cardiovascular disease development.The essential amino acid tryptophan (Trp) is metabolized by gut commensals, yielding in compounds that affect innate immune cell functions directly, but also acting on the aryl hydrocarbon receptor (AHR), thus regulating the maintenance of group 3 innate lymphoid cells (ILCs), promoting T helper 17 (T The gut microbiota is the assemblage of microbial communities colonizing the intestinal habitat, with the great majority belonging to the bacterial kingdom, but also comprising fungi, archaea, protists, and viruses , converting Trp to N-formyl-L-kynurenine Taleb  Escherichia coli, Clostridium sp., and Bacteroides sp., converts Trp into indole and its derivatives IAld, IAA, and indole-3-propionic acid (IPA). In the aromatic amino acid aminotransferase and indolelactic acid dehydrogenase–dependent pathway, the enzyme aromatic amino acid transaminase (ArAT) converts Trp into indole-3-pyruvate (IP), which is further metabolized into indole-3-lactic acid (ILA) through the enzyme indolelactic acid dehydrogenase (ILDH). Through the phenyl lactate dehydratase gene cluster (fldAIBC), ILA can further be converted into 3-indoleacrylic acid (IA), which is finally converted into indole-3-propionic acid (IPA)  is associated with the accumulation of the nephrotoxin indoxyl sulfate in the serum of uremic patients, which is due to impaired renal secretion  by correcting the composition of the gut microbiota  immunity  is one example of an autoimmune disease that has been linked to changed Trp metabolism , have direct anti-inflammatory effects on immune cells Fig.  Wlodars. For insCandida albicans and to rotavirus infection  cells  Taleb . IL-22 rEndothelial dysfunction precedes atherosclerosis, one of the most severe forms of vascular inflammation. Analysis of vascular dysfunction in germ-free mouse models indicated that the gut microbiota promotes vascular inflammation and oxidative stress . IPA could induce pregnane-X-receptor (PXR) expression in the aorta of mice and diminished endothelial-dependent vasodilator responsiveness , Lactococcus lactis subsp. lactis (IL1403), Lactobacillus plantarum (FI8595), Streptococcus thermophilus (NCFB2392), Escherichia coli K-12, Morganella morganii , Klebsiella pneumoniae  and Hafnia alvei  were described to produce serotonin directly .Hypertension is a common and serious medical condition that can lead to cardiovascular disease. Elevated blood pressure significantly increases the risks of several organ pathologies in heart, brain, or kidney, leading to major complications such as congestive heart failure, cerebral haemorrhage, and renal failure Doyle . ApproxiLactobacillus murinus. The presence of Lactobacillus has been shown to prevent the formation of TH17 cells and consequently reduce hypertension in mice. Lactobacillus depletion due to increased salt intake was accompanied by decrease in the Trp metabolites IAA and ILA, suggesting a link between Trp metabolites and cardiovascular health -model. Within this study, elevated blood pressure was found to decrease Lactobacillus species involved in gut homeostasis and Trp metabolism -deficient mouse atherosclerosis model, antibiotic treatment resulted in increased atherosclerosis, connected to a loss of intestinal microbiome diversity and alterations in microbial metabolic functional capacity with a major impact on the host serum metabolome  physiology on the molecular and cellular level.