Gene therapy has been evaluated for the treatment of prostate cancer and includes the application of adenoviral vectors encoding a suicide gene or oncolytic adenoviruses that may be armed with a functional transgene. In parallel, versions of adenoviral vector expressing the p53 gene (Ad-p53) have been tested as treatments for head and neck squamous cell carcinoma and non-small cell lung cancer. Although Ad-p53 gene therapy has yielded some interesting results when applied to prostate cancer, it has not been widely explored, perhaps due to current limitations of the approach. To achieve better functionality, improvements in the gene transfer system and the therapeutic regimen may be required. We have developed adenoviral vectors whose transgene expression is controlled by a p53-responsive promoter, which creates a positive feedback mechanism when used to drive the expression of p53. Together with improvements that permit efficient transduction, this new approach was more effective than the use of traditional versions of Ad-p53 in killing prostate cancer cell lines and inhibiting tumor progression. Even so, gene therapy is not expected to replace traditional chemotherapy but should complement the standard of care. In fact, chemotherapy has been shown to assist in viral transduction and transgene expression. The cooperation between gene therapy and chemotherapy is expected to effectively kill tumor cells while permitting the use of reduced chemotherapy drug concentrations and, thus, lowering side effects. Therefore, the combination of gene therapy and chemotherapy may prove essential for the success of both approaches. In the United States, prostate carcinoma is the most frequent cancer among men, accounting for 19% of cancers in 2017. Though incidence rates vary by region and are higher in developed countries, an estimated 1.1 million men were diagnosed with prostate cancer in 2012, making it the second most common cancer in men worldwide. The highest rates are in Australia, North America, and Northern and Western Europe, which are regions where testing for prostate-specific antigen (PSA) has become commonplace. In South America, the rate is 60.1 per 100,000 (age-standardized rate). In a global estimate, prostate cancer is the fifth leading cause of death from cancer in men. The prediction for 2020 is 1,392,727 new cases worldwide If detected early while locally confined, prostate cancer is largely curable by radical prostatectomy or radiotherapy Gene therapy using adenoviral vectors has been evaluated in 17 clinical trials for the treatment of prostate cancer . Here, we will address some of these approaches, emphasizing the modifications made to the vector. The use of a prostate-specific promoter to drive viral expression or control oncolytic viral replication assures specificity through transcriptional control. Alternatively, modifications in the fiber protein are expected to alter vector tropism at the level of transduction.Most of the prostate-specific promoters are derived from the PSA and prostate-specific membrane antigen (PSMA) enhancers The serotype 5 adenovirus (Ad5) initially makes contact with its cellular receptor  through the fiber protein; thus, its modification can direct viral tropism. For example, compared to an unmodified Ad5 vector, a shortened chimeric Ad5/35 hybrid fiber protein had an increased transgene insert size as well as increased transduction efficiency in different prostate cancer cell lines in vitroConditionally replicating viruses were tested in phase I/II clinical trials. These adenoviral vectors encode the E1 gene under control of a tissue-specific promoter, thus limiting viral replication. In a phase I clinical trial with 20 patients, different doses of the CG7060 virus , which contains the PSA enhancer, were well tolerated, and there was a correlation between viral dose and effect: the patients with the greatest reduction in PSA levels received the highest dose Oncolytic adenoviral vectors have also been armed with functional transgenes. A prostate-restricted replicative adenovirus (PRRA) using a prostate-specific enhancer (PSES) to control the expression of E1A, E1B and E4 was armed with the FasL gene. This conditionally replicating adenoviral vector induced apoptosis of PSA/PMSA-positive cells and was less toxic compared to an AdCMVFasL vector, which killed all mice in 16 hours due to multivisceral failure First-generation adenoviral vectors armed with a suicide gene were safe and well tolerated in phase I clinical trials for patients with local recurrent or metastatic prostate cancer. Only one patient who received the highest viral dose suffered grade 4 thrombocytopenia and grade 3 hepatotoxicity; some patients showed evidence of reduced PSA levels. Suicide gene therapy has also been combined with conditionally replicating adenoviruses in prostate cancer patients with locally recurrent disease, newly diagnosed and locally aggressive disease or metastatic disease. Even though it was shown to be safe and an initial PSA decline was observed, patients relapsed The p53 protein is an important tumor suppressor involved in a variety of cellular responses to stress. P53 degradation is mediated by murine double minute 2 (MDM2), and disruption of the p53/MDM2 complex frees p53 to promote the transcription of specific target genes that, in turn, direct cellular responses such as apoptosis Viral vectors expressing p53 have been in development for the last 25 years, and even though retroviral vectors were the first to be tested in patients, adenoviral vectors have been more broadly used, and different versions have been employed to express the tumor suppressor p53 In the 1990s, Ad-p53 was tested in prostate carcinoma cell lines and xenograft mouse models, showing varied results. Some groups have demonstrated that Ad-p53 can induce apoptosis and reduce tumor volume in vitro and in vivo than Ad-CMVp53, and in situ gene therapy resulted in reduced tumor volume and increased overall survival only with Ad-PGp53. In this same work, we observed that the PC3 prostate carcinoma cell line was not efficiently transduced by Ad5 in vitro and in vivo, inducing high levels of reactive oxygen species (ROS), DNA damage and alteration of mitochondrial membrane permeability and resulting in apoptosis We have developed an improved Ad-p53. Instead of using a constitutive promoter, we have developed a p53-responsive promoter (PG), which was initially incorporated in a retroviral vector. This modified expression system could surpass the parental unmodified vector by up to seven-fold For prostate cancer, the most commonly used chemotherapy agents are mitoxantrone, docetaxel and cabazitaxel. One of the first drugs used for treatment of CRPC was mitoxantrone, a synthetic anthracenedione derivative with immunomodulatory and antineoplastic activity that was approved by the FDA in 1987 for treatment of different cancers and in 1996 for prostate cancer. Clinical trials indicated that the combination of mitoxantrone plus prednisone and corticosteroid improved quality of life without affecting survival WAF-1 and induction of the phosphorylation of Bcl-X(L)/Bcl-2 members Docetaxel is the standard chemotherapeutic agent used to treat patients with prostate cancer Cabazitaxel is a taxane approved by FDA in June 2010 for treatment of prostate cancer subsequent to the use of docetaxel. Like docetaxel, cabazitaxel suppresses the dynamics of microtubules, resulting in inhibition of proliferation and cellular arrest by inducing mitotic spindle deformity. However, cabazitaxel is more efficient The combination of Ad-p53 with chemotherapy may benefit both approaches. In a phase II clinical trial, NSCLC patients were treated with Ad-p53, docetaxel or a combination of both, and the median survival time was 7.7 months for patients who received both therapies and 5.9 months for patients who received only docetaxel There is an intimate relationship between chemotherapy and p53, where the cellular p53 status is important for the prediction of drug efficacy. In ovarian cancer cells, the combination of Ad-p53 and docetaxel was positive only in cells expressing mutant p53, while the treatment was ineffective in cells with wild-type p53 status In vivo, the combination of antisense clusterin oligodeoxynucleotides, mitoxantrone and Ad-p53 eradicated subcutaneous and orthotopic PC3 tumors The combination of Ad-p53 and docetaxel resulted in enhanced antitumor effects in a murine model of HNSCC Prostate cancer is one of the most important tumors in men. Development of new treatments has shown promising results, including gene transfer approaches as an appealing alternative. Thus, gene therapy is slowly regaining lost territory in the treatment of prostate cancer. The use of prostate cancer-specific oncolytic viruses and suicide gene therapy has reached clinical trials. Adenoviruses expressing the tumor suppressor p53 are employed for HNSCC, but their use has been limited in prostate cancer. We have shown that improvements in the transgene expression system and alteration of viral tropism may improve the suppressor activity of an Ad-p53. Even so, gene therapy may work in cooperation with traditional chemotherapy, benefiting both approaches and bringing about synergistic activity as an effective prostate cancer treatment.Tamura RE, Luna IV, Lana MG and Strauss BE elaborated and reviewed text. Luna IV designed the figure.


In a diverse family of cellular cofactors, coenzyme A (CoA) has a unique design to function in various biochemical processes. The presence of a highly reactive thiol group and a nucleotide moiety offers a diversity of chemical reactions and regulatory interactions. CoA employs them to activate carbonyl-containing molecules and to produce various thioester derivatives , which have well-established roles in cellular metabolism, production of neurotransmitters and the regulation of gene expression. A novel unconventional function of CoA in redox regulation, involving covalent attachment of this coenzyme to cellular proteins in response to oxidative and metabolic stress, has been recently discovered and termed protein CoAlation (S-thiolation by CoA or CoAthiolation). A diverse range of proteins was found to be CoAlated in mammalian cells and tissues under various experimental conditions. Protein CoAlation alters the molecular mass, charge and activity of modified proteins, and prevents them from irreversible sulfhydryl overoxidation. This review highlights the role of a key metabolic integrator CoA in redox regulation in mammalian cells and provides a perspective of the current status and future directions of the emerging field of protein CoAlation. The. The1].  reduced . It has  reduced . The proThe biosynthesis and homeostasis of CoA is controlled at different levels: transcription of genes encoding biosynthetic enzymes, regulation of enzymatic activities by a feedback mechanism, signalling pathways, degradation of CoA and interconversion between CoA and its thioester derivatives. Various extracellular stimuli, such as nutrients, hormones of metabolic homeostasis, intracellular metabolites and stress, were found to regulate the total level of CoA in mammalian cells. It is reduced in to insulin, glucose, pyruvate and fatty acids, whereas glucagon, glucocorticoids and oxidative stress have an opposite effect –8. PANK Regulation of other enzymes in the CoA biosynthetic pathway, especially PPCS and PPCDC, is less understood. The PPAT and DPCK activities of CoA synthase were found to be strongly induced by phospholipids . The ideThe total cellular CoA content is also controlled by degradation, involving phosphodiesterases, phosphatases and pantetheinases  15]. Th. Th15]. The estimated CoA levels in mammalian cells and tissues span more than a 10-fold range. Liver, heart and brown adipose tissue have the highest CoA levels, followed by kidney and brain. The CoA pool is largely made up of CoASH, and acetyl CoA is the largest component of the acyl CoA pool. The subcellular distribution of CoA in mammalian cells reflects the variety of processes in which it is implicated. The concentration of CoA in mitochondria and peroxisomes are in the range of 2–5 and 0.7 mM, respectively, whereas levels of cytosolic and nuclear CoA are significantly lower, ranging from 0.05 to 0.14 mM ,21.CoA and its thioester derivatives play important roles in numerous biosynthetic and degradative pathways of cellular metabolism, allosteric interactions and the regulation of gene expression. These include synthesis and oxidation of fatty acids, the Krebs cycle, ketogenesis, biosynthesis of cholesterol and acetylcholine, degradation of amino acids, regulation of gene expression and cellular metabolism via protein acetylation and others  1,22,23,2322,23.de novo CoA biosynthesis is an evolutionary conserved biochemical process, significant structural and regulatory differences between microbial and human biosynthetic enzymes make the CoA biosynthetic pathway an attractive target for the development of novel antibiotics.Although The role of the CoA thiol group in the production and function of various thioester derivatives has been extensively studied since the discovery of this coenzyme in the middle of last century. In contrast, the contribution of the CoA thiol moiety towards redox regulation and antioxidant defence has yet to be established.Mammalian cells contain high levels of low-molecular-weight (LMW) thiols that provide protection against a variety of reactive oxygen, nitrogen and electrophilic species  generated inside cells by incomplete reduction of molecular oxygen, dysregulation of metabolic processes or produced during the detoxification of xenobiotic and endobiotic compounds. High levels of ROS, RNS and RES have the potential to damage cellular macromolecules, including proteins, nucleic acids, lipids and carbohydrates . In contIn mammalian cells and tissues, glutathione (GSH) is the most abundant LMW thiol with concentrations ranging from 0.5 to 10 mM . GSH is Ka of the CoA thiol (∼9.8) at physiological pH protects it from oxidation to the sulfenic acid state (CoASOH) [Ka of the CoA thiol needs to be decreased. The reactivity of CoA in cellular redox processes can be enhanced by complexing with the enzyme(s) which can reduce the pKa value of its thiol and facilitate covalent modification of cellular targets by CoA, as reported for GSH in complex with glutathione transferases [The redox functions of CoA in mammalian cells under physiological and pathophysiological conditions are not well understood. The relatively high p(CoASOH) . To perfsferases . The enzin vitro during prolonged storage in aqueous solutions or in the presence of free radicals. The redox potential of the CoASH/CoASSCoA couple at pH 7.0 is −234 mV and comparable to that of the GSH/GSSG couple (−240 mV), meaning that CoA can significantly contribute to the electrochemical potential inside the cell [The bulk of CoA in mammalian cells exists in reduced (CoASH) and thioster (acyl CoA) forms. CoA disulfides (CoASSCoA) or mixed disulfides with other LMW thiols (such as cysteine and GSH) have been identified in mammalian cells, but their cellular functions are largely unknown. CoASH can be oxidised to CoASSCoA the cell . In bactthe cell . Furtherthe cell ,38.The existence of CoA mixed disulfides with cysteine residues in proteins has been known for many years. They were reported in several biochemical and crystallographic studies, and a number of CoA-modified proteins identified as acetyl CoA acetyltransferase, glutamate dehydrogenase flavodoxin, phenol sulfotransferase and peroxide sensor OhrR (organic hydroperoxide resistance repressor) –42. HoweCysteine is one of the most evolutionarily constrained amino acids and the least commonly used in human proteome . Despitein vitro, cell-based and animal models, several research tools and methodologies have been developed. These include: (a) anti-CoA monoclonal antibodies, which specifically recognise CoA in various immunological assays, including ELISA, Western blotting, immunoprecipitation and immunohistochemistry; (b) a robust mass spectrometry-based methodology for the identification of CoAlated proteins; and (c) efficient in vitro CoAlation assay [2O2. In this experimental model, the patterns of protein CoAlation and protein glutathionylation diverged significantly, suggesting differential targeting of cysteine thiols in cellular proteins by CoA and GSH in response to oxidative stress [In the last decade, proteomics studies have revealed that cysteine modifications involved in protein redox regulation are more widespread in biological systems than previously estimated and exert a considerable influence on cellular processes, including cellular signalling, proliferation, differentiation and apoptosis. A novel mode of redox regulation involving covalent modification of cellular proteins by CoA has recently been discovered in a collaborative effort of several laboratories and termed protein CoAlation . To discon assay ,46. Anale stress .Protein CoAlation was also found to be modulated by metabolic stress induced by nutrient deprivation or overload. A significant increase in the level of CoAlated proteins was observed in cells cultured in medium lacking pyruvate and glucose or in rat liver after fasting for 24 h . FeedingExtensive protein CoAlation induced by oxidising agents and metabolic stress, and the developed methodology allowed the identification of over 500 CoA-modified proteins in mammalian cells and tissues [Ka values of cysteine residues are of critical importance in redox-regulated processes. At physiological pH, the pKa values of protein cysteine thiols vary from 8.2 to 9.9 for solvent-accessible cysteines and therefore have low deprotonation ability [Ka) are more susceptible to deprotonation and oxidation to sulfenic acid in the presence of ROS [in vitro CoAlation of metabolic and signalling proteins, we propose that under oxidative stress, CoASH can modify covalently cysteine residues within a basic amino acid environment, as they have lower pKa and higher reaction rates with H2O2 and other oxidising compounds.The p ability . Cysteine of ROS . Sulfeni2O2-perfused rat hearts clearly suggests that different regulatory cysteines can be specifically targeted by CoA and GSH in response to oxidative stress. Bioinformatics of experimentally identified glutathionylation sites revealed the presence of positively charged amino acids  flanking modified cysteines [A significant difference in the patterns of CoAlated and glutathionylated proteins in Hysteines . Redox-iThe question which arises from these findings is: how does covalent attachment of CoA affect the regulation and function of modified proteins ? First oWhat is the relevance of protein CoAlation in the function of mammalian cells? It is reasonable to speculate that under normal growth conditions, CoA functions to produce metabolically active thioesters, while it may act as a LMW antioxidant protecting protein cysteine thiols from irreversible overoxidation in cellular response to oxidative and metabolic stress.Maintaining a balance between oxidation and reduction reactions is essential for key cellular processes, such as growth, proliferation, differentiation and survival. Accumulating evidence indicates that dysregulation of redox-sensitive signalling and the thiol–disulfide homeostasis are associated with various human pathologies, including metabolic syndrome, cancer, cardiovascular and neurodegenerative diseases. Protein CoAlation is an emerging field of research that has the potential to become an integral part of redox sensing and regulation under physiological and pathophysiological conditions.in vitro by a non-enzymatic mechanism [Understanding how mammalian cells sense ROS and co-ordinate downstream biological responses is still a major challenge. The original findings on protein CoAlation raise a fundamentally important question: what are the molecular mechanisms of protein CoAlation/deCoAlation? The proposed redox-regulated CoAlation/deCoAlation cycle is shown in echanism . Howeverin vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase efficiently protects the catalytic cysteine 151 from irreversible overoxidation by H2O2 . The potential for CoA to function as a physiological antioxidant in cell-based and animal models has yet to be investigated.We have recently demonstrated that in situ visualisation of CoAlated proteins in cells and tissues will be of particular importance for advancing research in this emerging field of study.Developing new research tools and methods will be instrumental for promoting research on protein CoAlation. The quantitative measurement of protein CoAlation in proteome-wide studies and the Unravelling the roles of protein CoAlation and antioxidant function of CoA in human pathologies associated with oxidative stress and redox imbalance is an important goal for the future.


In specific dosages, the N-methyl-D-aspartate receptor (NMDA) antagonist ketamine can be used to model transient psychotic symptoms in healthy individuals that resemble those of schizophrenia. Ketamine administration also temporarily impairs cognitive functions, which can be studied by event-related potentials (ERPs). ERPs also allow dissecting what stages of information processing are affected by ketamine and what stages remain functional. For tasks requiring the differentiation of targets and non-targets, it has repeatedly been shown that ketamine administration in healthy individuals leads to decreased amplitudes of the ERP component P3b in response to target stimuli. However, it could be argued that this ketamine-induced P3b reduction is the consequence of an increased difficulty to differentiate targets from non-targets, primarily mediated by ketamine's psychotomimetic rather than pharmacological effects. The current review of ERP studies seeks to clarify the issue whether P3b effects of ketamine may indeed be explained as the consequence of an experienced increase in task difficulty or whether alternative mechanisms are perhaps more plausible. The review first summarizes the effects of task difficulty on ERP components related to intentional stimulus categorization (P3b), involuntary attention switches to distractors (P3a), as well as sensory processing . Secondly, the ERP effects of task difficulty are contrasted with those observed in ketamine studies in healthy individuals. Findings show that P3b amplitudes are consistently diminished by an increased task difficulty, as well as after ketamine administration. In contrast and as most important difference, increased task difficulty leads to increased P3a amplitudes to distractors presented in same modality as targets, whereas ketamine leads to reduced P3a amplitudes for such distractors. This dissociation indicates that the decreased P3b amplitudes after ketamine cannot be explained by a drug-induced increase in task difficulty. The conjoint reductions of P3a and P3b amplitudes instead suggest that working memory operations, in particular working memory updating are impaired after ketamine, which is in line with previous behavioral findings. Various studies have administered subanesthetic doses of ketamine, an N-methyl-D-aspartate receptor (NMDA) antagonist, to model psychotic symptoms and psychosis-like experiences in healthy individuals. Krystal et al.  were oneSome of the studies used event-related potentials (ERPs) in order to test whether such subanesthetic doses of ketamine lead to processing deficits in healthy individuals similar to those observed in patients with schizophrenia and to reveal the neurocognitive consequence of the drug administration , as well as the task accuracy.In the first result section, we present a selective review of previous studies showing how the ERP components P3a and P3b, P1, N1, and PN of healthy individuals are modulated by an increased task difficulty in active oddball tasks. Task difficulty in oddball experiments can be modulated by a range of factors , with reduced P1/N1 amplitudes after ketamine administration becoming more apparent at long ISIs  reported diminished task accuracy after ketamine (Ahn et al., Ketamine effects on the P3a were investigated in three active oddball studies (Watson et al., The current review evaluates empirical evidence whether the ketamine-induced ERP alterations in active oddball experiments can be explained by a drug-related decreased discriminability of targets and standards, resulting in an increased task difficulty, as suggested by Schwertner et al. . The majOverall, there is only weak empirical evidence that ketamine leads to decreased discriminability of standard and target stimuli, as suggested by Schwertner et al. . The patThis failure to update neural context representations might primarily be related to encoding deficits: Already in early studies, a disruption in episodic memory after ketamine has been reported (e.g., Krystal et al., In general, behavioral, neurophysiological, and neuroimaging studies on ketamine effects in healthy individuals have improved our understanding of a possible contribution of a deficient glutamatergic neurotransmission to symptoms and cognitive deficits in schizophrenia. On the basis of the NMDA receptor hypofunction hypothesis Javitt, , new treThe conjoint reduction of the target-related P3b and the distractor-related P3a after ketamine administration conflicts with the assumption that ketamine alters the perceived salience of different categories of stimuli, as proposed by Schwertner et al. . Rather,TR searched and reviewed the literature. TR drafted the initial version of the manuscript. TR and AS were involved in the finalization of the submitted manuscript and discussed the significance of the findings.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.


Furthermore, it is increasingly evident that most if not all leucocyte populations migrate in lymph and that such migration is not only important for immune modulation, but also for the timely repair and resolution of tissue inflammation. In this article, I review the latest research findings in these areas, arising from new insights into the distinctive ultrastructure of lymphatic capillaries and lymph node sinuses. Accordingly, I highlight the emerging importance of the leucocyte glycocalyx and its novel interactions with the endothelial receptor LYVE-1, the intricacies of endothelial chemokine secretion and sequestration that direct leucocyte trafficking and the significance of the process for normal immune function and pathology.The lymphatics fulfill a vital physiological function as the conduits through which leucocytes traffic between the tissues and draining lymph nodes for the initiation and modulation of immune responses. However, until recently many of the molecular mechanisms controlling such migration have been unclear. As a result of careful research, it is now apparent that the process is regulated at multiple stages from initial leucocyte entry and intraluminal crawling in peripheral tissue lymphatics, through to leucocyte exit in draining lymph nodes where the migrating cells either participate in immune responses or return to the circulation  MEM and TREG) and neutrophils to draining lymph nodes (dLNs) in the process of immune activation, modulation and peripheral tolerance  cells, recently re-defined as the recirculating memory (TRCM) subset  cells. Furthermore, more recent cell tracking studies using photoconvertible Kaede mice have revealed that a significant proportion (25%) of the CD4 population are FOXP3+ TREGs, thus uncovering a previously unrecognized role for the lymphatics in conveying these important immunoregulatory cells. By comparison, only low numbers of naïve T cells are usually present in afferent lymph, and despite the fact these can be shown to enter lymphatic capillaries after adoptive transfer in mice, their normally low frequency in tissue means they rarely do so in vivo. The likely functional significance of TEM cell migration in afferent lymph may be to allow the amplification and polarization of immune responses in the dLNs and maintenance of the T cell memory pool, as well as enabling the re-entry of these lymphocytes to the circulation to target pathogen dissemination in further tissue sites , followed by antigen presenting DCs and very small numbers of B cells which together account for most of the remaining 10–15%. Most of the T cells are antigen-experienced CD4) subset , 21, whiesponses –25. Notaesponses –28, whicue sites , 21.The second most numerous leucocyte population in afferent lymph are DCs, which ferry endocytosed antigens from the tissues, primarily for immune priming in dLNs. Although small numbers of DCs migrate in lymph under steady state conditions to maintain peripheral tolerance to self-antigens –33, the via the inflamed lymphatics to dLNs (1/2 6–12 h) and undergo early apoptosis before removal by macrophage efferocytosis  monolayers form only partial surrogates of discontinuous junctions , this traffic increases significantly. For practical reasons, most studies of leucocyte interstitial migration in the context of lymphatic trafficking have focused on DCs in the mouse dermis and epidermis. Although sessile in the steady state, these cells are subsequently induced to differentiate and crawl toward LVs, in response to inflammatory signals received from cytokines, prostaglandins and leukotrienes released from keratinocytes activated by microbial products and other Toll-like receptor (TLR) ligands , and heparan sulfate proteoglycans (HSPGs) . The rat ligands . The mov ligands , 57, 72. ligands . This dil domain . Moreovel domain . Migratiase ROCK , 74, 75.adhesion , it was n fibers . In the ex vivo crawl-in assays with dermal tissue explants  deficient mice reported that under normal resting conditions, DCs can enter lymphatic vessels and migrate to dLNs independently of all integrins, just as documented for interstitial migration, by means of chemokine-directed amoeboid motion  or treatment with bacterial LPS  upregulation of the key integrin counter-receptors ICAM-1 and VCAM-1 together with various other adhesion molecules associated with leucocyte endothelial transit such as E-selectin, and a range of different chemokines that are attractants for DCs, monocytes, lymphocytes and neutrophils  forms of β2 integrin (LFA-1) present in complementary projections on the DC surface , it therefore relies on homo-dimerization and clustering, as well as a high ligand density to achieve the multiplicity of co-ordinate binding interactions required for tethering of the polymer chains  controls the lymphatic entry step  population that remains resident within the tissues as sentinels, and have since been defined phenotypically as a (CCR7int/+ CD62Lint CD69− CD103+/− E-selectin ligand+) recirculating memory (TRCM) cell subset, based on their tracking in photoconvertible fluorescent Kaede mice (+ T regulatory cell (TREG) as well as inflammation-associated Th1 and Th17 cell subsets . As. Aset almembrane . Diapedee uropod . The CCLantibody , 90, 1353CR, respectively, and CXCL1, CXCL2, CXCL5, and CXCL8 that are chemotactic for neutrophils that express CXCR1 and CXCR2. In common with CCL21 these are all upregulated by exposure of the endothelium to inflammatory cytokines or other inflammatory stimuli (+ DC in skin and co-administration of a synthetic CXCR4 antagonist (4-F-benzoyl-TN14003) impaired their migration to dLNs and capacity to promote T cell proliferation after contact hypersensitization with the hapten DNBS  monolayers, in co-operation with CCL21, as well as entry to intact lymphatic vessels in ex vivo exposed skin , ACKR2, formerly known as D6, ACKR3 (CXCR7), and ACKR4 (CCRL1) bind and internalize inflammatory CC chemokines and prevent their inappropriate accumulation on the surface of lymphatic capillaries while also helping to establish the polarity of their gradients . Accordir access , 147. Itr access .+ T cell entry to initial lymphatics in inflamed peripheral tissues. Normally present at high concentrations in lymph and low concentrations in lymph nodes, the resulting gradients of S1P direct transit of T cells bearing the G-protein coupled receptor S1PR to efferent lymph , best known as a regulator of lymphocyte exit from lymph nodes but which appears also to be a negative regulator of CD4nt lymph , 150. Hoillaries . Moreoved VCAM-1 . WhetherREG population that migrates between tissues and lymph nodes to maintain peripheral tolerance and immune suppression have been shown to employ lymphotoxin (LT), a lymphokine member of the TNF superfamily, to transit inflamed lymphatic vessels by engaging its signal transducing receptor LTβR in lymphatic endothelium (via lymphoid tissue inducer (LTi) cells, the trimeric lymphotoxin molecule is expressed on the surface of TREG as a membrane-bound LTα1β2 heterotrimer. Moreover, genetic deletion of LTα in murine TREGs or treatment with soluble LTβR Ig fusion protein was shown to disrupt their entry to lymphatic capillaries in mouse skin, while leaving the transit of naïve CD4 and CD8 cells unaffected HETE), that together evoke local endothelial junctional retraction  fulfilled this role , fluorescent CCL21 secreted from the luminal surface of the endothelium underwent re-binding downstream to form authentic, directionally oriented gradients for DC migration (D 70 nM) in Biacore analyses  that likely renders intraluminal crawling redundant, and hence it is thought the cells are conveyed toward downstream lymph nodes by passive lymph flow.RCM cells which reside transiently before returning to the blood circulation via efferent lymphatics, most leucocytes reaching the nodes proceed no further and having fulfilled their immune function, ultimately die there. In either case the cells arrive into the subcapsular sinus (SCS), a labyrinthine compartment continuous with the afferent lymphatics that is situated just beneath the outer capsule of the node. From there, they transit across the SCS endothelium to access the T and B cell-rich cortex, in the case of DCs and neutrophils to prime or re-activate T cell immune responses, and in the case of TMEM and TREGs to influence or downregulate such responses, before they egress and circulate back to the tissues  lymphatics, it was found that DCs and CD4 the SCS . Whereas the SCS . Moreoverenchyma . Curioustor CCR8 . More resduction . Signifisduction .aka Pal-E, MECA 32) that forms diaphragm-like structures in the SCS and certain blood vessels and which primarily regulates the size selective entry of macromolecules to the underlying cortical fibrillar conduit network for presentation by LN HEVs  cells and innate lymphoid cells (ILCs). However, the notion of a synapse may not apply to neutrophils which use a unique mechanism of integrin-dependent proteolysis and lipoxin-mediated endothelial retraction to “invade” lymphatic vessels. This unusually specialized process may have evolved to enable these professional phagocytes to exit almost instantaneously from sites of infection and reach the dLNs well-ahead of slower migrating DCs. In comparison, we know little about how the docking and adhesion of transmigrating leucocytes is choreographed and how the many “accessory” adhesion molecules including the Mannose receptor, ALCAM, CLEVER-1, CD31, CD99, and others (via lymph is markedly increased, zippers replace buttons. It will be interesting to determine whether leucocytes have the ability to enter through zipper junctions and whether the process involves different molecular mechanisms to buttons and transit via a transcellular or paracellular route. A further priority will be to ascertain whether these mechanisms of lymphatic entry are universally applicable or vary between tissue beds such as the intestines, brain and central nervous system (As outlined in this text, we now have detailed insight into the first key step in such trafficking, the entry of cells to the lymphatic vessels. In the case of DCs, this has revealed an intricate and closely co-ordinated mechanism in which physical contact of migrating leucocytes with lymphatic endothelium triggers the local exocytosis of CCL21 and formation of LYVE-1el entry , 85, 101d others  integrats system , given t+ T cells and neutrophils actively crawl along the internal surface of initial vessels using guidance from CCL21 and transient integrin-mediated adhesion. Yet why such mechanisms should have evolved to deliberately slow the downstream progress of antigen presenting and immune effector cells is unclear. Might the intimate contact with endothelium imposed by intraluminal crawling enable for example en route uptake of foreign antigens or maturation signals by DCs, or MHC-mediated antigen presentation to recirculating T cells or TREGs for immune tolerance? Could it provide a platform for interactions between different leucocyte populations themselves? Or might intraluminal crawling provide DCs or T cells with the option to exit and re-enter lymphatic vessels prior to reaching dLNs in order to sample or respond to antigen in the surrounding tissues? Though not yet reported in initial lymphatics vessels, there is evidence that migrating leucocytes exit collectors in adipose tissue during bacterial infection, and that DCs which interdigitate the vessel endothelium to regulate vessel permeability and sample the surrounding tissue can subsequently detach and migrate to dLNs (Recent research has also provided surprising insight into how leucocytes, having entered the initial lymphatics, migrate within the vessel lumen toward downstream lymph nodes. Rather than being conveyed by passive lymph flow, it is now apparent that DCs, CD4 to dLNs .REGs, the inclusion of lymphotoxin blockade to impair their migration to lymph nodes for immune suppression might also provide an adjunct to checkpoint inhibitors for tumor immunotherapy. As a corollary, our understanding of the main factors regulating DC migration from inflamed tissues could be exploited to optimize vaccine delivery. For many years the notion of using DCs as adjuvants for adoptive cancer immunotherapy has fuelled efforts to enhance their maturation ex vivo using TLR ligands and inflammatory cytokines and to optimize antigen loading and presentation. Combining these approaches with pre-conditioning of vaccination sites to boost lymphatic vessel density, the efficiency of DCs for vessel entry and nodal transport should lead to much greater clinical efficacy. Likewise, boosting the exit of macrophages via lymphatics could aid in the resolution of inflammation and tissue recovery in conditions such as myocardial infarction where their delayed removal results in a failure of cardiomyocyte replenishment, and an increase in tissue scarring and fibrosis (With the many insights into lymphatic trafficking and its immune consequences that have been gained from basic research, there is increasing scope for clinical translation and the development of new immune based therapies based on migration blockade. Judicious targeting of integrins, ICAM-1, chemokine receptors, S1P and particularly lymph-specific adhesion molecules such as LYVE-1 by appropriate blocking mAbs may be envisaged as therapeutic strategies for transplant rejection, to prevent DC migration from engrafted tissues and consequently activation of alloimmune responses in host dLNs. This is particularly applicable in the case of corneal allografts, where such therapies could be applied locally, thus avoiding off-target effects associated with systemic antibody administration. In the case of Tfibrosis , 179–181The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine, which is thought to play a major role in the pathogenesis of inflammatory diseases, including allergy. TNF is produced at the early stage of allergen sensitization, and then continues to promote the inflammation cascade in the effector phase of allergic reactions. Consequently, anti-TNF treatment has been proposed as a potential therapeutic option. However, recent studies reveal anti-intuitive effects of TNF in the activation and proliferative expansion of immunosuppressive Tregs, tolerogenic DCs and MDSCs. This immunosuppressive effect of TNF is mediated by TNFR2, which is preferentially expressed by immunosuppressive cells. These findings redefine the role of TNF in allergic reaction, and suggest that targeting TNF-TNFR2 interaction itself may represent a novel strategy in the treatment of allergy. Highlights- Pleiotropic function of TNF in allergy is likely mediated by its two receptors, TNFR1 and TNFR2- Activation by TNFR1 results in the allergic inflammatory responses while TNFR2 plays a role in the immune tolerance to allergens- TNFR2 is preferentially expressed by highly suppressive and replicating Tregs and TNFR2 signaling leads to the activation and proliferation of Tregs- Targeting of TNFR2 to boost Treg activity may represent a novel strategy for treating patients with allergy.Allergy is an immune-mediated hypersensitivity to allergens. Exposure of allergens through inhalation, ingestion or skin contact leads to diseases such as asthma, allergic rhinitis, food allergy, and atopic dermatitis. Allergy is a complex disease, and both genetic and environmental factors contribute to its pathogenesis. It affects 30–35% of the population at some point in their life, with incidence continuing to rapidly grow each year. The past decades have exhibited large-scale anthropogenic changes which are currently considered the leading causes of the increasing burden of allergic diseases.The development of allergy can be divided into two phases: 1) the sensitization and memory phase, and 2) the effector phase, which can be further staged into immediate and late responses   . In one or Tregs  was dimior Tregs . In anotor Tregs . Becauseor Tregs . Furtheror Tregs .Overall a proper maturity and homeostasis of immune system of neonates during the first years of life is fundamental to minimize allergic development. Maternal allergy correlates well with impaired frequency and function of Tregs in neonates and consequently the increased susceptibility for the development of allergy in early childhood –29. A stTNF is a pleiotropic cytokine that plays important dual roles in maintaining immune homeostasis and in promoting the development of diseases. TNF is required for host defense against pathogens, immune surveillance against malignancies, as well as cell proliferation and survival . This cyDual biological functions of TNF are likely to be transduced through its two distinct receptors, TNFR1 and TNFR2. TNF and its receptors have both a membrane-bound form and a soluble form. TNFR1 shows high affinity toward both forms of TNF, while TNFR2 is only fully activated by membrane-bound TNF . Once acTNF is reported to play significant role in the pathogenesis of allergy and contributes to both early and late stages of allergy development. This is evident when allergy manifestations are inhibited in TNF-knockout (KO) mice as well as with anti-TNF treatments –37. FurtAlthough binding of TNF to its distinct receptors generally activates the same major signaling pathways (NFκB and MAPK), the distinct structure and motifs of TNFR1 and TNFR2 result in entirely different functional consequences. TNFR1 bears the death domain, which recruits several death signaling proteins such as TNFR1-associated death domain protein (TRADD), Fas associated protein with death domain (FADD) and the TNFR-associated factor (TRAF)-1 to induce inflammation and apoptosis .In the context of apoptosis, TNF-TNFR1 axis signals through caspase 3 and 8, and is mainly responsible for the host defense of pathogen  and antiUnlike TNFR1, TNFR2 lacks the death domain where the activation of TNFR2 recruits TNFR-associated factor 2 (TRAF2) that mainly promotes cell proliferation and survival . In alleDistinct functions of TNF and its receptor led to the prospective of selectively targeting TNFR1 to inhibit apoptosis, and TNFR2 to induce cell survival. This approach intends to achieve homeostasis in various autoimmune and inflammatory diseases including allergy. These disorders are primarily associated with defects in TNF signaling through TNFR2. Therefore, this particular receptor is of interest and important roles of TNF-TNFR2 interaction on various cell types are further discussed in the next section  have been established as therapeutic options in inflammatory diseases. Etanercept, a genetically engineered recombinant protein that comprises of TNFR2 and Fc portion of human IgG1, specifically binds to TNF and blocks its interaction with cell surface receptors . StudiesUnlike in allergy, TNF-TNFR2 axis in autoimmune diseases and cancer has been well established. Adalimumab has shown to expand functional Tregs through TNF-TNFR2 axis on monocytes in rheumatoid arthritis . Another+Foxp3+ Tregs after allergen challenge, partly via the activation of CD103+ DCs . It can be considered as an achievable long-term cure for allergy as basic principle in SIT, which involves inducing immune tolerance toward allergens by specifically and repeatedly administrating the causative allergen (Due to its restricted cellular expression and prominent role in immune regulation, TNFR2 is still a more attractive target for treatment in diseases, including allergy. Faustman and Davis  suggeste103+ DCs , 110. Thallergen .In allergy, TNF may have dual pro-inflammatory and anti-inflammatory activities, which are likely mediated by its two distinct receptors. TNFR2 signaling is attributable to the immunosuppressive effects of TNF and thus is protective against allergy. Consequently, targeting TNF-TNFR2 pathway may represent future direction to develop new therapies in allergy. However, as a pleiotropic cytokine, TNF and its effects on TNFR signaling is diverse and can either regulate allergic manifestations or to control disease pathogenesis. Implications of TNFR2 in health and diseases requires additional investigation to further elucidate their exact mechanisms and provide more insight for future strategies in manipulating TNFR2 for therapeutics.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glutaredoxins (GRXs) have at least three major identified functions. In apoforms, they exhibit oxidoreductase activity controlling notably protein glutathionylation/deglutathionylation. In holoforms, i.e., iron–sulfur (Fe–S) cluster-bridging forms, they act as maturation factors for the biogenesis of Fe–S proteins or as regulators of iron homeostasis contributing directly or indirectly to the sensing of cellular iron status and/or distribution. The latter functions seem intimately connected with the capacity of specific GRXs to form [2Fe–2S] cluster-bridging homodimeric or heterodimeric complexes with BOLA proteins. In yeast species, both proteins modulate the localization and/or activity of transcription factors regulating genes coding for proteins involved in iron uptake and intracellular sequestration in response notably to iron deficiency. Whereas vertebrate GRX and BOLA isoforms may display similar functions, the involved partner proteins are different. We perform here a critical evaluation of the results supporting the implication of both protein families in similar signaling pathways in plants and provide ideas and experimental strategies to delineate further their functions. Many cellular reactions and biological processes require metalloproteins, among which those containing iron (Fe) cofactors such as mononuclear and dinuclear (non-heme) Fe centers, hemes and iron–sulfur (Fe–S) clusters, are particularly crucial. Unlike other metals such as copper or zinc, there is no universal Fe chaperone described and so far, only poly rC-binding proteins (PCBPs) were shown to coordinate Fe entry in the cytosol and serve for the metalation of non-heme Fe enzymes in mammals . In contTwo GRX classes, I and II, are present in most organisms whereas additional classes are specific to some species/genus/kingdoms . GRXs ofEscherichia coli BolA in the regulation of cell morphology, possibly as a transcriptional regulator (Saccharomyces cerevisiae cytosolic Bol2/Fra2 (Fe repressor of activation 2) in the regulation of iron homeostasis  and large-scale interactomic analyses in various organisms   . Yeast Bproteins . So far,proteins . The cliand Bol3 . Additioand Bol3 .E. coli were recently shown as implicated in the maturation of the respiratory complexes I and II, but the effects are only visible when multiple genes are mutated  and both BolAs (BolA and IbaG) from  mutated . This roC operon , (ii) th protein , and  the corresponding yeast grx5 and bol1–bol3 mutants, indicating that they possess similar structural and functional determinants  localization is controlled by a Fra2-Grx3/4 inhibitory complex   . The curse Fra1) . By transe Fra1) .Cryptococcus neoformans, Fe repletion promotes Grx4 relocation from the nucleus to the cytoplasm allowing the regulation of Cir1, a master regulator of Fe-responsive genes ], and mitochondria  is altered  at high temperature or under long photoperiod  mutant displays no phenotype under control conditions and no change in the activity of typical Fe–S enzymes , a role in the maturation of Fe–S proteins still needs to be demonstrated in planta, despite they can functionally substitute to their mitochondrial yeast counterparts.Evidence obtained so far indicate that the class II mono-domain GRXs and BOLAs present in mitochondria of non-plant eukaryotes and in bacteria act as maturation factors for the biogenesis of Fe–S proteins. A similar role seems true for the plant mitochondrial GRXS15, but it is now mandatory to examine whether it also contributes to the maturation of extra-mitochondrial proteins. Another challenge will be to understand why it is essential in plants unlike in yeast. Also, the physiological consequences of bol2/fra2 mutant. Experimental evidence indicate that the involvement of GRX and/or BOLA in DRE2 maturation is likely also true in mammals and plants, but evidence supporting other functions are scarce.With regard to the cytosolic multi-domain GRXs and BOLAs, a role in Fe metabolism seems evolutionary conserved, but their contribution and partners differ. In yeast, their primary function is to regulate Fe-responsive transcription factors. Additional functions are to ensure a proper Fe distribution toward all types of Fe cofactors (including heme and non-heme Fe centers) and/or to serve for Dre2 maturation, thus contributing to the correct functioning of the CIA machinery. In this case, Grx3/4 have an exclusive or predominant role because the corresponding mutant is lethal or strongly affected, unlike the GRXS15, but also dominant negative mutant lines expressing mutated versions of GRX or BOLA unable for instance to ligate the Fe–S cluster, i.e., mutated for the catalytic cysteine of GRXs or the conserved histidine residue of BOLA.A first prerequisite to future molecular and physiological analyses is to generate the missing single knock-out lines but also multiple knock-out lines for possibly redundant proteins. This would be particularly important to obtain lines combining mutations for GRXS14 and GRXS16, for BOLA1 and BOLA4, but also for GRXS17 and the only other Fe–S ligating GRXs reported so far in the cytosol, namely GRXC1 , or BOLAgrxs17 mutants are only visible in specific conditions. For the BOLA2-GRXS17 couple, understanding their connection and discriminating between Fe- or redox-related functions will require in particular to assess the phenotypes of the corresponding mutants in the same experimental setup and conditions. Considering the described importance of GSH for ligating Fe–S cluster in GRX homodimer or GRX-BOLA heterodimer and for the maturation of cytosolic Fe–S proteins (At the physiological level, the growth of these plants should be analyzed under standard conditions, but also under environmental constraints as the shoot phenotypes of proteins , crossinIn other respects, an obvious strategy is to measure the abundance/activity of representative Fe–S proteins in these lines. However, performing quantitative proteomic and metabolomic approaches may be more informative and help obtaining a broader view of the molecular and cellular mechanisms affected and of the compensations established. It may also rapidly point to metabolic differences existing among mutants.In all cases, determining the identity of the direct and indirect targets of both GRXs and BOLAs would represent a mandatory information. For instance, the proteins involved in the Fe–S cluster maturation process may act at different steps. Various approaches complementary to quantitative proteomics proved valuable even for detecting supposedly transient interactions among Fe–S cluster donors and acceptors . Hence, In summary, the combination of genetic approaches, omics analyses and conventional biochemical tools should in principle allow better delineating the roles and specificities of GRX/BOLA couples in the maintenance of Fe homeostasis in plants.All datasets analyzed for this study are included in the manuscript and the Supplementary Files.All authors wrote the text and approved the final version 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.


Plasmodium parasites, is widespread throughout tropical and sub-tropical regions worldwide; it mostly affects children and pregnant woman. Eradication has stalled despite effective prevention measures and medication being available for this disease; this has mainly been due to the parasite's resistance to medical treatment and the mosquito vector's resistance to insecticides. Tackling such resistance involves using renewed approaches and techniques for accruing a deep understanding of the parasite's biology, and developing new drugs and vaccines. Studying the parasite's invasion of erythrocytes should shed light on its ability to switch between invasion phenotypes related to the expression of gene sets encoding proteins acting as ligands during target cell invasion, thereby conferring mechanisms for evading a particular host's immune response and adapting to changes in target cell surface receptors. This review considers some factors influencing the expression of such phenotypes, such as Plasmodium's genetic, transcriptional and epigenetic characteristics, and explores some host-related aspects which could affect parasite phenotypes, aiming at integrating knowledge regarding this topic and the possible relationship between the parasite's biology and host factors playing a role in erythrocyte invasion.Malaria, a disease caused by  Anopheles mosquito carrying Plasmodium parasites; the disease emerges mostly in tropical and sub-tropical regions around the world. Five Plasmodium species infect humans: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi   WHO, . The Worty) WHO, . Such inMost of malaria's clinical symptoms are related to its ability to select, invade, and proliferate inside erythrocytes. A pool of proteins acting as invasion ligands having affinity for receptors on erythrocyte surface would thus enable the recognition, attachment and invasion of the parasite in its merozoite form  and reticulocyte binding-like homologs   and some members of the Pf Rh family    . The former (invasion ligands) enable parasite adhesion and, during the first steps of invasion, ligand selection associated with the parasite's invasion phenotype ; however, they may change the expression of some virulence-associated genes. Strikingly, Tarr et al.  encoded by such var genes   1, 2, and 3, might be acting as a strategy for evading a host's immune response, since such high A-T composition was found to be associated with a reduced amount of hydrophobic amino acids and thus a relative increase in hydrophilic ones, these being good targets for antibody responses  revealed a high G:C to A:T transition rate and low complexity regions in all parasite genomes analyzed, thereby facilitating the appearance of indels. This showed that the parasite has genomic structural malleability, partly enabling its ability to adapt and evolve  , having intergenic regions and promoter sequences. Like other eukaryotes, the parasite's mRNA is processed in the nucleus and translated in the cytoplasm; the transcription machinery is partly responsible for regulating expression, being modulated in response to environmental stimuli like temperature, glucose levels and stress factors  and transcription factors (elements in trans) , describing 27 proteins associated with the transcription of specific genes in P. falciparum during precise phases of the parasite's life cycle  acting on genes associated with invasion by binding to the NGGTGCA upstream motif  to evaluate the motif's importance in gene transcription  for reanalysing Santos et al. (pfrh2b) and Pf EMP1-associated var gene in the mutated strain compared to the non-mutated one. The authors reported no significant changes regarding pfeba140, pfeba181, pfeba175, pfrh1, pfrh2a, pfrh2b and pfrh5 expression  is a Pf AP2 coactivator regulating transcription during invasion, possibly as a secondary effect after complex formation between these two proteins  and with the parasite's motility mechanisms    , binding DNA in a similar way to that of orthologs found in other eukaryotes  has repressive activity during transcription, is conserved in other eukaryotes, binds both DNA and RNA and could play a role in parasite transcription regulation  and P. falciparum calcium-dependent protein kinase 1 (Pf CDPK1)] until its transcription its needed (late schizont stage), suggesting fine-tuning of translation time  transcription factor, the pre-initiation complex (PIC) and nuclear factors related to the high-mobility-group (HMG) box  acting as cell cycle control and progression regulator), histone 3 (H3), and histone 2A (H2A)  and the transcription factor IIE (Pf TFIIE), coordinating transcriptional initiation, promoting loop formation enabling interaction with other transcription factors and favoring nucleosome assembly  and trophozoite stages   comprise the Pf HMGB1 and Pf HMGB2 have specificity for DNA, are located in the trophozoite and schizont nuclei and are involved in DNA bending, modifying the nucleosome structure and inducing transcriptional changes  and sexual (gametes) parasite forms has shown that both proteins are expressed in the nucleus during intra-erythrocytic cycle stages and that Pf HMGB2 was also present in gametocytes cytoplasm. This led to the conclusion that Pf HMGB1 could be associated with proliferation whilst Pf HMGB2 might be involved in the parasite's sexual differentiation  for targeting gene regulation promoter sequences   are directly correlated with transcription activation and the amount of transcriptional activity occurring during the intra-erythrocytic cycle ]  is important for chromatin remodeling and has been previously described in eukaryote organisms as being related to heterochromatin structure and gene silencing or activation; it has an ortholog in the parasite  or GYPB (S-s-) deletion) because these proteins are implicated in EBA175 (GPA) and EBL1 (GPB) ligand binding  and GYPB-A hybrid correlation ; this has previously been described as a malaria erythrocyte invasion protector antigen, as assessed by in vitro invasion assays  and Leach blood groups  , encoded by the Plasmodium species, such as P. vivax, has been thoroughly described; it is also known as the Duffy antigen receptor for chemokines (DARC), referring to antigens encoded by the FY gene, having alleles FYa and FYb, which are related to Fy+b−)(a antigens. Fy −b−) binding  interaction with DARC; it is important during invasion for merozoite attachment and tight junction formation between merozoite and erythrocytes  could appear during both ring and trophozoite stages and are associated with chromatin changes and transcriptional regulation by transcription factors  inhibits calcium release and also Pf EBA175 secretion  gene  .Histone acetylation may be a mechanism enabling repressed alternative gene transcription. This could apply to P. falciparum parasite must sense a targeted host's nutritional status, evade its immune response and bind to available erythrocyte receptors for efficient invasion and development   influence parasite invasion of erythrocytes, as such interactions may trigger modifications in the parasite's signaling pathways that could induce changes in chromatin architecture and the formation and activation of transcription factor complexes governing gene transcription programmes (i.e., inducing sexual differentiation and gametocyte formation, or the expression of the genes required during invasion such as P. falciparum's invasion of host cells. Further investigation is required regarding the signaling pathways activated by external stimuli which could play a role in the epigenetic and transcriptional changes affecting the expression of some invasion ligands related to invasion phenotypes. Such efforts will enable finding critical points which could be exploited for medical treatment and vaccines facilitating effective control of this disease.Studying such interactions and changes in the parasite-host relationship may facilitate a renewed approach to dealing with parasite infection; microarray, chromatin immunoprecipitation and sequencing technologies have accelerated most recent discoveries in this field, nevertheless, more studies are needed to characterize the complete set of factors affecting MA-S conceived the work, drafted the manuscript and designed the figures. MAP and HC critically revised the manuscript for intellectual content. All the authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The induction and action of type I interferon (IFN) is of fundamental importance in human immune defenses toward microbial pathogens, particularly viruses. Basic discoveries within the molecular and cellular signaling pathways regulating type I IFN induction and downstream actions have shown the essential role of the IFN regulatory factor (IRF) and the signal transducer and activator of transcription (STAT) families, respectively. However, the exact biological and immunological functions of these factors have been most clearly revealed through the study of inborn errors of immunity and the resultant infectious phenotypes in humans. The spectrum of human inborn errors of immunity caused by mutations in IRFs and STATs has proven very diverse. These diseases encompass herpes simplex encephalitis (HSE) and severe influenza in IRF3- and IRF7/IRF9 deficiency, respectively. They also include Mendelian susceptibility to mycobacterial infection (MSMD) in STAT1 deficiency, through disseminated measles infection associated with STAT2 deficiency, and finally staphylococcal abscesses and chronic mucocutaneous candidiasis (CMC) classically described with Hyper-IgE syndrome (HIES) in the case of STAT3 deficiency. More recently, increasing focus has been on aspects of autoimmunity and autoinflammation playing an important part in many primary immunodeficiency diseases (PID)s, as exemplified by STAT1 gain-of-function causing CMC and autoimmune thyroiditis, as well as a recently described autoinflammatory syndrome with hypogammaglobulinemia and lymphoproliferation as a result of STAT3 gain-of-function. Here I review the infectious, inflammatory, and autoimmune disorders arising from mutations in IRF and STAT transcription factors in humans, highlightning the underlying molecular mechanisms and immunopathogenesis as well as the clinical/therapeutic perspectives of these new insights. Several decades of research uncovering the basic biology, regulation; and functions of the machinery for induction and responses to type I interferon (IFN) have paved the way for an understanding of a number of very diverse human diseases arising when one or more molecules in these pathways are defective. In this manner, the study of humans with primary immunodeficiencies (PID)s provides important understanding of specific protective immunity in humans, an insight that cannot always be gained by studying experimental animal models. Moreover, the study of individuals with defects in these pathways may teach us valuable lessons about principles of basic cell biology and infection immunology. Finally, detailed knowledge on the fundamental immunopathogenesis allows for rapid and specific diagnosis and not least for targeted treatments for individuals with these rare inborn errors of immunity.Several PIDs have been associated to the defective expression or function of molecules belonging to innate immune signaling receptors or pathways , 2. ThesThe innate immune system utilizes pattern recognition receptors (PRR)s to detect pathogen–associated molecular patterns (PAMP)s to mount protective immune responses, including production of cytokines and IFN . ProductIn mammals nine different IRF family members have been described. All IRF proteins have a conserved amino-terminal DNA binding domain (DBD) with a helix-loop-helix structure and a motif containing five tryptophan residues. IRF-association domains (IAD)1 and 2 at the carboxyterminal region of IRFs mediate homodimeric and heterodimeric interactions with other IRF family members, transcription factors, and co-factors. Whereas IRF3 has restricted DNA binding properties, IRF7 exhibits broader DNA binding specificity, accounting for its capacity to induce several IFNα subtypes. The C-terminal signal response domain of IRF proteins contains several possible phosphorylation sites, of which phosphorylation at serine 386 for IRF3 or serine 477 and 479 at IRF7 are believed to represent the main activation sites  signaling pathway mediates responses to a number of important cytokines and growth factors  Figures, 2. The Type I IFNs act via the IFNα/β receptor (IFNAR)1 and IFNAR2, type III IFN signals through IFNLR1 and IL10R2, whereas IFNγ acts via IFNGR1 and IFNGR2  Figure , 22, 25.The STAT3 molecule was discovered by Akira et al. by purification and cloning and was found to bind to the interleukin (IL)6 responsive element of the acute phase response promoter . SimultaIn the second part of this review, presently known PIDs associated with defects in IRF and STAT transcription factors are described, including the clinical presentation, infectious phenotype and the genetic and immunological basis of disease. An overview of individual genetic defects and associated PIDs is given in Table IRF3 mutation resulted in change from the positively charged arginine to the neutral glutamine, causing functionally defective phosphorylation, dimerization, and transcriptional activation of IRF3  in protection against herpes simplex encephalitis (HSE) –37. Base of IRF3 . Since n of IRF3 . This vectively, , 38. Impctively, . Also ofctively, . Importactively, .Despite a number of single nucleotide polymorphisms (SNP)s identified by genome wide association studies (GWAS) in patients with severe disseminated influenza infection, as well as evidence from mouse studies of an essential role of IFN in antiviral defenses against Influenza virus, the first monogenic defect associated with severe influenza was only described in 2015 , 43. TheIrf7 knock-out mice which exhibit elevated susceptibility to a number of RNA- and DNA viruses  homozygous IRF9 deficiency  Figure . Since Influenza . AltogetListeria monocytogenes and salmonella species, as well as fungi have also been described  viremia , originally termed Job's syndrome, “cold” abscesses caused by  feature . Other c feature , 76. Fun viremia , 78. The viremia , 80. Wit viremia . Similar viremia .STAT3 locus in patients with HIES , explaining why STAT5B defect causes Growth hormone insensitivity syndrome (GHIS) with a complex infectious and somatic phenotype Figure . This me disease . Indeed,trations . Moreovetrations . Finallytrations   or hematopoietic stem cell transplantation (HSCT), where results are generally excellent, particularly in the case of early treatment, although depending on the underlying condition and genetic etiology. Thus, survival and cure is currently reached in up to 90% of patients undergoing HSCT for severe PIDs , 116. A I addition, general vaccination strategies are relevant in the case of severe influenza in IRF7 and IRF9 deficiency. Prophylaxis with antibiotics (for example co-trimoxazole) and antifungals (such as intraconazole) are broadly employed with good results in conditions, such as HIES, CMC, GHIS, and MSMD . ProphylNovel medications include use of selective JAK inhibitors for the treatment of excessive production of type I IFN in the group of interferonopathies caused by mutations in STING or other molecules within the DNA sensing pathways , 120. SuFinally, looking into the future, new powerful technologies, such as CRISPR/Cas editing of genetic defects in PIDs is a promising avenue, which has already demonstrated potential success for medical conditions such as β-globin expression in thalassemia, and more recently in the case of certain PIDs, most notably chronic granulomatous disease (CGD) and adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) –124. It Lessons learned from the fascinating decade-long unraveling of PIDs are numerous and have provided fundamental new insight into basic infection immunology and the correlates of protective immunity against bacterial, viral, and fungal pathogens in humans. More recently, it has also brought new knowledge about the pathogenesis of a number of conditions with underlying autoimmunity and autoinflammation. Importantly, these discoveries have been taken directly into clinical medicine and are thus the basis, upon which the invention and development of specific prophylactic and therapeutic strategies for patients are based.Notably, this journey has repeatedly demonstrated significant differences between mouse and human immunity, which underscores the importance and strength of studying human patients to understand basic innate and adaptive immunology in humans. Moreover, with the increasing awareness of the major contribution of autoimmunity and autoinflammation as part of many PIDs, there is now a need for the development of approaches to treating these complex pathologies . Again, In conclusion, improved understanding of the genetic defects and immunopathogenesis of various currently known PIDs is of fundamental importance to identify new targets and immunomodulatory agents in future management of conditions of immune dysregulation, infection, and inflammation. Given that this field of research is in a rapidly expanding phase, it is to be anticipated that many additional human inborn errors of immunity will be uncovered within the next few years. Finally, despite the extensive list of PIDs caused by mutations in IRFs and STATs as described in the present review, we are likely to learn even more in the future, thus providing new insights into both basic biology, signaling, and regulation of these transcription factors and the human pathologies their dysregulation may cause.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In 2018, there was a large West Nile virus (WNV) outbreak in northern Italy. We observed five atypical cases of WNV infection that were characterised by the presence of WNV RNA and WNV IgG at the time of diagnosis, but no IgM response during follow-up. Neutralisation assays demonstrated pre-existing Usutu virus immunity in all patients. Besides challenging diagnosis, the immunological crosstalk between the two viruses warrants further investigation on possible cross-protection or infection enhancement effects. During the large West Nile virus (WNV) outbreak that occurred in Northern Italy in 2018, we observed cases with WNV infection and an atypical antibody response, characterised by a strong ‘original antigenic sin’ effect. Here, we demonstrated that these cases of WNV infection occurred in individuals with previous immunity against Usutu virus (USUV), a closely related flavivirus that is also endemic in northern Italy. We describe here the clinical and virological features of these secondary WNV infections and discuss possible clinical implications.During the surveillance period 1 June–30 November 2018, cases of possible autochthonous arboviral infection were tested at the Regional Reference Laboratory of Veneto Region  for confirmation of West Nile virus (WNV) and Usutu virus (USUV) infection according to the surveillance programme of the Italian Ministry of Health [Among individuals with confirmed WNV infection, we observed five cases  that presented with WNV IgG antibodies at the time of diagnosis who lacked a specific IgM antibody response or had a blunted IgM response during follow-up .These cases included two asymptomatic blood donors (cases 4 and 5), two blood donors – who reported headache, asthenia and myalgia during the previous 2 months (cases 1 and 2) – and a woman in week 28 of pregnancy with rash and asthenia (case 3). The pregnant woman was vaccinated against yellow fever virus in 2004; no other case reported receiving vaccination against any flavivirus nor previous flavivirus infections.WNV lineage 2 was identified in the two asymptomatic blood donors and in the pregnant woman, while WNV was not typeable in the other cases 1 and 2 because their viral load was too low. In all five cases, WNV RNA was detectable in blood for a short time after index donation or symptom onset (up to 5 days). Neutralisation assays performed at baseline and follow-up serum samples showed low or absent WNV neutralising antibodies and high titres of USUV neutralising antibodies in baseline samples and a progressive increase of WNV neutralisation titres during follow-up, consistent with WNV infection following previous USUV immunity . For comWe also observed two cases who were WNV IgM-negative but WNV IgG-positive at the time of first evaluation, who developed high levels of WNV IgM antibodies during follow-up  and WNV RNA was still detectable in whole blood on day 20 after the index donation (i.e. at the time of delivery), while in case 1, WNV RNA was detectable only in the index donation but not in follow-up samples. In both cases, neutralisation assays showed increasing titres of both WNV- and USUV-neutralising antibodies during follow-up, without significant differences between the two viruses . These lWNV RNA and USUV RNA were detected in whole blood and urine samples by in house real-time RT-PCR methods ,3. WNV nThe titres of WNV- and USUV-neutralising antibodies were defined, respectively, as the reciprocal of the highest dilution of the serum that reduced by 50% the number of plaques in Vero cells (PRNT50) and as the reciprocal of the highest dilution of the serum that showed 100% neutralisation of cytopathic effect in microneutralisation assay.WNV and USUV are genetically related neurotropic mosquito-borne flaviviruses, which are endemic in several European countries . In theiIn Italy, the first human cases of WNV infection were detected in northern Italy in 2008 . Since tInfections from closely related flaviviruses, like WNV and USUV, may pose problems not only because of the induction of cross-reactive antibodies that challenge the differential diagnosis, but also because the immunological crosstalk between heterologous viruses may increase the risk of severe disease through a mechanism of antibody-dependent enhancement of infection.The 2018 transmission season recorded a substantial increase in the number of human WNV infections, with ca 1,500 confirmed cases in European Union countries, with Italy as the most affected country . The unpThe five cases of WNV infection with atypical immune response were characterised by the presence of WNV RNA in blood and WNV IgG antibodies at the time of diagnosis, but the absence of a specific IgM antibody response or a blunted IgM response during follow-up. All these cases had already USUV neutralising antibodies at the time of the first evaluation and developed WNV neutralising antibodies during follow-up. This pattern of antibody response, which has not been described so far in the literature in patients with WNV infection, is consistent with secondary WNV infection after primary infection with USUV and supports a strong ‘original antigenic sin’ effect between the two viruses. A similar pattern of antibody response has been observed in patients with dengue virus (DENV) infection following infection with a heterologous DENV serotype  and in pTo date, no studies have investigated the immunological interplay between WNV and USUV , but expHere, the cases of WNV infection that were secondary to USUV infection were asymptomatic or had a mild febrile disease, suggesting that disease enhancement had not occurred. Further investigation including a larger number of cases is needed to better define the clinical and virological features of WNV infection in individuals with pre-existing flavivirus immunity and to understand if USUV infection provides cross-protection against WNV disease or whether it might increase the risk for more severe disease through antibody-dependent enhancement. Finally, two cases of secondary WNV infection were pregnant women who delivered healthy babies: while WNV infection is generally not associated with adverse pregnancy outcome , the eff


Objective: To identify the pressure-related features of isolated cerebrospinal fluid hypertension (ICH) in order to differentiate headache sufferers with ICH from those with primary headache disorder.Methods: In this prospective study, patients with refractory chronic headaches and suspected of having cerebrospinal fluid-pressure elevation without papilledema or sixth nerve palsy, together with controls, underwent 1-h lumbar cerebrospinal fluid pressure monitoring via a spinal puncture needle.Results: We recruited 148 consecutive headache patients and 16 controls. Lumbar cerebrospinal fluid pressure monitoring showed high pressure and abnormal pressure pulsations in 93 (63 %) patients with headache: 37 of these patients with the most abnormal pressure parameters  had the most severe headaches and associated symptoms ; 56 patients with the less abnormal pressure parameters  had less severe headaches and associated symptoms.Conclusions: Nocturnal and postural headache, and abnormal pressure pulsations are the more common pressure-related features of ICH in patients with chronic headache. Abnormal pressure pulsations may be considered a marker of ICH in chronic headache. Isolated cerebrospinal fluid (CSF) hypertension refers to the occurrence of CSF-pressure elevation in the cranial-spinal compartment without an identifiable cause. As we investigated the abnormalities of CSF pressure in patients with chronic headache by using a novel method of lumbar CSF pressure monitoring, and the pressure parameters evaluated for diagnosing elevated CSF pressure in the cranial-spinal compartment are not included in the ICHD-3 diagnostic criteria of idiopathic intracranial hypertension , the terICH manifests with symptoms and signs such as papilledema and sixth nerve palsy. However, such symptoms may be also lacking in patients with high CSF pressure , 3. In sCSF pressure measurement creates further confusion. The technique of continuous recording of CSF pressure found that patients with intracranial hypertension due to brain tumor or hydrocephalus had rapid large pulsations in pressure in form of B waves and plateau waves. Whereas, small pulsations in CSF pressure synchronous with respiration (ranging 10–30 mm CSF) and with systole (ranging 20–30 mm CSF) were observed in normal subjects . It is nGiven the difficulty of diagnosing headache attributable to isolated high CSF pressure, and the fact that a single-spot CSF opening pressure measurement may be insufficient to identify high CSF pressure in headache sufferers, recognition of headache attributable to high CSF pressure in the absence of papilledema or sixth nerve palsy is subject to much debate in clinical practice . As a reThis highlights a need to identify the characteristics and the pressure-related features of ICH in patients with chronic headache who had no evidence of papilledema and sixth nerve palsy in order to differentiate headache sufferers with ICH from those with primary headache disorder. In this study, our focus was therefore on headache sufferers suspected of having isolated high CSF pressure.Institutional Review Board approval was obtained for this prospective, single-center, cohort study, and written informed consent was obtained from all the participants.Screening of headache sufferers with refractory chronic headaches and associated symptoms  who were suspected of having high CSF pressure, and patients with non-organic neurologic disorder (psychogenic movement disorders or psychiatric diseases) who had undergone a lumbar puncture for a reason other than chronic headache was conducted between December 2012 and February 2018 at the referral center for Headache and Intracranial Pressure Disorders of the Institute of Neurology in Catanzaro. Patients were enrolled in the study if they met the following inclusion criteria: absence of papilledema  and of sixth nerve palsy; normal gadolinium enhanced MR of the brain; no history of systemic disease or drug intake known to be associated with elevated CSF pressure; and no evidence of current or prior cerebral venous thrombosis. The criteria for exclusion from the study were: abnormal neurological examination; MR evidence of structural brain lesions; cerebral venous thrombosis; hydrocephalus; and arterial hypertension.Participants were asked to complete standardized forms so as to provide us with details of their general medical history (including body mass index: weight in kilograms divided by the square of height in meters), and of their neurological, ophthalmological, and family histories. All patients were interviewed and examined by the same two neurologists, and headaches were diagnosed according to the criteria established by the IHS . PosturaThe participants underwent magnetic resonance (MR) of the brain and a cerebral MR venography (MRV) before lumbar puncture (LP). They underwent LP so that CSF opening pressure could be measured and CSF pressure could be monitored for 1 h via a spinal puncture needle.The validated 0–10 visual analog scale (VAS) and the Migraine Disability Assessment (MIDAS) questionnaire were administered both at baseline and during the follow-up for assessing pain and disability. Higher scores on VAS and MIDAS served to indicate the severity of headache. Mood and anxiety were assessed with Beck Depression Inventory-II (BDI-II), and Hamilton Anxiety Rating Scale (HARS) at baseline and during the follow-up. Besides, we asked patients to fill in a headache diary throughout the study. A respondent to medical treatment (good outcome) was considered a subject who achieved a 50% or greater reduction in number of headache days per month.ICH was diagnosed if the patient met the following diagnostic criteria: (i) at least two or more of the following symptoms of raised CSF pressure: postural or nocturnal headaches, headache aggravated by coughing or worse on awakening, transient visual obscurations, photopsias, vertigo, pulsatile tinnitus, intracranial noises; (ii) at least two or more of the following neuroimaging signs: bilateral transverse sinus stenosis, empty sella, posterior sclera flattening, perioptic subaracnoid space distension; vertical tortuosity of the orbital optic nerve, tonsillar ectopia; (iii) altered pressure parameters for stage 1 or stage 2 of CSF hypertension; (iv) normal CSF contents; (v) symptoms relieved by a reduction in CSF pressure.3-4 or L4-5 interspaces. The pressure transducer  was attached to the hub of needle via a 10-cm long flexible tube without loss of CSF. It was placed at the level of the same horizontal plane as the estimated level of the spinal puncture needle in the subarachnoid space. It was then zeroed. A pressure cable linked the transducer to the monitor . At the start, opening pressure was recorded for 4 min to be certain that the pressure values had stabilized. Immediately after, CSF pressure was monitored for a 60-min period in order to record the mean pressure, highest peak pressure, mean pulse amplitude, and abnormal fluctuations in pressure. Physician observation and blood pressure evaluation were continuous in order to analyze CSF pressure only during artifact-free epochs, and to exclude arterial hypertension. At the end of the procedure the closing pressure was measured. A CSF removal (about 15 ml) was performed only in patients with altered CSF pressure parameters.The procedure outlined below has also been described in a previous report . The sam2O to 680 mm H2O . Brain MR consisted of sagittal T1- and transverse T2-weighted spin echo sequences with a slice thickness of 5 mm and an interslice gap of 1 mm. MRV was performed using 3D-PC techniques as described elsewhere . All brat-test. All p-values were corrected according to Bonferroni. Logistic regression models were used to assess linear relationships linking the odds of symptoms to pulse amplitude and to the presence of pressure pulsations. Models were chosen according to minimum Akaike Information Criterion. Statistical analysis was performed with R Statistical software .Differences in sex distribution and in the distribution of binary variables have been assessed by means of the Fisher's exact test, followed by pairwise proportion test. The Shapiro-Wilk test was used to check for normality before performing comparisons. The Kruskal-Wallis test, followed by the pairwise Wilcoxon rank sum test, was used to assess differences in age at examination, opening pressure, pulse amplitude, BDI-II and HARS and MIDAS questionnaire between groups. Body-Mass Index, mean pressure, and highest peak pressure were compared using the ANOVA test followed by the pairwise Participant characteristics are set out in Table 2O, highest peak pressure above 270 mm H2O, and mean pulse amplitude >65 mm H2O. The remaining 55 patients and 16 controls had both normal pulsations in CSF pressure, with both opening pressures and mean CSF pressures below 200 mm H2O , normal mean CSF pressure  and with no evidence of abnormal pressure pulsations , elevated mean pressure  associated with abnormal pressure pulsations , elevated mean CSF pressure  associated with severe abnormal pressure pulsations , patients were divided into three groups Table . Group 12O had the most severe headaches (highest scores on VAS and MIDAS) and associated symptoms . In Group 2, patients with less severe abnormal pressure pulsations and mean pressure >200 mm H2O, had less severe symptoms; in Group 1, patients who had no abnormal pressure pulsations and mean pressures below 200 mm H2O had primary chronic headache disorder  and a reduction in number of headache days (mean reduction 67%). It should be noted that none of the patients with ICH developed postural headache after LP or reported a worsening of headache during follow-up. We recommended an informal weight reduction diet program in all obese patients and prescribed acetazolamide  and topiramate (50–100 mg/day) simultaneously for the majority of patients with ICH. The rationale for using topiramate in this condition is its broad mechanistic profile (including an inhibitor effect on carbonic anhydrase isoenzymes II and IV). Hence, the simultaneous administration of topiramate and acetazolamide may have a synergic effect on headache and high CSF pressure in these patients . Patient ± 2 and To improve patient compliance during the lumbar CSF pressure monitoring, local anesthesia and a mild sedation were given before LP. This avoided confounding factors such as patient pain and anxiety and pressure artifacts during continuous pressure recording. Nine (6%) hence complained of discomfort during the procedure. While there were no major adverse events associated with lumbar CSF pressure monitoring, 15 (27%) headache sufferers without altered CSF pressure parameters experienced temporary (2–3 h) discomfort and mild headache soon after the procedure.Our results provide evidence that nocturnal and postural headache, and abnormal pressure pulsations are the most common pressure-related features of isolated high CSF pressure in patients with chronic headache. Moreover, abnormal pressure pulsations are the main pressure findings of ICH, and they may be considered a marker of ICH in patients with chronic headache who have no evidence of papilledema or sixth nerve palsy. In contrast, the lack of the abnormal pressure pulsations is associated with normal CSF pressure in both patients with primary chronic headache and non-headache controls. These findings suggest that abnormal CSF pressure pulsations may differentiate headache sufferers with high CSF pressure from those with primary headache disorder.There was indeed an important difference in patients' clinical manifestations. Symptoms such as nocturnal attacks of pulsating pain and postural headache, vertigo, headache aggravated by coughing or pulsatile tinnitus were more common in patients with ICH than in headache patients without altered CSF pressure parameters. These differences may be explained by the occurrence of particularly abnormal pressure pulsations in patients with more severe symptoms. Indeed, nocturnal attacks of pulsating pain and postural headache were more common in patients with ICH. The abnormal pulsations in CSF pressure occurring during sleep may be the cause of nocturnal headache, while rapid pulsations in CSF pressure precipitated by posture changes may trigger the positional headache –21. This2O , even though in 5 of these patients the baseline CSF pressure was below 200 mm H2O . Moreove2O  may be clarified by reference to the theory that CSF pressure is derived from the circulation of cerebral blood and cerebrospinal fluid . In fact2O. Although a previous study reported an upper normal limit of 200 mm H2O for CSF opening pressure, irrespective of body mass index  were found to present normal appearances of TS or unilateral TS stenosis. However, the presence of altered CSF pressure parameters during pressure monitoring was associated with symptoms of raised pressure, which were relieved by reducing CSF hypertension in these patients. This fact indicates a symptomatic elevation of CSF pressure, causing a chronic headache. Moreover, in the patients and controls with no abnormal pressure pulsations and with opening and average CSF pressures below 200 mm H2O, MRV revealed normal appearance of TS or unilateral TS stenosis. These data provide evidence for the first time that abnormal pressure pulsations and high CSF pressure may also occur without sinovenous stenosis.It is worth noting that on MRV some headache sufferers with stage 2 CSF hypertension (an opening and average pressure above 250 mm HOur results show that 1-h lumbar CSF pressure monitoring via a spinal puncture needle is a safe method of CSF pressure measurement, and also indicate that 60 min of pressure recording is sufficient to detect abnormal pressure pulsations when assessing headache sufferers suspected of having high CSF pressure. Continuous pressure recordings in these patients with ICH also demonstrated that CSF pressure may vary considerable over time. This suggests that CSF pressure assessment by measuring the height of the fluid column in the manometer line connected to a spinal puncture needle may be misleading in the case of headache sufferers. In everyday practice, lumbar CSF pressure monitoring via a spinal puncture needle for 60 min may identify either “false-positive”  CSF pre (CSF preThere are limitations to this study, in part linked to the fact that we investigated the CSF pressure-related features in patients with chronic headache by using a new method of lumbar CSF pressure measurement. The CSF pressure parameters considered for diagnosing isolated CSF hypertension in our patients with chronic headache are not included in the diagnostic criteria of idiopathic intracranial hypertension . We did Finally, our data demonstrate that the presence and the severity of abnormal pressure pulsations are related to clinical manifestations of ICH in patients with chronic headache. Our data highlight the effectiveness of lumbar CSF pressure monitoring via a spinal puncture needle over a 1-h period for detecting high CSF pressure in patients with chronic headache with no papilledema or sixth nerve palsy. They also suggest that CSF pressure is a continuous, not discrete, variable.FB: study concept and design, acquisition, analysis, and interpretation of the data, drafting, revising the manuscript, including its theoretical contributions, final approval of the version to be published. MC, LR, CB, DM: acquisition of data and analysis of data, final approval of the version to be published. BV: statistical analysis, final approval of the version to be published. UA and AQ: revising the manuscript, final approval of the version to be published.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.