The neuroimmune mechanism underlying neuropathic pain has been extensively studied. Tumor necrosis factor-alpha (TNF-α), a key pro-inflammatory cytokine that drives cytokine storm and stimulates a cascade of other cytokines in pain-related pathways, induces and modulates neuropathic pain by facilitating peripheral (primary afferents) and central  sensitization. Functionally, TNF-α controls the balance between cell survival and death by inducing an inflammatory response and two programmed cell death mechanisms (apoptosis and necroptosis). Necroptosis, a novel form of programmed cell death, is receiving increasing attraction and may trigger neuroinflammation to promote neuropathic pain. Chronic pain is often accompanied by adverse pain-associated emotional reactions and cognitive disorders. Overproduction of TNF-α in supraspinal structures such as the anterior cingulate cortex (ACC) and hippocampus plays an important role in pain-associated emotional disorders and memory deficits and also participates in the modulation of pain transduction. At present, studies reporting on the role of the TNF-α–necroptosis pathway in pain-related disorders are lacking. This review indicates the important research prospects of this pathway in pain modulation based on its role in anxiety, depression and memory deficits associated with other neurodegenerative diseases. In addition, we have summarized studies related to the underlying mechanisms of neuropathic pain mediated by TNF-α and discussed the role of the TNF-α–necroptosis pathway in detail, which may represent an avenue for future therapeutic intervention. Massive unregulated release of pro-inflammatory cytokines (cytokine storm) such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) due to infection, nerve injury or immunotherapy causes organ damage and unbearable pain . NeuropaMicroglia in the healthy brain continuously palpate the surrounding tissue for subtle disturbances  and can TNF-α is important for mammalian immunity and cellular homeostasis. The role of TNF-α as a master regulator in balancing cell survival and death has been extensively studied in various cell types and tissues. As shown in When TNF-α binds to TNFR1 on the membrane surface, the conformation of TNFR1 changes, and TNFR1 complex I is rapidly formed via the recruitment of various proteins, including TRADD, RIPK1, TNF receptor-associated factor 2 (TRAF2) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2). TRADD is very important for the recruitment of TRAF2 and ubiquitination of RIPK1 and complex I. The TGF-activated kinase 1 (TAK1) binding protein (TAB) complex and IκB kinase (IKK) complex consisting of IKK1 can prevent cell death, whereas cIAP1/2 can prevent TNFR1-mediatednecroptosis and promote ubiquitination of RIPK1 ,61. UbiqDeubiquitination of RIPK1 results in the formation of either complex IIa or complex IIb. If caspase-8 is present in cells, TNFR1 complex I recruits TRADD and caspase-8 to form complex IIa , activating a caspase cascade and leading to RIPK1-independent apoptosis (RIA). When receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein are fully expressed and caspase-8 is present, ubiquitination of RIPK1 is inhibited, and it interacts with RIPK3 to form complex IIb , which activates caspase-8 and triggers RIPK1-dependent apoptosis (RDA) ,64.Stimulation of the Fas/TNFR family can not only trigger a canonical ‘extrinsic’ apoptotic pathway but also activate necroptosis inhibited by necrostatin-1 (Nec-1), a specific and potent small-molecule substance, in the absence of intracellular apoptotic signalling . When caTNF-α, which primarily activates a cascade of other cytokines , is upreStudies have shown that TNF-α facilitates heat-induced CGRP release from nociceptor terminals in the skin  and sensSCN8A), another major TTX-S VGSC in the mammalian nervous system, regulates neuronal activity at the axon initial segment [Nav1.6  has been intensively studied in the sensory system. In humans, the loss of function of Nav1.7 leads to a complete inability to sense pain [p-p65 non-transcriptionally gates Nav1.7 channels in the membrane of neurons in rat DRG, and TNF-α contributes to the protein–protein interaction between p-p65 and Nav1.7 within several minutes [TTX-S Nav1.7  , transie+ ions . In respIn the spinal cord, TNF-α enhances excitatory synaptic transmission and increases AMPA- and NMDA-evoked excitatory postsynaptic currents (EPSCs) to induce pain hypersensitivity . After nChanges in higher-order functions, such as learning and memory disorders, anxiety or depression, are critical components of pain phenotypes, especially in a chronic pain state ,117,118.At the molecular level, supraspinal TNF-α modulates excitatory and inhibitory synaptic transmission in different ways . OverproIn addition to TNF-α-mediated neuroinflammation and excitotoxicity , apoptosNecroptosis is characterised by necrotic cell death and autophagy activation ,146. In The literature published on necroptosis in the field of neuroscience from 2012 to 2021 was quantitatively and qualitatively analyzed using the Bibliometrix R Package and VOSviewer software ,150, andKeyword co-occurrence analysis performed using VOSviewer revealed four categories of keywords related to necroptosis, which are shown in red, blue, green and yellow in As mentioned above , necroptNecroptosis, a mode of programmed cell death similar to necrosis and conventional apoptosis, is usually accompanied by plasma membrane rupture, organelle swelling and inflammatory cell infiltration ,147. RIPNeuroinflammation owing to abnormally elevated TNF-α levels in the primary sensory afferent, spinal cord and ACC following peripheral nerve injury contributes to neuropathic pain ,21,71. PChronic pain is often accompanied by adverse pain-associated emotional reactions such as anxiety and depression ,180. Sevα-induced oligodendrocyte necroptosis through interaction with TNFR1 [Earlier studies have confirmed that in patients with severe depression, the loss of glial cells and reduction of neuronal size occur in the deeper cortical layers in ACC and the dorsolateral prefrontal cortex ,200. RIPth TNFR1 . AlthougDepression is closely related to a decline in cognitive abilities such as concentration and memory difficulties  with a dUpregulation of TNF-α ,227,228,The neuroimmune mechanisms underlying neuropathic pain are complex and involve many factors, including inflammatory and anti-inflammatory imbalances that in the most severe form is called cytokine storm. For example, spinal formyl peptide receptor type 2 (FPR2/ALX), a member of the formyl peptide receptors family, plays an analgesic role by reducing cytokines and BDNF . This re


Many patients with heart failure with preserved ejection fraction (HFpEF) are undiagnosed, and UK general practice registers do not typically record heart failure (HF) subtype. Improvements in management of HFpEF is dependent on improved identification and characterisation of patients in primary care.To describe a cohort of patients recruited from primary care with suspected HFpEF and compare patients in whom HFpEF was confirmed and refuted.Baseline data from a longitudinal cohort study of patients with suspected HFpEF recruited from primary care in two areas of England.A screening algorithm and review were used to find patients on HF registers without a record of reduced ejection fraction (EF). Baseline evaluation included cardiac, mental and physical function, clinical characteristics, and patient reported outcomes. Confirmation of HFpEF was clinically adjudicated by a cardiologist.In total, 93 (61%) of 152 patients were confirmed HFpEF. The mean age of patients with HFpEF was 79 years, 46% were female, 80% had hypertension, and 37% took ≥10 medications. Patients with HFpEF were more likely to be obese, pre-frail or frail, report more dyspnoea and fatigue, were more functionally impaired, and less active than patients in whom HFpEF was refuted. Few had attended cardiac rehabilitation.Patients with confirmed HFpEF had frequent multimorbidity, functional impairment, frailty, and polypharmacy. Although comorbid conditions were similar between people with and without HFpEF, the former had more obesity, symptoms, and worse physical function. These findings highlight the potential to optimise wellbeing through comorbidity management, medication rationalisation, rehabilitation, and supported self-management. HFpEF is common  but the condition is often unrecognised and poorly managed. To the authors’ knowledge, no previous studies have provided a detailed characterisation of patients with HFpEF within primary care HF registers. This study confirmed diagnosis and phenotyped a cohort of patients recruited from primary care with possible HFpEF, comparing patients in whom HFpEF was confirmed with patients in whom HFpEF was refuted. Patients with HFpEF were differentiated from patients not meeting HFpEF diagnostic criteria by higher levels of obesity, frailty and symptoms, and worse physical functioning. Self-management and self-monitoring of worsening signs and symptoms of HF were extremely limited in patients with HFpEF. Management of comorbidities in HFpEF is essential but complex. It needs to incorporate medication reviews, and increased use of non-pharmacological interventions such as self-management support and exercise training or cardiac rehabilitation. Polypharmacy could be decreased by better differentiation between patients with HFpEF and heart failure with reduced ejection fraction (HFrEF). Current evidence suggests HFpEF is driven by comorbid conditions, especially obesity, hypertension, diabetes, and kidney disease, leading to systemic inflammation and endothelial microvascular dysfunction. Despite its prevalence, HFpEF remains poorly diagnosed, managed, and researched. Under-recognition of HFpEF relates to lack of awareness and uncertainty regarding its pathophysiology, treatment, and diagnostic criteria. Pathways to HF diagnosis are variable, and limited knowledge of HFpEF and a lack of relevant echocardiographic information leads to under-identification in primary care.HFpEF accounts for half of all HF and 70% of those with HF aged >65 years. Evidence for effective pharmacological treatment specific to HFpEF is sparse. Current recommendations are to control comorbid conditions and use diuretics to manage volume overload. Lack of pharmacological treatment is thought to relate to patient heterogeneity, leading to interest in defining phenotypes that might respond to specific therapy. Phenotyping has been based on populations recruited into clinical trials with limited comorbidity or admitted for acute HF, and thus not representative of most patients in the community. Characterising patients with HFpEF in primary care is an essential step towards improving diagnosis and management, as well as recruiting into trials.Most patients with HF are managed in primary care, especially those with HFpEF who may not be referred to specialists, or if referred not provided with a treatment plan. Patients were recruited based on a search of primary care HF registers for patients with no record of reduced ejection fraction. In this report, the baseline characteristics of those patients in the cohort who were confirmed as having HFpEF are described, and they are compared to patients in whom HFpEF was not confirmed.This analysis presents baseline data from a longitudinal observational study that is a component of the Optimising Management of Patients with Heart Failure with Preserved Ejection Fraction in Primary Care (OPTIMISE HFpEF) study.Study participants were recruited from 30 general practices in two regions of England: East of England and Oxfordshire, Thames Valley. Practices were included from cities, towns, and semi-rural areas varying by Index of Multiple Deprivation (IMD) score from high deprivation to more affluent areas (IMD 2–9). Owing to slow accrual, patients were also recruited from an older persons’ clinic in London and a HF service in Cambridgeshire receiving primary care referrals. The study is supported by an active patient advisory group, and patients were involved in development and analysis.Patients with possible HFpEF were identified via an electronic medical record screening algorithm of HF registers in general practices and physical record screening in the outpatient clinics. The algorithm was designed to screen out patients with codes for left ventricular systolic dysfunction (LVSD) and cardiomyopathy. Patients identified in the electronic search were screened by GPs against study criteria. Exclusion criteria included an EF <50%, moderate to severe systolic dysfunction, significant cognitive impairment, or end-of-life care. Patients deemed eligible were invited to participate by letter from the practice. Those interested attended a baseline assessment where informed consent was obtained.HFpEF diagnosis was clinically adjudicated by a cardiologist based on a global evaluation of the available history of any heart failure symptoms, signs of HF, consideration of natriuretic peptide levels, and evidence of relevant structural heart disease and/or diastolic dysfunction on transthoracic echocardiogram (TTE), as per European Society of Cardiology (ESC) guidelines criteria at start of recruitment . A more Box 1Signs and symptoms of heart failureEjection fraction >50%Elevated natriuretic peptides:NT-proBNP >125 pg/mlBNP >35 pg/mlEvidence of relevant structural heart disease and/or diastolic dysfunction:2 for males and >95 g/m2 for femalesLeft ventricular hypertrophy: left ventricular mass index >115 g/m2Increased left atrial volume index: >34 ml/mEarly diastolic tissue velocity Ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/e’ >13)E/A ratio <1 or >2Deceleration time of mitral valve early diastolic inflow m/s Isovolumetric relaxation timeBNP = brain natriuretic peptide. HFpEF = heart failure with preserved ejection fraction. NT-proBNP = N-terminal pro B-type natriuretic peptide.Variables included: physical characteristics; past medical history and comorbidities; heart function (12-lead electrocardiogram and transthoracic echocardiogram [TTE]); oedema assessment; breathlessness and fatigue (modified Borg); frailty assessment by Clinical Frailty Scale (CFS); Survey of Health, Ageing and Retirement in Europe Frailty Instrument (SHARE-FI); cognition assessment ; physical functioning  and physical activity levels (7-day accelerometer wear); laboratory testing ; anxiety and depression ; HF quality of life (Kansas City Cardiomyopathy Questionnaire [KCCQ]); HF self-care ; HF symptoms (Symptom Status Questionnaire — Heart Failure); and health-related quality of life (EQ-5D-5L). Validated assessments, standardised equipment, and a detailed manual of operations and procedures promoted consistency across sites. to give a sample of 200 patients. It was anticipated that 40% of patients on HF registers would be identifiable as possible HFpEF.The target sample size was 270 recruited, with an estimated 25% not being confirmed as HFpEF2 for categorical variables and t-tests for normally distributed continuous data. Where data are missing, values are reported as such. Statistical analyses were performed using R (version 3.63) and IBM SPSS Statistics (version 27).Patient characteristics and assessments were described using frequencies, measures of central tendency, and proportions as appropriate. Normality for continuous data were assessed using the Shapiro-Wilk test and Q-Q plots. Normally distributed data are presented as mean±standard deviation, non-parametric as median and interquartile range (25%–75%), and categorical data as absolute number and per cent. Descriptive statistics are presented for the cohort, and comparisons according to confirmed HFpEF versus non-HFpEF using χIn primary care, 52% of patients on HF registers were considered eligible. Between July 2018 and November 2019, 152 patients were recruited, 16% of those were eligible . Ninety- Patients with HFpEF walked 65 m less than the non-HFpEF group, and took >2 seconds longer to walk 10 m. Sarcopaenia was more prevalent in the HFpEF versus non-HFpEF group  , although the investigations were only intended to diagnose patients without HFpEF. When delineating by confirmation of HFpEF, patients with HFpEF were more likely to be pre-frail or frail, and have greater functional impairment based on 6MWD and gait speed. Patients with HFpEF were less physically active and spent more time in very low levels of activity compared with those not confirmed HFpEF.= 0.176) .P = 0.085), and mean HbA1c in 39 patients known to have diabetes was 56.4±16.7 mmol/l.Laboratory tests were available for 131 (86%) participants . Values Patient reported outcome measures  showed nIn this cohort of patients recruited mainly from HF registers in primary care (86%), the predominant characteristics of patients with HFpEF were a greater proportion of females, advanced age, and multimorbidity. Significant differences by group were found, as patients with HFpEF had more obesity, pre-frailty or frailty, functional impairment by 6MWD and gait speed, demonstrated lower levels of activity, and had greater likelihood of reporting symptoms, such as dyspnoea and fatigue, than those not confirmed HFpEF.As might be expected in an older multimorbid sample, patients were taking multiple medications. Sixty-five per cent of patients with HFpEF were taking diuretics, but many presented with signs and symptoms of volume overload such as peripheral oedema. Although few abnormalities were found in laboratory values, HbA1c levels in patients with diabetes indicated that glycaemic control was less than optimal. Findings on the EHFScB indicated that few patients with HFpEF agreed with statements that they regularly performed actions recommended for self-management such as monitoring weight gain or notifying a healthcare provider for signs and symptoms of worsening HF. Patients did not report high levels of depression or anxiety symptoms, and quality-of-life scores were moderately high on both the KCCQ and EQ-5D-5L visual analogue scale. Over half of the eligible sample did not respond to the study invitation, and 58% of those responding declined participation. Information about non-responders or those declining was not collected, but it is plausible that some may have had poorer health or not thought the study was relevant to them. Limited recruitment may have introduced bias in the sample; however, it is notable that the sample was older, multimorbid, functionally impaired, and came from both low and high areas of deprivation. The sample was limited by a high proportion of patients not confirmed as HFpEF. Confirmation of HFpEF was clinically adjudicated using symptoms, signs, NT-proBNP, and echocardiogram data, following European Society of Cardiology (ESC) guidelines criteria. Future studies may include additional testing to determine diagnosis.This study presents a well-phenotyped cohort of patients with HFpEF recruited mainly from primary care practices in two regions in England, indicating the challenges and problems faced. Recruitment was slow, and likely limited by focusing on patients on practice HF registers, so patients not yet diagnosed with HF or with HF not added to the register were excluded. Future studies may find more patients by searching the practice adult population for those on diuretics or combinations of medications used for HF. The older patients with HFpEF with multiple comorbid conditions, such as obesity, hypertension, diabetes, and kidney disease, has been described as ‘garden variety’ HFpEF, indicating that this is a frequent phenotype encountered in clinical practice. However, this common phenotype contrasts with HF clinical trials where limited reporting of comorbid conditions and low prevalence of obesity and multimorbidity is usual in recruited patients with HFpEF. Studies have attempted to delineate patients into distinct phenotypes based on clinical and diagnostic characteristics using patient samples from secondary care centres and clinical trials. Currently there is no agreement on distinct phenogroups, and others have called for simpler designations using single characteristics such as sex, obesity, and atrial fibrillation. This analysis fills a gap in the literature by detailing the characteristics of the prevalent patient who is older and multimorbid with HFpEF in primary care, revealing areas of need in their management.The prevalence of comorbidities has been reported to be higher in HFpEF than HFrEF, consistent with the idea that comorbid conditions drive the inflammatory response leading to HFpEF. Overweight and obesity is highly prevalent in patients with HFpEF (up to 80%), as is frailty. A recent analysis of 4605 older patients (mean age 80.3 years) with HFpEF hospitalisation found that 41% had frailty, and that frailty was the most important predictor of re-hospitalisation, and second (after age) for mortality.Multiple studies have shown a greater prevalence of women among populations with HFpEF, although it is unclear whether this is related to higher survival rates of women at older ages, or factors such as the stronger relationship between obesity and incident HFpEF among women compared with men. The average 6MWD difference between groups was 65 m. A recent meta-analysis found each 50 m 6MWD reduction was significantly associated with increased risk of all-cause mortality, readmission rates, and combined death or readmission. Although all patients had low activity levels, the average vector magnitude was lower in those with HFpEF compared with patients without HFpEF, and less than in a UK Biobank sample of patients with HF and in another study of HFpEF.Exercise intolerance in HFpEF is owing to both cardiac and peripheral factors, with pro-inflammatory factors, fatty infiltration, and impaired oxidative metabolism leading to decreased muscle strength. The overall score on the EHFScB Scale was low compared with a sample of 1192 patients with either HFpEF or HFrEF , indicating fewer self-care behaviours among the cohort in the present study.Somewhat surprisingly, despite symptoms and limited functional status, quality-of-life scores were moderately high. The developers of the KCCQ define scores from 50 to 74 as fair to good health status, and ≥75 as good to excellent. Current recommendations to manage comorbid conditions and to use diuretics are not trivial given the number of co-existing conditions, detrimental effects of polypharmacy, and challenges of fluid balance in older adults with renal and functional impairment.The study demonstrates that multimorbidity, polypharmacy, obesity, pre-frailty and frailty, poor physical function, low activity levels, and symptoms are prevalent in patients with HFpEF and present key management challenges. Patients with HFpEF often sit outside of specialist HF services in the UK owing to commissioning restrictions, and primary care therefore takes the lead in managing patients. but is important in ensuring appropriate treatment. For example, a decrease in polypharmacy in HFpEF could be enhanced by differentiation of HFpEF from HFrEF. Medications indicated for HFrEF should not be prescribed unless there is another indication , as they do not exert the same protective effect in HFpEF. Medication reviews in primary care provide the opportunity to consider the necessity for specific medications.Implications for primary care practice begin with the identification of patients with HFpEF, which likely needs specialist support, Scores on the EHFScB indicated that many patients did not practice behaviours related to self-management. Teaching and supporting self-management should be a component of HF reviews, and all providers need to facilitate this partnership with patients.Over half of the patients in both groups were prescribed diuretics, which often limit their ability and willingness to leave the house. The challenge of managing diuresis is further complicated if patients have incontinence, as reported by almost 20% of patients in the sample. Managing fluid balance also requires consideration of patient behaviours and support to enable patients to monitor signs and symptoms, limit fluid and excessive salt intake if appropriate, and know when to contact a healthcare provider. Home-based targeted rehabilitation, such as in the REACH-HFpEF pilot study, may improve patient and carer outcomes and be key to ensuring patient participation. The Rehab-HF trial demonstrated that patients who were recently hospitalised and very frail with HF benefit from rehabilitation.Interventions to improve general health status, such as physical activity, dietary enhancement, and management of breathlessness, should be introduced. Exercise training or bespoke cardiac rehabilitation could be developed and commissioned given the evidence of benefit. The American Heart Association and American College of Cardiology have made a limited recommendation for the use of spironolactone in some patients with HFpEF.New therapies to treat HFpEF may be added to current medication regimens in the future. Indications from recent studies are that medications such as sodium-glucose co-transporter-2 (SGLT2) inhibitors, spironolactone, and sacubitril with valsartan may be effective, even if in specific subgroups.Patients recruited from primary care with confirmed HFpEF demonstrate marked impairment across a range of domains including multimorbidity, functional impairment, and frailty. These findings highlight the need to recognise and record HFpEF as a diagnosis, which would enable clinicians to identify patients and work together to optimise wellbeing through comorbidity management, medication rationalisation, rehabilitation, and self-management.


The adoption of Antiretroviral Therapy (ART) substantially extends the life expectancy and quality of HIV-infected patients. Yet, eliminating the latent reservoirs of HIV to achieve a cure remains an unmet need. The advent of nanomedicine has revolutionized the treatment of HIV/AIDS. The present study explores a unique combination of Tenofovir (TNF) with gold nanoparticles (AuNPs) as a potential therapeutic approach to overcome several limitations of the current ART.TNF-tethered AuNPs were successfully synthesized. Cell viability, genotoxicity, haemolysis, and histopathological studies confirmed the complete safety of the preparation. Most importantly, its anti-HIV1 reverse transcriptase activity was ~ 15 folds higher than the native TNF. In addition, it exhibited potent anti-HIV1 protease activity, a much sought-after target in anti-HIV1 therapeutics. Finally, the in vivo biodistribution studies validated that the AuNPs could reach many tissues/organs, serving as a secure nest for HIV and overcoming the problem of deficient drug delivery to HIV reservoirs.viz. anti-HIV1 and anti-HIV1 protease. These findings are being reported for the first time and highlight the prospects of developing AuNP-TNF as a novel next-generation platform to treat HIV/AIDS.We show that the combination of TNF and AuNPs exhibits multifunctional activity, The online version contains supplementary material available at 10.1186/s12951-022-01750-w. Timothy Ray Brown (the Berlin patient), Adam Castillejo (the London patient), and recently, a woman in New York. The focus, after that, shifted to intense investigation on eradicating the virus. Several human body parts are HIV niches, i.e., HIV reservoirs. Researchers soon realized that eliminating reservoirs of HIV would be a stumbling block to attaining the goal of virus eradication, especially on a massive scale.The cure for Human Immunodeficiency Virus (HIV) indeed became a real possibility after the precedence set by three patients, It is well established that incorporating Antiretroviral Therapy (ART) into clinical platforms increases life expectancy and quality. Furthermore, combination Antiretroviral Therapy (cART) targets distinct phases of the HIV replication cycle and improves overall antiviral efficacy to > 80%, even in drug-resistant cases –3. HowevGiven these limitations, many patients require lifelong ARTs, which entails long-term drug toxicity. Also, there is a lot of patient reluctance to treatment adherence, which increases the risk of developing drug resistance. In addition, ARTs notoriously fail to penetrate specific tissues, allowing viral reservoirs to form. Therefore, despite all the advantages of ARTs, further substantial improvement in their usage is the need of the hour.+ T cells, the periodic homeostatic proliferation of cells that sustain the latent viral reservoir, and low penetration of ARTs into the tissue where the virus replicates  adenine] (TNF), a precursor of Tenofovir Disoproxil Fumarate (TDF), is marketed commercially under VIREADsyndrome . TNF is syndrome –40. The syndrome , 42. Fursyndrome . The admsyndrome . Also, isyndrome . TNF is syndrome .It is evident from the chemical structure of TNF that it exhibits a negative charge facilitating improved systemic transport and disposition to various tissues/organs –50. TherAdequate structural and chemical characterization of the AuNPs is essential while considering their utilization as a drug cargo. Hence, the free TNF, AuNPs, and TNF conjugated AuNPs (AuNP-TNF) were characterized for their size, morphology, structure, and distribution of their elements.The hydrodynamic size of AuNPs was determined to be 26.53 nm (Polydispersity Index (PDI)- 0.144) with a net negative surface charge of -41.1 ± 5.94 mV which could be attributed to the carboxyl groups of the citrate ions capping the AuNPs  with a negative charge of - 47.0 ± 5.77 mV  results , TNF being tethered to the AuNPs surface as determined from the UV-Vis spectroscopy was used to examine the optical characteristics of the AuNPs-based nanoconjugates that had been synthesized. The visible spectrum analysis was from 400-700 nm wavelength. The AuNPs solution with a wine-red tint indicated the maximum Surface Plasmon Resonance (SPR) peak at 524 nm , Peripheral Blood Mononuclear Cells (PBMC), and MΦ cells (5 days). Remarkably, even after a prolonged incubation period, the viability of the AuNP-TNF-treated cells was significantly greater than those treated with free TNF (p ≤ 0.05). However, with regard to AuNPs, higher cell viability was observed as compared with the free drug (TNF) at the various tested concentrations  cell proliferation assay was used to evaluate the cellular viability of free TNF, AuNPs, and TNF conjugated AuNPs, and the data were interpreted as CC50 Fig. . Data prThe results  incubated for 24 and 48 h. Figure VB28 (R5) or HIV1UG070 (X4)) were exposed to various sub-toxic concentrations of free TNF, AuNPs, and AuNP-TNF to assess potential anti-HIV1 efficacy. Table 50) values for free TNF, AuNPs, and AuNP-TNF against HIV1VB28 and HIV1UG070 strains in TZM-bl cells and PBMCs, respectively. It was confirmed that the TNF anti-HIV1 activity was significantly enhanced upon conjugation with AuNPs in TZM-bl cells and PBMCs. , it achieves a greater TI over free TNF (p ≤ 0.0001). These results confirmed that the synthesized nano-ART could inhibit both HIV-1 primary isolates as well as pseudovirus efficiently compared to free TNF . The IC50 values of free TNF, AuNPs, and AuNP-TNF were found to be 0.98 ± 0.072, 5.32 ± 0.860, and 0.067 ± 0.005 µg/mL, respectively, which demonstrated that the conjugation of TNF with nanoparticles has a substantial inhibitory effect on the RT-ase enzyme. In vitro inhibition of HIV-1 reverse transcriptase by free TNF, AuNPs, and AuNP-TNF was investigated. The ICThe inhibitory impact of free TNF, AuNPs, and AuNP-TNF on HIV-1 protease was examined using His tagged-HIV protease. Figure Confocal microscopy was used to examine the internalization capacity of Cyanine5.5 NHS ester tagged AuNPs (Cy5.5-AuNPs) by the TZM-bl cells. The cells had a distinctive red puncta hue, indicating an elevated accumulation of Cy5.5-AuNPs within the treated cells Fig. A; in con® in vivo imaging system and Near-infrared (NIR) fluorescence images of major ex vivo organs  were obtained at different time points . All injected mice survived, and there were no visible signs of aberrant behavior or illness. At the injection location, there was no erythema or edema, indicating that there was no irreversible impairment to the endothelial cell structure or vessel wall due to the injection. Figure For in vivo biodistribution analyses, the behavior of Cy5.5-AuNPs was evaluated in healthy male BALB/c mice by IVISConversely, the accumulation of the targeted Cy5.5-AuNPs was more in the lung than in the kidney. In addition, the NIR fluorescence intensity increased in the brain over time. Targeted Cy5.5-AuNPs, on the other hand, tended to be more prevalent in the liver, which has higher metabolic activity.2/sr] / [µW/cm2] was determined  was recorded 7 days post-administration that showed fluorescently tagged AuNPs were predominantly retained in the abdominal region  was used to synthesize AuNPs spherical AuNPs with narrow size distribution . The AuNUV-Vis spectroscopy of the AuNPs showed a shift in the SPR peak from 524 to 526 nm and a change in the amplitude after TNF conjugation. Similar observations have been reported by many researchers / [W/cm2] was determined. The harvested organs were preserved in 10% neutral buffered formalin  for histopathology analysis. FLIT was also recorded, which leverages the signal's geometry, depth, and intensity to create a 3D reconstruction of the mouse and provides anatomical localization of the fluorescence signal  of AuNP-TNF.Additional file 2. 3D Fluorescent Imaging Tomography (FLIT) record of the abdominal region of a BALB/c mouse, 7 days post-administration of fluorescently tagged AuNPs.


Due to the key role of tumor necrosis factor-alpha (TNF-α) in the pathogenesis of immunoinflammatory diseases, TNF-α inhibitors have been successfully developed and used in the clinical treatment of autoimmune disorders. Currently, five anti-TNF-α drugs have been approved: infliximab, adalimumab, golimumab, certolizumab pegol and etanercept. Anti-TNF-α biosimilars are also available for clinical use. Here, we will review the historical development as well as the present and potential future applications of anti-TNF-α therapies, which have led to major improvements for patients with several autoimmune diseases, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn’s disease (CD), ulcerative colitis (UC), psoriasis (PS) and chronic endogenous uveitis. Other therapeutic areas are under evaluation, including viral infections, e.g., COVID-19, as well as chronic neuropsychiatric disorders and certain forms of cancer. The search for biomarkers able to predict responsiveness to anti-TNF-α drugs is also discussed. One of the main breakthroughs of the past 50 years in medicine has been the development of biologics. The invention of hybridoma technology goes back to 1975, when Kohler and Milstein [mAbs may exert two types of therapeutic effects: first, by binding an antigen, they can neutralize its biological functions; second, by opsonizing target cells, they may induce an effective Fc receptor-mediated immune response.In 1986, the first murine mAB (muromonab CD-3) was approved by the USA Food and Drug Administration (FDA) for preventing acute kidney transplant rejection . This drA further and highly significant improvement in the pharmacology of mAbs was achieved with the production of humanized mAbs that were developed by transplanting murine hypervariable segments of the complementary-determining regions (CDRs) into a human Ab structure ,11,12. IAnother innovative approach to obtain fully human mAbs is based on transgenic animals that are genetically modified by inserting human immunoglobulin genes into their genome sequence deprived of immunoglobulin loci. Hence, plasma B cells of transgenic animals can produce fully human mAbs after exposure to a specific antigen ,19. AlthThe advent of these new technologies propelled interest in the production of recombinant Fc-fusion proteins, which are characterized by the Fc domain of the Ab conjugated with another protein such as an enzyme, cytokine or a receptor through a peptide linker, therefore increasing the stability and half-life of the bound protein and prolonging its biological effects . The firThe development of these Abs was based on the discovery of Abs with only two heavy chains (containing a single variable domain referred to as a VHH or nanobody) in the serum of camelids  in 1993 . AlthougIn 2019, the first bivalent nanobody directed against the A1 domain of the von Willebrand factor (caplacizumab) received FDA and EMA approval to treat patients with acquired thrombotic thrombocytopenic purpura (aTTP) ,28. ThreCurrently, different nanobody therapies are investigated in the clinical setting. It appears likely that as the breadth of nanobody targets is expanded, their utility as diagnostics or therapeutics will grow as well.In the present review, we will discuss the historical development, as well as the present and potential future applications of all five FDA-approved biological anti-TNF-α agents , which have led to major improvements for patients with RA and other autoimmune diseases, such as ankylosing spondylitis, Crohn’s disease, ulcerative colitis and psoriasis. More recently, other therapeutic areas are under evaluation, including viral infections, e.g., COVID-19, neuropsychiatric disorders and cancer .The search for biomarkers for predicting anti-TNF-α responsiveness is also discussed.The tumor necrosis factor-alpha (TNF-α) gene is located in chromosome 6. It consists of four exons, and it encodes for a pro-inflammatory and immunomodulatory transmembrane protein made up of 233 amino-acid residues . TNF-α iAn abnormal production of TNF-α is associated with several chronic, immunoinflammatory diseases, such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis (PS), psoriatic arthritis (PsA) and autoimmune uveitis ,46,47,48To date, five anti-TNF-α drugs have been approved: infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), certolizumab pegol (Cimzia) and etanercept (Enbrel). Several studies have demonstrated the efficacy and safety profiles of these agents in different diseases. In particular, Burr and colleagues demonstrated that patients with severe ulcerative colitis treated with infliximab (10 mg/kg) showed superior endoscopic improvement compared with the placebo group (with a response rate of 0.61) . AnotherOverall, these studies highlight that the use of TNF-α inhibitors is safe and well tolerated in a large percentage of patients suffering from various diseases.Infliximab is a chimeric mAb, generated by Vilcek and Le , consistβ, which is a cytokine involved in the regulation of bowel immune cells [Etanercept is a recombinant Fc-fusion protein that comprises two human TNF-R2 conjugated with the CH2 and CH3 domains of the Fc region of human IgG1, which mimics soluble TNF-R activity . It was ne cells . Furtherne cells .In 2002, adalimumab, a fully human mAb generated using phage display technology, was approved by the FDA to treat patients with RA . SubsequGolimumab is a fully human mAb produced using Medarex’s UltiMab transgenic mouse platform, in which human IgG genes are inserted into the genome of engineering mice. It has a bivalent Fab region, which can bind the soluble and transmembrane form of TNF-α proteins with a higher affinity than infliximab and adalimumab, reducing both the circulating TNF-α protein levels and the binding of TNF-α with its receptors . In compIn 2008, certolizumab pegol was approved by the FDA to treat patients with CD . Its effThe toxicity profile of TNF-α blockers is acceptable; however, patient surveillance continues to be warranted. Common side effects range from gastrointestinal to behavioral disturbances. Rarely, patients may also experience symptoms, such as fatigue, myalgia, nausea and anorexia which often disappear after the discontinuation of therapy ,76,77. FFor years, researchers have been trying to understand whether patients treated with TNF-α blockers are at a greater risk of developing serious infections than the general population, reaching conflicting results. The discrepancies among the different studies could be related to several factors, such as the duration of the treatment and follow-up, the patients’ prior therapies, and the severity of the disease ,82,83. TAmong the neurological manifestations associated with biological TNF inhibitors, headaches and behavioral disturbances are the most common . HoweverAnother adverse effect of TNF-α inhibitors is the onset of induced iatrogenic lupus . Many people who develop ATIL show variable symptoms, such as skin rashes, thrombocytopenia, leukopenia, pericarditis, pneumonia and rarely, hemolytic anemia ,99. ThesAlthough all anti-TNF-α were reported to potentially induce ATIL, several studies have shown that the onset of ATIL occurs mainly in patients taking infliximab and, in some cases, after treatment with etanercept and adalimumab ,103,104.Nowadays, it is still unclear whether TNF-α inhibitors play a role in the exacerbation of systemic lupus erythematosus. In order to ensure an accurate diagnosis, it is recommended that a thorough immunological screening is always performed before starting the treatment with TNF-α inhibitors.The risk of malignancy associated with the use of TNF-α inhibitors is still widely debated. In fact, treatment-induced suppression of the immune system may increase the risk of cancer development. According to Bongartz and colleagues, high doses of infliximab or adalimumab increases by 3.3-fold the risk of malignancy in RA patients compared with placebo groups. However, this study did not analyze any person–year incidence rate . In contRecently, an increased risk of cancer, especially for non-melanoma skin cancer and lymphoma, was described in patients taking anti-TNF-α agents compared with the control population ,110. In Although these studies have reported an increased cancer risk in patients taking TNF-α blockers, no definitive conclusions can be drawn, as many studies have not confirmed this trend ,112,113.In light of the enormous interest attracted by immune checkpoint inhibitors (ICI) in the treatment of cancer and the frequent occurrence of immune-related adverse events (irAEs) associated with their use, it has also been debated whether the use of biological TNF inhibitors may impact the pharmacological response to ICI and the development of irAEs. Emerging clinical and preclinical evidence suggests that at least short courses of TNF inhibitors are safe for the treatment of irAEs in patients with cancer undergoing ICI therapy. Data from preclinical studies also propose that TNF inhibition might augment the antitumor effect of ICI therapy while simultaneously ameliorating irAEs ,116.The clinical data indicate that not all patients respond to TNF-α blocker therapy. In fact, the use of these agents is associated with a lack of clinical response in about 30–40% of naïve patients (primary therapeutic failure) . It is aManeiro and colleagues showed up to a four-fold increased risk of infusion reactions in ADA-positive patients compared with ADA-negative patients . InflixiTo avoid the immunogenic effects of TNF-α inhibitors, two therapeutic approaches are available: one is to switch to a second TNF-α inhibitor or agents with different mechanisms of action; another alternative strategy may be the use of immunosuppressive agents in combination with TNF-α inhibitors. In particular, several studies have shown that the co-administration of immunosuppressants  reduces immunogenicity and increases the serum levels of TNF-α inhibitors ,128,129.Although numerous studies have shown that the development of ADA can be associated with a loss or reduction of the therapeutic action of TNF-α inhibitors, these data should be considered with considerable caution. In fact, the presence of ADA cannot be considered a condition necessarily associated with the loss or reduction of efficacy of the respective biotechnological drug, as some patients who develop ADA against a specific TNF-α inhibitor show a good persistence of therapeutic response to treatment with the same agents . The devOverall, the mechanisms underlying the development of ADA and their effects on therapeutic efficacy are not fully understood yet. Further studies are needed to identify possible biomarkers that could predict the development of ADA in patients after treatment with TNF-α inhibitors.Based on several clinical studies, TNF-α inhibitors seem to play a key role in the evolution and development of various pathologies. Indeed, there are post-marketing reports of the worsening of congestive heart failure (CHF) in patients treated with these agents. In particular, a randomized study suggests that infliximab increases the risk of hospitalization in patients with CHF . SimilarRegarding the role of TNF-α inhibitors in pregnancy, there is no evidence to suggest that treatment with these drugs, before or during pregnancy, is associated with increased risks of preterm births, congenital anomalies or poor pregnancy outcomes ,137. HowFinally, some adverse events may be induced by interactions between TNF-α inhibitors and other drugs. Although, most studies have observed no clinically relevant drug interactions, some evidence suggests that TNF-α inhibitors may affect cytochrome p450 (CYP450) activity . In partDrug interactions should be considered as one of the possible causes of any adverse events. When unexpected clinical responses occur, the physician should determine the serum concentrations of the drugs and adjust the dosage until the desired effect is obtained. If the dosage adjustment is ineffective, it should be replaced with one that does not interact with the other agent.During viral infections, inflammation plays a key role in the fight against viral agents. However, TNF-α can also lead to excessive activation of immune cells and a hyperproduction of other pro-inflammatory cytokines, damaging different organs . RecentlIn particular, the Inflammatory Bowel Disease Registry (SECURE-IBD) has reported the beneficial effects of TNF-α blockers for treating patients with inflammatory bowel disease (IBD) affected by COVID-19. In particular, among 2307 IBD patients treated with anti-TNF-α monotherapy before and during COVID-19 disease and 2088 patients receiving sulfasalazine/mesalazine treatment, 8% were hospitalized and <1% died in the anti-TNF-α group, while 20% were hospitalized and 3% died in the other group .In addition, the Global Rheumatology Alliance (GRA) registry has reported the beneficial effects of TNF-α blockers for treating COVID-19 patients with RA. On 12 April 2021, 1388 patients with RA received TNF-α inhibitors at the onset of COVID-19. Of these, 103 patients (7.4%) were hospitalized with ventilation, and only 36 (2.6%) died. Notably, treatment with other drugs (rituximab and Janus kinase inhibitors) has shown a higher probability of developing a severe disease than treatment with TNF-α inhibitors . No assoOverall, these data have shown that the clinical use of TNF-α blockers is significantly associated with a lower COVID-19-related hospitalization rate compared with other drugs. However, further studies are needed to better define an immune profile of both the disease course and therapy with TNF-α antagonists. For instance, some data show that the use of TNF-α inhibitors seems to be associated with a mild humoral response to the SARS-CoV-2 vaccines and a faster lowering of the antibody titre compared with patients treated with other drugs ,150. FurSeveral studies have recently demonstrated that a persistent inflammatory state plays a predominant role in the pathogenesis of psychiatric and neurodegenerative diseases ,154,155.Other studies have shown how TNF-α is positively associated with the severity of depressive symptoms . In addiBased on the studies described above, several studies have recently focused on evaluating TNF-α inhibitors as potential therapeutics for behavioral disorders, obtaining encouraging results. Indeed, some studies have reported a significant reduction in anxiety and depressive-like behaviour and an improvement in cognitive function in murine models after anti-TNF-α treatment ,169. In Although TNF-α inhibitors are unable to cross the BBB due to their molecular weight, they can bind to peripheral TNF-α, also reducing its levels and the overall activated immune cells in the brain parenchyma . To bypaOverall, although no data on the role of other TNF-α inhibitors are available, these studies highlight that the use of TNF-α as an adjunctive antidepressant therapy exhibits only partial positive results in a small group of patients with behavioral disorders. In addition, a recently study demonstrated that the use of TNF-α inhibitors could be a potential risk factor for the development of a manic episode in patients with or without psychiatric disorders .The therapeutic use of anti-TNF-α has led to important advances for patients with several chronic inflammatory diseases. However, there are still autoimmune/immunoinflammatory diseases for which there is still no evidence of the efficacy of TNF-α inhibitors.Numerous clinical trials are ongoing for the evaluation of TNF-α blockers in a wide range of disorders that may be characterized by an abnormal production of pro-inflammatory cytokines . Most noAlthough TNF-α blockers are the main therapeutic option for patients with immune-mediated inflammatory diseases, they are characterized by high costs and immunosuppressive activity. Moreover, 40% of patients treated with them do not respond or show a loss of response over time . For theFinally, some studies have also shown that genetic variants could influence the response to TNF-α blockers. In particular, it was found that the presence of the IFNGrs2069705C allelic variant in patients with RA resulted in a better responsiveness to TNF-α inhibitors compared with patients harboring the wild-type allele . MoreoveUnfortunately, to date, no biomarker for anti-TNF responsiveness has been validated due to the heterogeneity of the results obtained. Hence, further studies using independent and large cohorts are needed.Anti-TNF-α therapy has paved the way for a revolution in the approach to treating chronic inflammatory diseases. In fact, they represent the first biotechnological drugs used in rheumatology after the failure of conventional synthetic drugs, such as methotrexate, sulfasalazine and mesalazine. The efficacy and safety of these biological agents have been confirmed in various fields, and novel potential applications range from psychiatric and neurological disorders to viral infections, such as COVID-19. Novel TNF-α blockers are also currently under development, including AVX-470, L19TNFa, Ozoralizumab, 99mTc and XPro1595. Despite considerable progress, there is still an area of unmet need, for instance, cases in which the therapeutic response is insufficient or in which there are specific clinical conditions of patients or the need to control specific aspects of the disease. This explains the wide range of drugs available and the attention of the reference guidelines to the specificities of the patient, focusing on the increasingly tailored therapy. Due to this, a great effort is being made in the identification of biomarkers to be used as prognostic tools in patients eligible for treatment with TNF-α inhibitors. Secondly, biotechnological therapy is burdened by the high direct costs of the drug. The lack of patent protection for the first TNF-α inhibitors has allowed the introduction of economically affordable biosimilars, such as ABP 501 , ABP 710 , BI 695501 , CHS-1420 , CT-P13 , FKB327 , GP2017 , PF-06410293 , PF-06438179 , SB2 , SB4  and SB5 . The availability of biosimilar agents has allowed a drastic and important reduction in pharmacological expenditure, making TNF-α blockers widely available and accessible to the population. To date, they are an integral part of the biological therapies available. However, although biosimilars are approved on the basis of similar mechanisms of action, efficacy and safety with their reference products, many clinicians remain hesitant to suggest biosimilars as a viable treatment option due to the nocebo effect that could occur in patients following the switch to biosimilars. For this reason, it is necessary that clinicians have full and complete knowledge of the scientific principles that support these agents, their clinical development, approval and safety monitoring.


Normal pressure hydrocephalus (NPH) has been a topic of debate since its introduction in publications. More frequent in the elderly population, it is characterized by gait disturbance, urinary urge incontinence and cognitive decline. Therefore, it is a clinical-radiological entity with relatively common findings for the age group, which together may have greater specificity. Therefore, its diagnosis must be careful for an adequate selection of patients for treatment with ventricular shunt, since the symptoms are potentially reversible. The tap test has a high positive predictive value as a predictor of therapeutic response, but a negative test does not exclude the possibility of treatment. Scientific efforts in recent years have been directed towards a better understanding of NPH and this narrative review aims to compile recent data from the literature in a didactic way for clinical practice. It is a syndrome defined by the presence of gait disturbance, urinary incontinence and cognitive decline (Hakim's triad), with progressive onset, radiological evidence of ventricular dilation and clinical improvement after shunting,Classically, it is divided into secondary NPH  - resulted in an average prevalence of NPH of 1.1% in the elderly,-,Similar prevalence has been found in Western studiesWhen the motor symptoms are investigated, the numbers can be higher. NPH was responsible for 19% of suspected cases of parkinsonism in a German studyClassically defined as communicating hydrocephalus, NPH has normal opening pressure on lumbar puncture and a disproportionate ventricular dilation to the cortical atrophy degree.As already mentioned, NPH is divided into an idiopathic and a secondary form. The latter is often related to subarachnoid hemorrhage, meningitis, intracranial tumors, traumatic brain injury, among other possible causes of poor cerebrospinal fluid reabsorption. It is assumed that these conditions lead to an inflammatory process of the arachnoid granulations, with reduced CSF reabsorption and alteration of the CSF flow dynamics, resulting in ventricular dilatation.Regarding the so-called idiopathic NPH, the anatomopathological studies are heterogeneous. Among the findings described are thinning and fibrosis of the meninges and arachnoid membranes, inflammation of arachnoid granulations, vascular alterations, AD pathology, rupture of the ventricular ependymal and subependymal gliosisAlthough no increase in intracranial pressure is observed during lumbar puncture, it is believed that there is a local pressure effect on the periventricular white matterAlso, apart from changes directly exerted by the local pressure, there is an increase in intracranial pulsatility pressure,Recent associations between NPH and the glymphatic system (a CNS metabolic clearance pathway) may explain the frequent association with neurodegenerative comorbidities and further studies may elucidate the order of events in the pathophysiology of the entity The diagnosis is primarily based on history and neurological examination. The classic triad, described by Hakim, Adams and Fisher in 1965When considering the average age at diagnosis, it is important to keep in mind that these symptoms can be manifestations of many other conditions unrelated to NPH. Likewise, the patient with NPH may present other symptoms not described in the disease due to associated comorbidities, which makes the diagnosis challenging.,,Gait disturbance is the most frequent symptom and, in most cases, the earliest. Labeled mainly as “apraxic gait” or “magnetic gait”, heterogeneity is observed among patients. Characteristically, the patient moves more slowly, with short steps, wide base and changes in direction using several steps, with the movement being fragmented or en bloc. There may be a posture of anterior inclination of the trunk, difficulty in climbing stairs and in performing transitional movements, such as sitting and standing up. The patient can more easily reproduce the gait movement in the sitting or supine position; however, he cannot perform this when in orthostasis,,Cognitive decline is secondary to frontal-subcortical pathway dysfunction, which mainly leads to a slowing of information processing speed and executive dysfunction. The patient may present with dementia itself, as well as mild cognitive impairment. Difficulty in sustained attention, abstract thinking, planning, decision making, and problem solving is observedUrinary symptoms are defined as an uninhibited neurogenic bladder, with urgency, increased frequency, with or without incontinence in the early stages. This type of finding should be well defined in history, as it is also a very prevalent symptom in the population aged over 60The two most used NPH guidelines are the InternationalThe diagnosis of NPH is rarely performed in isolation - it requires a broad investigation to rule out its multiple possibilities of differential diagnosis. The elderly patient may have several comorbidities that can lead to NPH cardinal symptoms. Cervical or lumbosacral spondylosis, large joint osteoarthritis, peripheral neuropathy, vestibular dysfunction, reduced muscle mass and even visual impairment are examples of reported etiologies for gait disturbance. Similarly, neurologic and non-neurologic causes of urinary incontinence should be considered and evaluated, such as urinary tract infection, prostatic hypertrophy, myelopathy, pelvic organ prolapse, bladder outlet obstruction and side effects of medication.However, the clinician should be aware of the main neurodegenerative diseases that present signs and symptoms that can lead to a misdiagnosis of PNH, especially when cognitive impairment is a prominent symptom. The main features of each of these are:Cognitive decline with predominant change in ability, visuospatial and executive dysfunction, complex visual hallucinations, Parkinsonism, and fluctuating consciousness. Intolerance to neuroleptics and good response to cholinesterase inhibitors.Parkinsonism evident in the early stages and with unique characteristics , markedly asymmetrical, an important response to levodopa that maylead to dyskinesia and cognitive manifestation of late evolution.Asymmetric findings, focal deficits, early pyramidal changes , acute onset and evolution in steps or cognitive stability when the disease is controlled.Presence of Parkinsonian symptoms that predominate in the lower limbs and gear change. Neuroimaging investigation demonstrates microangiopathy and/or ischemic lacunar lesions in the periventricular and basal ganglia regions.May have early impairment of gait and is associated with frequent falls. However, it evolves with Parkinsonian symptoms with axial rigidity, alteration of extrinsic ocular motricity , cervical and facial dystonia, which lead to expression of consternation characteristic of the disease (omega sign).May be associated with NPH, especially when the condition presents prominent amnestic changes and deficits suggestive of cortical dysfunction, such as loss of visuoperceptual and visuospatial functions, language alteration and apraxia. A change of gait is not common in the early stages of AD alone.Neuroimaging showing ventriculomegaly is essential for diagnosis, as well as for excluding NPH mimics.CT is able to show enlargement of lateral and third ventricles, as well as other typical findings of the disease; however MRI is more accurate. Several findings are described and together they help to define the diagnosis.,,,Evans index, one of the first signs described, is able to diagnose and quantify ventriculomegaly. The calculation is made through the ratio between the largest diameter between the frontal horns of the lateral ventricles over the largest diameter of the cranial vault cavity observed in the same axial section. A ratio greater than 0.3 indicates ventricular dilatation,Flattening of the cortical sulci in high convexity with widening of the sylvian fissure and ventricular dilation can be observedMarginal hypersignal to the lateral ventricles on T2-weighted or FLAIR sequences due to ependymal CSF transudation ;Dilation of the third ventricle,Reduction of the callosal angle in the coronal section,Increased CSF flow in the Sylvius aqueduct associated with flow void33. CSF flow obstruction, such as aqueduct stenosis or Chiari malformationThere used to be a supposition that NPH patients had inverted CSF flow in the cerebral aqueduct, but this finding was not confirmed in a recent study using phase contrast MRI. However, an increase in the variability and pulsatility of the CSF flow in the aqueduct was demonstrated, with a reduction in the flow pulsatility in the cervical region. These findings may be incorporated in the future to aid diagnosis and assessment of the post-operative prognosisFinally, it is important to remember that ventriculomegaly does not imply NPH. SPECT and PET-CT can aid the diagnosis, revealing hypometabolism in the periventricular and frontal regions, respectively.29.PET-PiB plays a minor role in the differential diagnosis, considering the advanced mean age of these patients, 32% of the patients demonstrate amyloid deposits on examinationSome parameters of magnetic resonance spectroscopy (MME) have been suggested as potentially useful in the diagnosis and follow-up of patients with NPH .6.Currently, the value of spectroscopy for diagnosis and follow-up of response in iNPH is controversial,,,Cisternography involves the injection of a radioisotope or contrast into the subarachnoid space, via lumbar puncture, with subsequent acquisition of serial images to observe the cerebrospinal fluid progression. Gadolinium MRI cisternography is the most modern methodThe treatment of NPH is surgical and carries a potential risk of complications, therefore, tests that can predict the therapeutic response after surgery are indicated, in addition to detailed clinical analysis and comorbidities.Tap Test (TT) - it is considered important as a pre-surgical evaluation and helps in the diagnosis when the response is positive. It is simple, easy to perform, with low complication ratesLumbar puncture is performed in the usual way, with the withdrawal of 30-50 ml of CSF. Before and after the procedure, the patient is evaluated for his cognition and gait. In this respect, there is a lack of standardization in the literature regarding the evaluation methods, as well as regarding the evaluation time after the puncture, and the studies are heterogeneousRegarding cognitive assessment, the well-known Mini-Mental State Examination can be used, with the aim of comparing pre- and post-puncture and posterior longitudinal follow-up. The clock drawing test and phonemic and semantic verbal fluency can be used to complement cognitive assessment. Moreover, several other assessment methods can be usedA recent systematic review,The CSF infusion test (CSF-IT)CSF analysis is often normal, notably opening pressure, and can be used to rule out other pathologies. Regarding biomarkers, a reduction in proteins derived from the amyloid precursor  stands out, in contrast to the decline specific to the Aß42 found in the AD Despite the name normal pressure hydrocephalus, transient increases in intracranial pressure are seen in the patients, especially during the night. Among the related alterations, the increase in the frequency of B waves is marked, particularly during REM sleep External lumbar drainage consists of the constant removal of CSF from the lumbar subarachnoid space, with a drainage system connected to a valve and a collection bag. As the point of the procedure is to predict the response to the shunt, the drainage rate is similar to that of the ventriculoperitoneal shunt with a medium-pressure valve (10 ml/hr) A positive response to the external lumbar drainage has been associated with an increased chance of responding to the shunt, but the negative predictive value is low Some factors limiting the routine application of this procedure are its invasive profile (with risks of hematoma and infection) and the need for hospitalization, since it is performed in the operating theater.,-Ventricular shunting is the treatment of choice in iNPH but, in this subset of patients, the experience with the shunt can be replete with some difficulties, such as variable or short-term responses, as well as the risk of complications-The shunt can be done either to the peritoneum  or to the atrium , with no differences in the prognosis between the two. In those with contraindications to ventricular shunting, lumboperitoneal (LP) shunting can be performed-There are different types of valves used. The fixed-pressure valve was most used in the past. Nowadays, the new adjustable valves are more appropriate, mainly because of fewer associated complications, such as subdural fluid collections and corrective surgeryIt is not clear if the effect of multiple comorbidities can negatively influence the response after the procedure, The immediate response to the ventricular shunting is good. In six and 12 months after the procedure, there is evident clinical benefit in 90 and 80% of the patients, respectively The evaluation parameters of response are not standardized, but in general they include a disability scale, such as the modified Rankin scale, and/or an evaluation of the severity of the symptoms of iNPH. The most used scales attribute points to gait, urinary continence and cognition. Gait is usually the alteration that is most sensitive to shunting, with around 83% of the patients presenting improvement in the gait evaluation  There are no studies investigating the long-term outcome of patients not eligible for shunting, and there is still no evidence that the temporary benefit of ventricular shunting can be sustained over yearsIn conclusion, NPH is a relatively prevalent medical condition, especially in the older age group. Gait disturbance is the most common and usually the first to manifest itself. It is also a reversible cause of dementia, therefore the correct diagnosis should be pursued in order to properly indicate the therapy. Although the triad: gait disturbance, cognitive impairment and urinary incontinence is well known, it is not present in many patients and shouldn’t be anticipated in treatment of the patient.Neuroimaging is essential to determine the typical findings of NPH, such as ventriculomegaly, DESH and narrow callosal angle. Nonetheless, even with clinical and radiological features suggesting NPH, it is important to objectively evaluate the clinical response to other tests, such as the tap test, before indicating shunting. In the suggestive cases of NPH with good response to the test, shunting (ventricular or lumbar) should be performed after careful discussion with the patient and family regarding the possibility of improvement of the symptoms and quality of life, but also the comorbidities, risks associated and the possibility of a transient response to the treatment. In patients with no response to the test but with suggestive features of HPN, it should be personalized as to whether the patient should or should not receive the invasive treatment.


Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established  RNA silencing is one of the most efficient antiviral defences in plants. It is activated by a viral double-stranded RNA (dsRNA) that is then processed by Dicer-like (DCL) ribonucleases producing 21 to 24-nt primary virus-derived small interfering RNAs (vsiRNAs). Subsequently, in the amplification step, host-encoded RNA-dependent RNA polymerases (RDRs) synthesize dsRNA from viral single-stranded RNA producing secondary vsiRNAs that, together with the primary vsiRNAs, are responsible for the spreading of the silencing through the plant. vsiRNAs bind to different Argonaute (AGO)-containing effector complexes and provide targeting specificity for RNA dicing  or chromatin modification . Viruses counterattack by producing one or several proteins that suppress this defence by targeting different steps in the pathway ,4.Geminiviruses are a group of insect-transmitted plant viruses that cause destructive diseases in major crops worldwide. The virions of this family are twinned and contain a single copy of circular single-stranded DNA, ranging in size from 2.5 to 3.0 kb ,7.During infection, geminiviruses must confront both types of silencing, PTGS, and TGS. As counter-defence, they encode more than one viral RNA silencing suppressor, being that the V2 proteins exhibit the most efficient PTGS suppression activity ,9.V2 (AV2) is a multifunctional protein that has been described as: a (i) PTGS or a TGS suppressor in most of the geminivirus species tested to date ,17,18,19Becurtovirus [Mastrevirus, it contains two intergenic regions, one large (LIR) and another small (SIR), located at opposite sides of the genome. BCTIV LIR includes a sequence capable of forming a stem-loop structure and a novel nonanucleotide (TAAGATT/CC) with a unique nick site. The BCTIV genome comprises three virion-sense  and two complementary-sense (C1 and C2) ORFs. Based on a comparison of nucleotide sequence identity of individual genes, the three virion-sense ORFs V3, V2 and V1 are similar to their positional homologs in the curtoviruses, whereas C1 and C2 are more similar to the ones from mastreviruses [Beet curly top Iran virus (BCTIV) is one of the causal agents of beet curly top disease (BCTD) in Iran, described recently as the type species of the genus rtovirus ,27. Its eviruses ,29  phosphorylation motif (hereafter named P1), present in all V2 proteins and two putative CK2 and PKC phosphorylation motifs. These were predicted in BCTV V2  and are also present in BCTIV V2, but not in begomoviral proteins B. Besides genera ,30. Besis genera ,32.N. benthamiana plants. Leaves were co-infiltrated with Agrobacterium cultures expressing GFP (GFP) and BCTIV V2 (thereafter named IVV2). As controls, leaves were co-infiltrated with GFP, and the empty vector (EV) (negative control) or a plasmid expressing the viral silencing suppressor V2 from BCTV (thereafter named BCV2) (positive control) were considered. Leaves were observed under UV light at 6 dpi. Leaves co-infiltrated with GFP and IVV2 displayed high intensity of GFP fluorescence, as leaves co-infiltrated with GFP and BCV2. Conversely, in the patches infiltrated with the empty vector, the intensity of fluorescence had disappeared almost completely . Similmpletely C. On thempletely D. Expresmpletely E.N. benthamiana plants. Co-infiltration with GFP and P19 [To determine the effect of BCTIV V2 on systemic PTGS, the constructs expressing GFP and IVV2 or BCV2 were agroinfiltrated in leaves of transgenic 16c  and P19  or the eFluorescence was monitored under UV light to detect the initiation of systemic silencing in newly emerging leaves, from 4 to 30 dpi. To evaluate the silencing progress, we applied the arbitrary silencing index, described in ref  Figure B. SystemAgroinfiltrated 16c plants were also employed to study the effect of BCTIV V2 on the short-range movement of the RNA silencing. For that, GFP expression was monitored with UV light in the cells surrounding the infiltrated tissues at 6 dpi to check if the silencing signal triggered by the GFP overexpression had been able to exit from the infiltrated area and cause the appearance of a red ring around the patch . In the N. benthamiana [N. benthamiana plants (PVX-IVV2). Agroinfiltration with the empty PVX vector was used as a negative control, whereas infiltration with PVX expressing either BCTV-V2 (PVX-BCV2) or the nonstructural protein (NSs) from tomato spotted wilt virus (PVX-NSs) were used as positive controls [As mentioned above, ectopic expression of V2 proteins from begomoviruses and curtoviruses from a potato virus X (PVX)- derived vector causes localized cell death in the infiltrated tissues and produces systemic necrosis associated with a hypersensitive response-like (HR-like) phenotype in thamiana ,16,24,34controls ,37.At 5 dpi, tissues infiltrated with the empty vector developed a local yellowing, typical for PVX, while tissues infiltrated with PVX-IVV2 showed intense local necrosis, similar to the infiltrated with the positive controls: PVX-NSs and PVX-BCV2 A. At 16 N. benthamiana leaves. BCTV V2 fused to GFP in the amino-terminal region of the viral protein (GFP-BCV2), and free GFP (GFP) were used as controls. Thirty-six hours after the agroinfiltration, tissues were collected and visualized using a confocal microscope. BCTIV V2 seems to have a similar subcellular localization to other V2 proteins, including its curtoviral counterpart [To obtain additional information on the function of BCTIV V2, we examined the subcellular localization of the protein, expressing GFP-fused versions of BCTIV V2  in wt nterpart ,40,41,42N. benthamiana plants were agroinoculated with the V2 mutant BCTIV (BCTIV-V2stp) or with the wild-type BCTIV (wt BCTV) as a positive control. Agroinoculation, with the empty vector, was used as a negative control. Symptoms were detectable in plants infected with wt BCTIV at 10 dpi, while plants infected with the V2 mutant remained did not show any symptom. At 28 dpi, wt BCTIV-infected plants showed the typical BCTIV symptoms , whereas those agroinoculated with BCTIV-V2stp did not develop any symptoms . No BCTIV DNA was detected in the apical leaves of plants inoculated with V2 mutant BCTIV B, indicaEscherichia coli strain DH5-α was used for subcloning. The Agrobacterium tumefaciens GV3101 strain was used for the agroinfiltration and agroinoculation/infection assays.Bacterial transformation and plasmid purification were carried out according to standard methods . EscheriNicotiana benthamiana and 16c N. benthamiana line .The ClustalW algorithm was used to align V2 and V3 homolog proteins. The prediction of the nuclear localization signals was made by the NLS Mapper online tool  and the attB PCR products. Subsequently, these entry clones were used for LR recombination reactions with Gateway™ destination binary vectors: pGWB2 yielding pGWB2-IVV2, for their expression in plants from a 35S promoter, and pGWB5 and pGWB6 yielding pGWB5-IVV2 and pGWB6-IVV2, to express V2 fused to GFP in the carboxyl- or the amino terminal region of the viral protein respectively .ClaI V2 F and SalI V2 R, and they were subcloned into the pGEMT-easy vector  to yield pGEMT- IVV2. Restriction fragments ClaI-SalI from this plasmid containing the complete ORF were cloned into PGR107 [Generation of PVX expression vectors: To generate the PVX expression vector, BCTIV V2 complete ORF was amplified using primers o PGR107 , yieldinKpnI  to release a monomer, and subsequently with EcoRI and EcoRV  to release a 0.3 mer fragment. This 0.3 mer fragment was cloned into KpnI/EcoRI sites of pGreen0229 binary vector [KpnI digestion was cloned into the KpnI site of pGreen 0.3 BCTIV, to obtain the 1.3 mer genome-length infectious clone pGreen 1.3 BCTIV.Generation of V2 mutant BCTIV virus: As a first step, a 1.3 mer genome-length infectious BCTIV clone was obtained from the previously constructed BCTIV dimer infectious clone (accession number: JQ707949) . The dimy vector , yieldinBCTIVV2 mutant virus was then generated by site-directed mutagenesis using the Nyztech Site-Directed Mutagenesis kit   with speN. benthamiana leaves were agroinfiltrated, as previously described [For local and systemic PTGS suppression assays and PVX infection, wild-type (wt) and 16c escribed . A long-N. benthamiana plants at two leaf stage were agroinoculated, as described by [For BCTIV infection, wt ribed by , Elmer eA. tumefaciens was transformed with binary vectors containing BCTIV V2 or BCTV V2 fused to GFP in the carboxyl- or the amino terminal region of the viral protein. Wt N. benthamiana leaves were agroinfiltrated with cultures at OD600 0.5–1. Fluorescence was detected in epidermal cells 36 h after infiltration using a confocal microscope (Zeiss LSM 880).For subcellular localization, DpnI to remove bacterial DNA in the infiltrated tissues  and then was subjected to qPCR analysis using primers BCTIV RTF and BCTIV RTR primers (N. benthamiana Elongation Factor α (EF1 α) as normalizer [Agrobacterium containing the BCTIV infectious clone was used as a negative control.For BCTIV replication and infection analyses, plant DNA was extracted from the infiltrated  or the apical (systemic) leaves of the infected plants at 6 or 28 days post-infiltration (dpi), respectively. Plant DNA extraction was carried out following the method using CTAB buffer: 100 mg of agroinfiltrated tissue was homogenized into 500 μL of extraction buffer (2% cetyl trimethylammonium bromide (CTAB), 1.5 M NaCl, 100 mM Tris pH 8, 100 mM EDTA, pH 8) and incubated at 65 °C for 15 min. After cooling, the mixture was extracted with chloroform/isoamilic acid (24:1), and the nucleic acids were precipitated with isopropanol. DNA was finally resuspended in water and treated with RNase (10 mg/mL) (invitrogen). This DNA was digested with  primers , and N. rmalizer . A bacteEF1 α for 30 s, 72 °C for 45 s; final extension 72 °C for 5 min). EF1 α was used as an internal gene control.Expression of BCTIV V2 and BCTV V2 in agroinfiltrated tissues was determined by semiquantitative RT-PCR. Total RNA was isolated using Trizol reagent (invitrogen) from the infiltrated leaves, treated with RNase-free Turbo DNaseI (Ambion), cleaned up by a phenol:chloroform treatment, and subjected to reverse transcription using Super-Script II RT reagent (Invitrogen) and oligo(dT) primers, according to the manufacturer’s instructions. Synthesized cDNA was subjected, then, to PCR amplification using viral specific primers   from the infiltrated leaves, and it was used for hybridizations as described . Probe V/V) dilution.For Western blot analysis, 120 mg of leaf tissue per sample was used. Total protein was extracted by 2X Laemmli buffer and separated using sodium dodecyl sulfate (SDS)–10% polyacrylamide gel electrophoresis (PAGE), then transfered to a PVDF membrane  by a semi-dry electrotransfer  . It was Curtovirus and Mastrevirus. Up to date, no studies have been conducted to study the function of any of the ORFs identified in the viral genome [BCTD is one of Iran’s most damaging sugar beet diseases caused by beet curly top virus, beet curly top Iran virus, turnip curly top virus and turnip leaf roll virus. These viruses share hosts, transmission modes, and vectors, and, as a result, the occurrence of mixed infections in the field is very frequent . BCTIV il genome ,28. In oThe conservation of protein domains/regions  present in all geminiviral V2, and involved in PTGS suppression and pathogenicity ,39, reinN. benthamiana, such as the ones produced during the HR, both at local and systemic levels in the wt N. benthamiana plants inoculated (N. benthamiana leaves (similar results are observed when expressing BCTV V2) [As BCTV V2, BCTIV V2 expression in a PVX heterologous system caused the induction of necrotic lesions in wt oculated A,B. ThesBCTV V2) . This diBCTV V2) . The difDepending on the silencing pathway steps that the viral suppressor is targeting, the protein needs to be in a specific localization inside the cell . AccordiN. benthamiana with a BCTIV V2 mutant showed that V2 or V3 is required for systemic infection. Although the mutation in V2 generated a change in a non-conserved aminoacid of V3 (The results from the infection of wt id of V3 , we cannConsidering all the results, we conclude that BCTIV V2 is a functional homolog of curtoviral and begomoviral V2.


Reported 5-year mortality rates among patients diagnosed with heart failure are upwards of 50% , 2. ReceIn the 2021 ESC update, advances in the pharmacological management of HFpEF are discussed and conclude that there are currently no convincing studies supporting morbidity/mortality benefits with HFpEF treatment as all studies have failed to achieve their primary endpoints . As prevp = 0.072 before adjustment vs. p = 0.047 after adjustment) (The role of ACEI/ARB therapy in HFpEF stems from the PEP-CHF (perindopril), I-PRESERVE (irbesartan) and CHARM-Preserved (candesartan) studies. The PEP-CHF trial did not show any statistically significant differences in mortality/HF-related hospitalizations between those who received perindopril vs. placebo and was underpowered for the primary outcome. Notably, the study showed a benefit in the reduction of HF hospitalizations favoring perindopril at 1 year, but this benefit was not sustained as the difference was negligible when compared to placebo thereafter . The CHAustment) . ReductiThe role of ARNI in HFpEF was assessed in the PARAMOUNT-HF and PARAGON-HF trials. These trials compared sacubitril/valsartan vs. valsartan. While the PARAMOUNT-HF trial demonstrated a reduction in NT-proBNP , the clinical relevance of this surrogate outcome is not clear and the PARAGON-HF trial demonstrated no difference in cardiovascular deaths or HF hospitalizations , 10. A sThe role of beta-blocker therapy has not been extensively studied in patients with HFpEF. The SENIORS trial reported a reduction in all-cause mortality or cardiovascular-related hospitalizations associated with the use of nebivolol vs. placebo. However, the generalizability of this study to patients with HFpEF is limited as only ~15% of participants had a LVEF >50% . CoupledThe role of spironolactone in HFpEF was assessed in the Aldo-DHF trial, which demonstrated an improvement in diastolic function (reported as an estimate of filling pressure), but not maximal exercise capacity at 12 months compared with placebo and the clinical relevance remained in question . NotablyThe EMPEROR-PRESERVED trial demonstrated fewer events in the composite outcome of cardiovascular death and HF hospitalization with empagliflozin vs. placebo, regardless of diabetes. However, this effect was driven by the reduced incidence of HF-related hospitalizations with zero difference in all-cause mortality . This isAdditional trials have evaluated whether medications from other therapeutic classes play a role in the management of HFpEF. Digoxin was evaluated in the DIG-PEF trial and whilst a potential reduction in the composite outcome of mortality and hospitalizations was observed at 2 years, the benefit was not sustained at the conclusion of the study (37 months) . Cyclic The pharmacological management of HFpEF continues to be an area of uncertainty due to multiple studies failing to show a clear benefit associated with therapies that have otherwise proven useful in the management of other HF subtypes. Currently, there are no approved medications that have demonstrated improved survival in patients with HFpEF. The initiation of therapies such as ACEI/ARB or beta-blockers is primarily based on their efficacy in the management of other comorbid conditions. The 2021 ESC guideline update recommends that “in the absence of recommendations regarding disease-modifying therapies, treatment should be aimed at reducing symptoms of congestion with diuretics.” Likewise, the strong recommendation in the recently published 2022 AHA/ACC/HFSA guideline for use of diuretics, as needed in this patient population are concordant with those in the 2021 ESC guideline.One of the most significant limitations in the current literature is the lack of data pertaining to those exclusively with EF ≥50% and future studies should aim to recruit only patients who meet this definition of HFpEF. In order to best evaluate the clinical implications of these therapies, studies should attempt to measure outcomes such as mortality, hospitalization, exercise tolerance and quality of life with adequate duration of follow up to ensure that any benefits seen early on are sustained. As we continue to search for therapies that may provide mortality/morbidity benefits to those with HFpEF, it is important for us as clinicians to understand the results and limitations of these pivotal clinical trials as they continue to emerge in order to make informed decisions in the interest of balancing risks and benefits to our patients. Until then, I guess we will stick with diuretics as our mainstay of therapy.Information was gathered by both LT and LH. Both authors have read and agreed to the content of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Programmed cell death (PCD), including necroptosis, has emerged as a significant pathway in cardiovascular diseases. The infection of viral myocarditis (VMC) could cause cardiomyocytes degeneration, necrosis, and immune-inflammatory myocardial response. In this review, we summarized and evaluated the available evidence on the pathogenesis, molecule mechanism, diagnosis, and potential treatment strategies of viral myocarditis, with a special focus on the novel mechanism of necroptosis for cardiomyocytes death. Studies have shown that tumor necrosis factor-alpha (TNF-α) is an important cytokine involved in the activation of necroptosis; an elevated level of TNF-α is continually reported in patients suffering from VMC, implicating its involvement in the pathogenesis of VMC. It is of great interest to explore the clinical implication of TNF-α. We subsequently conducted a meta-analysis on the efficacy of serum TNF-α expression level and its diagnostic accuracy on acute viral myocarditis detection. Taken together, the review demonstrates a compelling role of necroptosis involved in the pathogenesis of VMC. Further, applying TNF-α as a serological marker for the diagnosis of VMC may be a useful strategy. Functional activities of all multi-cellular organisms, maintenance of a homeostatic balance between new cells and those damaged or unrequired cells, or to speak simply, regulating the number of cells, demand a constant effort of cell death . Cell deUnderstanding the molecular mechanism of any form of PCD and its roles in disease pathogenesis and development is the key point of current research, which may contribute to effective biomarkers for diagnosis, new therapeutic targets, treatment strategies, and management options.The word necroptosis is renamed to distinguish from necrosis that can be seen as accidental (unprogrammed) cell death that occurs in response to overwhelming chemical or physical insult, extreme physical temperature, pressure, chemical stress, or osmotic shock . NecroptIn 1997, Vercammen et al. found that triggering of the TNF receptors leads to necrosis of L929 cells while the Fas antigen results in apoptotic cell death, implicating that cell necrosis may also be regulated by a programmed form, and either necroptosis or apoptosis can occur in the same cell type within the pathways sharing the same cellular components . In addiBroadly speaking, necroptosis is confirmed as a new form of programmed cell death, distinct from necrosis that necrosis is not an uncontrollable passive process at all, but there is a possibility for a controllable form of cell death involving in PCD. Apoptosis was long thought to be the only regulated cell death pathway and the subject of intensive research for decades. On the contrary, its counterpart, necroptosis, has been investigated until recently that it can also be induced by TNF-α following its activation on tumor necrosis factor receptor 1 (TNFR1)  Pennica. WhereasThe understanding of specific viruses affected myocarditis is still unfolding and its pathogenesis remains further investigation. Myocarditis is one of the clinically important cardiovascular diseases induced predominantly by viruses and also by other infectious agents including bacteria, protozoa, and fungi. The disorder is associated with poor prognosis when complicated by left ventricular dysfunction, heart failure, or arrhythmia . BecauseThere are five common viruses associated with viral effect myocarditis that can be categorized as follows: the primary viruses are adenoviruses and enteroviruses such as coxsackie B or echoviruses; viscerotropic viruses, for example, parvovirus B19 have lifelong persistence , though Research indicates that infection on the myocardium by enteroviruses occurs in three phases . In PhasCardiomyocyte is a kind of terminal differentiation cell with limited regenerating ability, and damage factors can induce cardiomyocyte death leading to cardiomyopathy and heart failure. Overwhelming cardiac inflammation and the loss of myocardium is an important pathogenic mechanism in the progress of myocarditis yet the induction of this mechanism is unclear . It was Established models and clinical data have demonstrated a causal role for TNF-α in virus-induced myocarditis. . In 2004The TNF-induced signaling has multiple outcomes: the mode of gene induction, survival, apoptosis, and necroptosis suggesting the existence of different molecule mechanisms of signaling complexes or pathways  Figure . Firstlyc from mitochondria, regulated by the BCL-2 family, initiator caspases , and effector caspases . (L (FLIP) blocks caspase-8-dependent apoptosis, and at the same time, the resulting caspase-8-FLIP heterodimers block RIPK3-dependent signaling requiring for necroptosis in the analysis of FADD, RIPK3 and FLIP, RIPK3 double-knockout mice. . . In necrut mice. . High inut mice. ; p53 prout mice. ; the BCLut mice. .Accordingly, TNF-α plays a prominent role in damaging multi-organs by inducing inflammatory cell death and perpetuating cytokine storm. TNF-α is largely responsible for the timing of iNOS induction by inducing a rapid response. . ElevateRelevant studies were identified through cross-checking of references and by electronic search of databases including PudMed, Embases, Cochrane Library, Scopus, ScienceDirect, and Chinese National Knowledge Infrastructure (CNKI). The following terms were used in the search strategy: (“necroptosis” OR “programmed necrosis” OR “necrotic cell death”), “viral myocarditis,” and “Tumor Necrosis Factor alpha” as well as their abbreviations and synonyms and all possible combination. The detailed search strategy is shown in the supplementary material.The studies were included in this analysis if they met the same condition and inclusion criteria as follows: 1) investigation of the TNF-α level and its diagnostic potential for viral myocarditis; and 2) studies provided sufficient data including exact data of the serum TNF-α measurement, sensitivity, and specificity. The exclusion criteria were as follows: 1) studies contributed no data to our analysis; and 2) studies were not in the form of original articles.Data were extracted from all eligible studies: the first author’s name, year of publication, number of participants, patient characteristics, age, the expression level of TNF-α, diagnostic accuracy data for VMC detection, the optimal cut-off values, etc. Meta-analysis was carried out by STATA 15.0 software. Continuous data of serum TNF-α expression levels in different periods were summarized using STD mean difference (SMDs) and 95%CIs as their effect sizes and a 95%CI excluding the point of no effect indicates statistical significance. The pooled sensitivity, specificity, and area under the curve (AUC) of the area under the summary receiver operating characteristic (SROC) were estimated to show the diagnostic accuracy of serum TNF-α. Heterogeneity across studies was assessed using the I2 statistic, and an I2 > 50% with a p-value < 0.05 was considered as having significant heterogeneity.We conducted a meta-analysis according to the guidelines set forth by the 2020 PRISM statement and discussed the diagnostic efficiency of serum TNF-α in viral myocarditis. A flow diagram of the study selection is displayed in During our meta-analysis on the existing literature of the clinical researches, serum TNF-α was significantly increased for the myocarditis group with an SMD of 3.54  and specificity of 86% (95% CI 0.74–0.93). The pooled positive likelihood ratio (PLR) was 5.7  and the pooled negative likelihood ratio (NLR) was 0.24 . Using a random-effects model, the DOR was 24 . There was no threshold effect, as indicated by Spearman correlation analysis . We used Deek’s funnel plot to evaluate the publication bias of included studies and the shape of the funnel plot showed no between-study heterogeneity (p = 0.19), and no publication bias was found (p = 0.30). The AUC was 0.90 (95% CI 0.87–0.92) implicating that the use of serum TNF-α can provide reliable performance for the diagnosis of VMC .The meta-analysis further confirmed that TNF-α was significantly higher in patients with viral myocarditis and gradually decreased as the disease progressed. Serum TNF-α could be a useful marker for identifying VMC in acute and recovery periods. The virus infection in myocarditis is characterized as a substantial production of TNF-α. Under the strong stimulation of TNF-α, complex II was formed and ultimately caused cardiomyocyte degeneration, necrosis, apoptosis, myocardial interstitial hyperplasia, and an immune-inflammatory response depending on whether caspase-8 is inhibited . OverexpDuring the evaluation of CVB3-infected myocarditis, serum concentrations of creatine kinase, CK-MB, and cardiac troponin I (cTnI) were significantly increased in the CVB3-infected mice than the noninfected group and the model group with Nec-1, suggesting severe damage to cardiomyocytes in the VMC mice models. Histopathologic features also showed distinct changes between groups. In the CVB3 infected mice, necrosis of most cardiomyocytes was observed, as well as myocardial swelling, unclear and strong acidophil staining, pyknosis, karyorrhexis, karyolysis, cell outline disappearance, and inflammatory cell infiltration. Myofilament and myotome were normal under an electronic electroscope of the control group; the cardiac muscle fibers were well-arranged. In the CVB3 group, mitochondrial swelling and severity vacancy-like denaturation with visible infiltrating mononuclear cells and leukomonocyte revealed that necroptosis is involved in viral myocarditis. Necroptosis has been certified to determine a crucial effort on the pathophysiological change of various diseases. Among patients suffering acute critical illnesses such as MI, septic shock, or acute viral myocarditis, the activation of caspase is inhibited due to the inadequacy of adenosine triphosphate (ATP) a cell can produce. Once the activation of caspases  was prevented, the necrotic signaling is initiated. Necrostatin-1, a necroptosis pathway inhibitor, can dramatically reduce inflammatory infiltration in cardiomyocytes and promote mice survival via blocking the inhibition of RIP1 kinase . The stuin vitro model of CVB3-induced myocarditis, investigating the molecular mechanisms of viral myocarditis via iCell® Cardiomyocytes (The current research progress is the application of the human-induced pluripotent stem cells (hiPSCs) in establishing an myocytes . It is smyocytes . Not onlmyocytes , suggestNecroptosis is not equivalent to the concept of necrosis but illustrates a new concept that cell death can be regulated through a series of signal transductions instead of dying passively, therefore suggesting a new therapeutic option, and blocking the necroptosis pathway may serve as a cardiovascular protective role in acute viral myocarditis. Direct evidence has implicated that necroptosis is the significant contributor to myocardial injury in VMC using Nec-1 to block the necroptosis signaling pathway and prevents myocardial injury via downregulating RIP1 and RIP3 expression . Necrostin vivo. Also, the role of necroptosis in diseases across the body including virus induced-cancer is warranted. Optimal window period and markers are poorly defined when marking cell death. Identifying these unknown mechanisms and biomarkers will lead to a novel understanding of necroptosis and new therapeutic treatments for their related diseases. The application of Nec-1 is worth investigating as it can not only contribute to the comprehension of necroptosis but also contribute to identifying the potential therapeutic targets.The updated investigation on programmed cell death provides insight into the pathology mechanism of myocarditis. Necroptosis plays an important role in cardiomyocyte death in acute viral myocarditis. Low sensitivity of viral detection and diagnostic methods hinders the treatment for acute myocarditis and may result in the development of dilated cardiomyopathy in partial patients . For the