Diffusion-weighted imaging (DWI) has revolutionised stroke imaging since its introduction in the mid-1980s, and it has also become a pillar of current neuroimaging. Diffusion abnormalities represent alterations in the random movement of water molecules in tissues, revealing their microarchitecture, and occur in many neurological conditions. DWI provides useful information, increasing the sensitivity of MRI as a diagnostic tool, narrowing the differential diagnosis, providing prognostic information, aiding in treatment planning and evaluating response to treatment. Recently, there have been several technical improvements in DWI, leading to reduced acquisition time and artefacts and enabling the development of diffusion tensor imaging (DTI) as a tool for assessing white matter. We aim to review the main clinical uses of DWI, focusing on the physiological mechanisms that lead to diffusion abnormalities. Common pitfalls will also be addressed.DWI includes EPI, TSE, RESOLVE or EPI combined with reduced volume excitation.• DWI is the most sensitive sequence in stroke diagnosis and provides information about prognosis.• DWI helps in the detection of intramural haematomas .• In diffusion imaging, ADC is inversely proportional to tumour cellularity.• DWI and DTI derived parameters can be used as biomarkers in different pathologies.•  Diffusion-weighted imaging (DWI) has revolutionised stroke imaging since its introduction in the mid-1980s, but it has also become a pillar of current neuroimaging. Diffusion abnormalities represent alterations in the random movement of water molecules in tissues, revealing their microarchitecture, and occur in many neurological conditions. DWI provides useful information, increasing the sensitivity of MRI as a diagnostic tool, narrowing the differential diagnosis, providing prognostic information, aiding the treatment planning and evaluating response to treatment.There have been several technical improvements in DWI, leading to reduced acquisition time and artefacts and enabling the development of diffusion tensor imaging (DTI) as a tool for assessing white matter. DTI allows tractography for surgical planning in brain tumours and other advanced techniques such as intravoxel incoherent motion (IVIM)  and kurtWe aim to review the main clinical uses of DWI, focusing on the physiological mechanisms that lead to diffusion abnormalities. Common pitfalls will also be addressed.Echo-planar imaging (EPI) is the method of choice for clinical diffusion-weighted imaging with MRI because of its low sensitivity to the motion-induced phase errors that occur during diffusion sensitisation of the MR signal. EPI is able to capture an entire image in a very short time. However, this method is prone to artefacts because of susceptibility changes at tissue interfaces and has limited spatial resolution. Major technical advances in diffusion MRI have enabled superior data fidelity, reduced image acquisition time and an improved signal-to-noise ratio (SNR) .Turbo-spin echo (TSE) is an adequate sequence to minimise susceptibility artefacts and distortion, and it increases the signal-to-noise ratio. Unfortunately, it also increases acquisition times and is therefore more sensitive to motion throughout the procedure.Readout segmentation EPI techniques (e.g. RESOLVE) allow DWI and DTI to be simultaneously generated, thus reducing the level of susceptibility-based distortion compared with the single-shot EPI technique . These aEPI combined with reduced volume excitation  is a focused excitation method that utilises two orthogonal pulses for slice selection in such a way that only the spins belonging to the intersection of the slab give rise to signal. This obviates the need for oversampling or distortion reduction  and alloLiney et al. compared EPI, RESOLVE and EPI combined with reduced volume excitation, concluding that EPI provided significantly higher ADC values than the other two sequences and that RESOLVE had the best repeatability ratio .Parameters derived from DWI and DTI studies allow a quantitative assessment, which in turn leads to improved accuracy and reproducibility. However, it should be noted that some of these parameters are still subject to clinical validation .ADC is the most widely used parameter derived from the conventional DWI sequence, representing the level of restriction to the motion of water molecules in the extracellular compartment. The term “apparent” relates to the fact that this motion is also influenced by other physiological processes, such as heartbeat, breathing or CSF pulsation. The main advantage of ADC relies on its widespread availability .DTI is based on the application of diffusion gradients in at least six different directions in space, enabling the evaluation of the movement of water molecules in 3D and whether there is a dominant direction to diffusion restriction. This allows tractography studies to be performed, which are multiplanar reconstructions of white matter tracts according to the dominant direction of water movement in each voxel.DTI provides several quantitative parameters, most of which are useful in the assessment of white matter conditions. Fractional anisotropy (FA) reflects how dominant one particular water movement direction in a voxel is. It varies from 0 to 1 and can be considered a biomarker of axonal integrity . Mean diffusivity (MD) represents a more exact value than ADC because it considers the three main directions of water movement. Axial diffusivity (AD) quantifies water movement along the main longitudinal direction, evaluating axonal integrity, while radial diffusivity (RD) reflects myelin integrity (myelin injury leads to increased RD).As we will discuss, all these quantitative parameters improve the diagnostic possibilities of MR in the evaluation of tumours, demyelinating and infectious diseases, and vascular pathologies.+/K+ ATPase and other ionic pumps. Water movement inside the cell becomes more limited resulting in cytotoxic oedema. In addition, the intracellular environment becomes more viscous, further restricting diffusion and decreasing the apparent diffusion coefficient (ADC).Brain ischaemia leads to variations in the intra- and extracellular space due to a failure of the NaSubsequently, inflammatory mediation factors are released, affecting the integrity of the blood-brain barrier and leading to water extravasation and vasogenic oedema with expansion of the interstitial space and an increase of the total water content of the tissue. Subsequently, ADC values gradually increase to their baseline. In chronic ischaemia gliosis eventually develops, which does not result in restricted diffusion.p = 0.0019) [DWI improves detection of ischaemia because it reveals alterations in water diffusion before the net increase of water content in the subacute phase of the infarct. DWI hyperintensity , 9 and c 0.0019) .Fig. 1AcDWI is far more sensitive than conventional imaging in the detection of early ischaemia. T2WI—including fluid-attenuated inversion recovery (FLAIR) imaging—can only detect ischaemia typically 1–4 h later, since signal abnormalities on these sequences relate to the subsequent increase in total water content in the infarcted area .In the setting of acute ischaemia, the mismatch between DWI and FLAIR has been suggested to enable identification of patients with acute ischaemic stroke who are likely to be within 4.5 h of symptom onset with high specificity and high positive predictive value , which iADC alone can be considered a biomarker of brain ischaemia as it allows differentiation between the two main causes of increased DWI signal in acute stroke : “true” The area showing diffusion restriction is usually taken as the “infarct core” , but some diffusion abnormalities may be reversible, mainly if there is early (< 4.5 h) spontaneous or therapeutic recanalisation of the occluded vessels . HoweverThrombolytic or endovascular therapies are the treatment options for acute stroke. Selection of patients eligible for these therapies is critical to achieve good outcomes and avoid fatal complications. Both time from stroke symptom onset to treatment and the individual therapeutic time window secondary to variations in collateral circulation determine the long-term functional outcome after stroke. Therefore, imaging—including DWI—has great prognostic value and is crucial to treatment decision making because it allows early diagnosis and an estimation of both the time of onset and the infarct-salvageable brain proportion.2 and a slice thickness of 3 mm is recommended [Despite some technical challenges , DWI is useful in assessing cord ischaemia. Although signal changes were reported on both T2 and DWI in the majority of cases, DWI showed a clear benefit over conventional MRI in some patients, in whom T2 failed to identify an abnormality , 20. Accommended  The new definition of TIA is based on a biological concept (tissue injury). A TIA is characterised by a transient episode of neurological impairment caused by focal ischaemia without acute infarction .Previously, TIA was defined based on arbitrary timing (duration ≤ 24 h). However, permanent tissue damage (infarction) is possible even when focal transient neurological symptoms last < 1 h [The current definition takes imaging into account as DWI must be normal for the diagnosis of TIA to be upheld. If MRI shows areas of restricted diffusion in a patient whose symptoms have resolved and lasted less than 24 h, the term cerebral infarction with transient symptoms is preferred . Once a The “crescent sign”, representing an intramural haematoma, can be an imaging criterion for diagnosing arterial dissection. It consists of an eccentric rim of hyperintensity surrounding the hypointense arterial lumen on MRI. It has traditionally been described on T1-weighted fat-saturation MRI sequences , but mayThe signal intensity of intramural haematoma in arterial dissection is influenced by the paramagnetic effects of blood breakdown products . The haeDWI using EPI usually shows susceptibility artefacts at the air-tissue or bone-soft tissue interfaces. Artefacts present as a fuzzy linear hyperintensity or diffuse hypointensity, which are usually easy to differentiate from the focal round or linear well-demarcated hyperintensity typical of intramural haematoma . Correla+/K+ ATPase pump failure, cytotoxic oedema and restricted diffusion. Therefore, diffusion imaging helps to define the stage of the disease because it allows differentiation between irreversible venous infarction (DWI hyperintense with restricted diffusion) and reversible venous oedema (DWI isointense with increased diffusion). In contradistinction to arterial stroke, DWI-positive venous stroke may be completely reversible.Ischaemia in the setting of venous thrombosis has a different physiology from arterial infarction . InitialIn venous ischaemia, DWI may be able to show the actual thrombus as increased signal intensity  because In previous studies, the approximate frequency of the increased DWI signal within the venous thrombus has been reported as 41% , thus noTaking into account that venous infarctions have a higher tendency to bleed, and haemorrhage can also restrict diffusion, imaging features in venous infarction may be considerably variable, depending on the time after onset and the sequence of events.Other vascular pathologies in which diffusion may be helpful are transient global amnesia, with a typical transient focal dot-like area of diffusion restriction in the hippocampus and/or parahippocampal gyrus , or postRegarding extraaxial tumours, DWI allows distinction between epidermoid and arachnoid cysts. Epidermoid cysts show diffusion restriction, as opposed to arachnoid cysts, which follow CSF signal intensity on all sequences. Other MR sequences can also aid in the diagnosis, since epidermoid cysts are bright on FLAIR, but not arachnoid cysts . In meniConcerning glial tumours, quantitative assessment with DWI has mainly targeted an estimation of cellularity based on its inverse relation with water diffusivity in the extracellular compartment. Hence, ADC can be considered as a tumour biomarker in gliomas: the higher the tumour grade, the lower the mean tumour ADC values . HoweverAnother use of DWI on imaging of CNS tumours is to aid in the distinction between glioblastoma and primary CNS lymphoma, two entities with markedly different treatment strategies and not always easy to differentiate with conventional imaging in patients presenting with an enhancing brain mass. Due to its higher cellularity compared with GBM, primary CNS lymphoma has been shown to exhibit lower ADC values . There aAttempts to differentiate glioblastoma from metastasis based on the FA values of the surrounding oedema have been made  based onDTI and tractography reconstruction can be helpful for surgical planning in patients with brain or spinal cord masses. It shows white matter tracts as well as displacement or interruption of these tracts secondary to different pathologies. Slow-growing tumours displace the fibres surrounding the lesion, whereas in ischaemia or trauma, representing more acute insults, the fibres are usually interrupted  Fig. 6)6).Fig. 6An additional important use of DWI after surgical resection of a tumour is to detect areas of diffusion restriction in the cavity margins on MRI performed in the first 48 h, corresponding to small postsurgical infarctions. The importance of this imaging time window lies in the fact that the majority of these infarcts will enhance after 1 or 2 weeks and could be potentially interpreted as residual tumour or tumour progression Fig. .Fig. 7Po3 mm2/s for juvenile pilocytic astrocytoma and < 0.9 × 103 mm2/s for medulloblastoma have been suggested with a high specificity [Regarding paediatric tumours, DWI can non-invasively add valuable information that can be used to narrow the differential diagnosis of the three most common tumours of the posterior fossa in children . Several studies have demonstrated that ADC values in the enhancing, non-necrotic, non-oedematous, solid parts of cerebellar tumours are negatively correlated with tumour grade   can be similar to primary cystic or necrotic tumours Fig.  and subaReddi et al. showed that diffusion-weighted imaging had a sensitivity and specificity of 96% for the differentiation of brain abscesses from primary or metastatic cancers   Fig. 1010. A stuFungal abscesses can have either restricted or elevated diffusion in the centre (depending on the content), in opposition to pyogenic and tuberculous abscesses that typically show restricted diffusion in the core of the cavity . RestricAspergillus CNS infections tend to be haemorrhagic, which is an additional cause of restricted diffusion. In general, it is important to include fungal abscesses in the differential diagnosis in immunocompromised patients because of different targeted antifungal treatment.Parasitic abscesses may also show both restricted and elevated diffusion . For exaIn the postsurgical setting, the accuracy of DWI in the diagnosis of CNS infectious complications is low, showing a high false-negative rate. There are often postoperative changes with diffusion restriction due to haemorrhage/ischaemia, which can hinder the distinction between purulent content and haemorrhagic/ischaemic components. The absence of restricted diffusion is not sufficient to exclude the presence of pyogenic postcraniotomy infection and should not be used as the main determinant in patient management in this clinical setting .Diffusion restriction is commonly seen in viral encephalitis, and the location of the affected area may guide the radiologist in the identification of the infectious agent. For example, the limbic system  is the typical involved area in herpes simplex encephalitis. Viruses more frequently causing potentially reversible lesions in the splenium of the corpus callosum are the influenza virus , EpsteinProgressive multifocal leukoencephalopathy (PML) is secondary to the infection of oligodendrocytes by the JC polyoma virus, which typically remains latent until reactivation in the context of an immunocompromised state . Due to its ability to investigate white matter architecture and diseases, DWI shows regions of active infection and cell swelling with high signal and allows distinction of two parts in the lesions: a central core  and a peripheral rim  .Key poinCreutzfeldt-Jakob disease (CJD) is the combination of progressive dementia and pyramidal, extrapyramidal, and/or cerebellar signs caused by a prion (proteinaceous particle without DNA or RNA but capable of causing infection). DWI MR has high diagnostic accuracy and is the most sensitive neuroimaging technique for striatal and cortical lesions from an early stage . TypicalThe pulvinar sign , “hockey stick” sign  or similar changes in the periaqueductal grey matter were reported to be specific for variant (vCJD) , but canIn multiple sclerosis, the majority of active plaques show normal or increased diffusivity, but some may show restricted diffusion, often at the margins . It is iImmune-mediated encephalitis includes two different types of conditions: paraneoplastic encephalitis syndromes, comprising limbic encephalitis, paraneoplastic cerebellar degeneration and brainstem encephalitis, and the encephalitis syndromes associated with antibodies against neuronal cell surface/synaptic proteins, which are commonly referred to as autoimmune encephalitis.Diagnosis is based on electroencephalography (EEG), lumbar puncture and serological testing for appropriate biomarkers . Neuroimaging should be performed despite yielding a high proportion of false negatives  . When poAlthough limbic encephalitis has been known as a paraneoplastic syndrome, there have been recognised forms characterised by serum autoantibodies, directed against glutamic acid decarboxylase (anti-GAD) and voltage-gated potassium channels (VGKC), not related to neoplastic conditions.On DTI studies, a reduction of fractional anisotropy and increased mean diffusivity have been shown in the white matter of patients suffering from NMDAR encephalitis , suggesting that white matter damage is a contributor to the pathophysiology and clinical phenotype, even correlating with disease severity .Another study aiming to assess white matter integrity in the two forms of non-paraneoplastic limbic encephalitis characterised by serum autoantibodies (GAD and VGKC) concluded that GAD-limbic encephalitis presented with FA reduction, probably reflecting demyelination, whereas VGKC-limbic encephalitis caused no changes in the diffusion properties of white matter, probably because this condition is more limited to grey matter .Abnormal signal intensity of the optic nerve due to diffusion restriction may be seen in ischaemic or traumatic optic neuropathy , 75. DWIWith the exception of the orbital apex region, DTI of the optic nerve is facilitated by its anatomy and antero-posterior orientation. This can be useful in assessing tumours such as neurofibromas, schwannomas or meningiomas  as well Other cranial nerves can also be studied with high-resolution DTI in some clinical situations. Demonstration of atrophy or loss of anisotropy in cases of trigeminal neuralgia and fibre displacement in vestibulocochlear schwannomas are examples of this .The DWI and DTI techniques allow quantitative analysis of microstructural changes in neurodegenerative diseases, even when no abnormalities are seen on conventional MRI sequences, because they are able to identify changes in brain tissue integrity. These changes are characterised by a decrease in the number of barriers that restrict the movement of water, thus causing ADC to increase in brain areas where neurodegeneration occurs. This has led to an increase in the use of DWI in the diagnostic investigation of neurodegenerative parkinsonian syndromes. Attempts have been made to use DTI of the substantia nigra as a diagnostic marker for Parkinson disease, but while some studies found highly significant FA reduction in the substantia nigra , others ADC values of the putamen have been used to discriminate between Parkinson’s disease and atypical parkinsonism, including the classical phenotype of progressive supranuclear palsy and corticobasal syndrome. Putaminal ADC values were significantly higher in both atypical parkinsonism syndromes compared with Parkinson’s disease .In other neurodegenerative diseases, such as Friedreich’s ataxia, DWI may be an appropriate non-invasive tool to systematically quantify the extent and distribution of brain changes. DWI has also highlighted the involvement of new areas in the pathophysiology of this condition, such as the optic radiations and middle cerebellar peduncles .Diffusion-weighted sequences currently play a central role in neuroimaging. The already widespread qualitative assessment allowed by these sequences improves sensitivity in the depiction of several central nervous system conditions, whereas new models of diffusion sequences provide quantitative parameters, allowing potential biomarkers for diagnosis, prognosis and follow-up. The future of DWI will undoubtedly include technical improvements to enhance data fidelity, to achieve high isotropic resolution  for 3D acquisitions and to reduce the image acquisition time. Furthermore, advances in ultrahigh field technology are already being applied to DWI.


Notable sex-differences exist between neural structures that regulate sexually dimorphic behaviors such as reproduction and parenting. While anatomical differences have been well-characterized, advancements in neuroimaging and pharmacology techniques have allowed researchers to identify differences between males and females down to the level of the synapse. Disparate mechanisms at the synaptic level contribute to sex-specific neuroplasticity that is reflected in sex-dependent behaviors. Many of these synaptic differences are driven by the endocrine system and its impact on molecular signaling and physiology. While sex-dependent modifications exist at baseline, further differences emerge in response to stimuli such as stressors. While some of these mechanisms are unifying between sexes, they often have directly opposing consequences in males and females. This variability is tied to gonadal steroids and their interactions with intra- and extra-cellular signaling mechanisms. This review article focuses on the various mechanisms by which sex can alter synaptic plasticity, both directly and indirectly, through steroid hormones such as estrogen and testosterone. That sex can drive neuroplasticity throughout the brain, highlights the importance of understanding sex-dependent neural mechanisms of the changing brain to enhance interpretation of results regarding males and females. As mood and stress responsivity are characterized by significant sex-differences, understanding the molecular mechanisms that may be altering structure and function can improve our understanding of these behavioral and mental characteristics. Sex differences in behavior can be observed through both scientific inquiry and casual observations in social settings. Although gender roles have been proposed to account for some of the variability in behavior observed between males and females . Based on the influence of estradiol demonstrated in females, one may hypothesize that estradiol would be equally efficacious in males; however, direct administration of estradiol is not sufficient to restore spine density in gonadectomized males  α, Erβ and G-protein coupled estrogen receptor 1 (GPER1). These receptors are localized throughout the brain of males and females providing estradiol a site of action to modify neuronal structure  signaling, however, in females LTP appears not affected in αNOS1 knockouts  similarly enhances neural transmission, suggesting the mμ-opioid receptor is specifically down-regulating neural firing in females. Females also exhibit low frequency LTP during proestrus, suggesting that activity threshold is reduced when estradiol is elevated (Harte-Hargrove et al., It is evident that sex differences in synaptic plasticity are driven by both direct and indirect mechanisms of steroid hormones. While these mechanisms exist at the molecular and synaptic levels, they are further reflected in structural differences that ultimately result in sexually-dimorphic behaviors. Recent advances in human neuroimaging have provided insights into the translatability of these sex-differences in synaptic plasticity across mammalian species. Overall changes in white matter, gray matter and cerebral spinal fluid have been observed across the menstrual cycle in human women. Generally, gray and white matter volume appear to increase during the luteal phase when estrogen and progesterone are elevated in women (Catenaccio et al., MH outlined, researched and wrote this review article. In addition, she composed the figure. LP provided editorial comments and suggestions on the research areas to include. GN assisted in the drafting and editing of this manuscript and advised MH, a postdoctoral trainee, during the process.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.


Environmental Tobacco Smoke (ETS) is a major, preventable cause of morbidity and mortality, disproportionately impacting vulnerable populations. Policy measures, guided by the WHO’s Framework Convention of Tobacco Control, have focused on the broad adoption of smoke-free laws. While smoke-free policies are effective in reducing ETS exposure, limited policy dissemination and suboptimal implementation strategies have limited their impact.New research reported by Berman and colleagues in this journal brings these issues into sharper focus. Substantial advances in tobacco control policy have been achieved in Israel, including widening of smoke-free laws, since the passing of a Knesset bill in 2012. However, Berman and co-authors present found no reduction in ETS exposure in a nationally representative sample of non-smoking Israeli adults in 2016 compared with an earlier benchmark measured in 2011. In line with research from international settings, they found that ETS exposure was higher among a traditionally vulnerable subpopulation. The findings serve to remind us that the mere adoption of a policy will not translate into meaningful public health impact without applying best practice implementation strategies. Above all, this work emphasizes the continual need for new research to improve existing policies and inform new policy approaches in pursuit of an end to the harm arising from the global tobacco epidemic. The global tobacco epidemic continues to exact a massive toll on human health. While the harms caused directly by smoking are now widely recognized, the harms associated with exposure to secondhand smoke are often overlooked. Secondhand smoke, also referred to as environmental tobacco smoke (ETS), contains some 7000 chemicals, of which some 250 are known to be toxic and 69 carcinogenic , 2. ETS With the guidance of the WHO’s Framework Convention on Tobacco Control — the first global public health treaty — smoke-free laws have been adopted in many national and subnational jurisdictions. While smoke-free laws were initially conceived as a strategy to protect workers in occupational settings such as hospitality venues, they have steadily expanded to encompass a diverse range of public settings, including airports, health and educational facilities, and many business and recreational facilities . As a reN = 194 adults who participated in the 2015–16 National Health and Nutrition Survey (RAV MABAT), they found that 63% of non-smoking Israelis were exposed to ETS. This finding is particularly disappointing in light of evidence that there has been no reduction in the proportion of non-smokers exposed to ETS or the concentration of cotinine, an ETS biomarker, since 2011, when these investigators analyzed the same outcomes in an earlier Israeli cohort [New research reported by Berman and colleagues  underscoi cohort . Of partThese perplexing findings raise the question: have hard fought tobacco policy gains, delivered via Knesset vote, produced little benefit to the health of the public? Experience has taught us that political will is predicated on public support, which is more likely to arise when policies are perceived as beneficial. Could these findings prove to be a setback in Israel’s ongoing policy efforts to curb the tobacco epidemic?adoption is not sufficient without effective policy implementation, encompassing better public communication and engagement, strategies to assist compliance, and rigorous enforcement [While Berman and colleagues provide only limited insights on the possible reasons why ETS exposure has not declined, they nonetheless point to a forward path. The authors appeal for increased enforcement of Israel’s far-reaching laws to support greater policy impact. PoorlyP enforced smoke-free policies are less likely to yield the benefit of ETS exposure reduction, and enforcement of existing policies requires constant vigilance . Smokingorcement . EffectiIsrael stands at a critical point in tobacco control policy. Difficult battles have yielded a raft of valuable policy initiatives, including the smoke-free laws recommended under the FCTC. However, Berman and colleagues show us that there is much progress still to be made. As those working in tobacco control research and policy consider the tobacco “end game” — the steps necessary to finally rid the globe of the tobacco epidemic — lessons from Israel emphasize that we must improve existing policies, develop new policy strategies, and improve policy implementation, if we are to overcome this pervasive public health problem.


In fact, both processes are associated with functional decay and similar molecular modifications. On epigenetic level, replicative senescence and aging evoke characteristic modifications in the DNA methylation (DNAm) pattern, but at different sites in the genome. Various epigenetic signatures, which are often referred to as epigenetic clocks, provide useful biomarkers: Senescence-associated epigenetic modifications can be used for quality control of cell preparations or to elucidate effects of culture conditions on the state of cellular aging. Age-associated epigenetic modifications hold high expectations to determine chronological age in forensics or to identify parameters that impact on biological aging. Despite these differences, there are some striking similarities between senescence- and age-associated DNAm, such as complete rejuvenation during reprogramming into induced pluripotent stem cells (iPSCs). It is yet unclear what makes epigenetic clocks tick, but there is evidence that the underlying mechanisms of both processes are related to similar modifications in the histone code or higher order chromatin. Replicative senescence therefore appears to be a suitable model system to gain better insight into how organismal aging might be governed epigenetically.Replicative senescence of cells  Primary cells, which are taken directly from living tissues, can only be culture expanded for a limited number of cell divisions before entering irreversible proliferation arrest . Since tThe phenomenon of cellular senescence is quite diverse. Decreasing proliferation of cells in culture is not only evoked by long-term expansion, but it can also be triggered, e.g., by irradiation or oncogene induced senescence . While tThere is a growing perception that culture expansion toward replicative senescence and aging are both reflected by specific modifications in the DNAm pattern, which might indicate that they are evoked by epigenetic processes . DNAm plAlmost 30 years ago, it has been suggested that cell culture is associated with a continuous loss in the general DNAm level, and that this loss might be related to the number of cell divisions . These ain vitro expansion or the number of cumulative population doublings (cPDs)  and fibroblasts, which at the time were analyzed with 27k Illumina BeadChip arrays, to select six CpGs that revealed almost linear hyper- or hypo-methylation with subsequent passages . Thus, Ds (cPDs) . This res (cPDs) . In comps (cPDs)  or stains (cPDs) , the DNAs (cPDs) . This exs (cPDs) .Already in 1973 it was described that aging is reflected by a global decrease in the 5-methylcytosine content of various tissues, and this led to the assumption that DNAm may regulate gene activity and somehow trigger the process of aging  – a hypoThe first epigenetic clocks to estimate donor age were again derived from the 27k Illumina Bead Chips . These sin vitro expansion on the epigenetic makeup . While in pluripotent state, iPSCs can be passaged virtually infinitively without any signs of cellular aging. Furthermore, iPSCs derived from elderly organisms can give rise to young organisms that pursue normal aging, as for example demonstrated in mice . Also onUpon re-differentiation of iPSCs toward other cell types the senescence-associated DNAm patterns are continuously reacquired as observed during culture expansion of primary cells . In contTo investigate if overexpression of the catalytic subunit of the human telomerase (TERT) would be enough to stop aging, we have immortalized fibroblasts and MSCs with TERT. While this facilitated elongation of telomeres and long-term expansion without any signs of replicative senescence, it did not reset senescence-associated DNAm changes – in fact, these epigenetic modifications were even further acquired during culture expansion of immortalized lines . This wade novo DNA methyltransferases: DNMT3A and DNMT3B. We have recently demonstrated that different splice variants of DNMT3A have transcript specific effects on the DNAm pattern  to compare senescence-associated DNAm in different subpopulations of MSCs. Notably, in clonally derived subpopulations, the DNAm levels of neighboring CpGs differed extensively, indicating that these genomic regions are not synchronously modified during senescence . In a moIt is still controversially discussed if aging is governed by a purposeful program or if it rather resembles an accumulation of stochastic, accidental events . While sin vitro has several strategic advantages for aging research: Effects of relevant genes or drugs can be systematically screened in a high throughput manner and with large compound libraries. It appears to be plausible that such findings can then be extrapolated to understand how to modulate age-associated epigenetic modifications – and possibly the process of aging.Epigenetic clocks for aging and senescence are certainly ticking independently, but the striking similarities indicate that the processes are regulated by similar means. There is evidence that the epigenetic modifications are not governed by targeting of regulatory complexes but that the DNAm changes may rather be orchestrated indirectly by other types of chromatin organization. Studying of senescence-associated changes The author confirms being the sole contributor of this work and has approved it for publication.www.cygenia.com), which can provide service for Epigenetic Senescence Signatures and Epigenetic Aging Signatures to other scientists.WW is cofounder of Cygenia GmbH (


This report describes the results of recruitment efforts and the subsequent participation of pregnant women in study activities in a 2010–2012 observational study focused on influenza illness and vaccination in California and Oregon, USA.Socio-demographic and health characteristics extracted from electronic medical records were compared among pregnant women who enrolled in the study, refused to participate, or were never reached for study invitation. These characteristics plus additional self-reported information were compared between women who enrolled in two study tracks: a prospective cohort vs. women enrolled following an acute respiratory illness (ARI) medical encounter. The characteristics of women who participated in weekly ARI surveillance  and a 6-month follow-up interview  were also examined.In year one, we reached 51%  of the potential participants we tried to contact by telephone, and 20% (1374/6938) of the women we invited agreed to join the prospective cohort. Women with chronic medical conditions, pregnancy complications, and medical encounters for ARI (prior to pregnancy or during the study period) were more likely to be reached for recruitment and more likely to enroll in the cohort. Twenty percent of cohort enrollees never started weekly surveillance reports; among those who did, reports were completed for 55% of the surveillance weeks. Receipt of the influenza vaccine was higher among women who joined the cohort (76%) than those who refused (56%) or were never reached (54%). In contrast, vaccine uptake among medical enrollees in year one  and two  was similar to other pregnant women in those years. Study site, white race, non-Hispanic ethnicity, and not having a child aged < 13 years at home were most consistently associated with joining as a cohort or medical enrollee and completing study activities after joining.We observed systematic differences in socio-demographic and health characteristics across different levels of participant engagement and between cohort and medical enrollees. More methodological research and innovation in conducting prospective observational studies in this population are needed, especially when extended participant engagement and ongoing surveillance are required.The online version of this article (10.1186/s12884-019-2280-0) contains supplementary material, which is available to authorized users. Prospective, community-based studies of the epidemiology of infectious diseases and the effectiveness of treatment and prevention strategies including vaccination are rare, in large part due to their high costs and many logistical challenges , 2. Yet,This lack of information was especially evident to us in 2009 as we planned a two-year observational study of influenza vaccine effectiveness (IVE) in preventing laboratory-confirmed influenza illness during pregnancy . Althougmedical enrollees (year one) and medical enrollees (year two). Because of uncertainty regarding where and how often pregnant women sought medical care for ARI, we also recruited pregnant women prospectively into a cohort prior to the initiation of influenza season in year one; this also gave us an opportunity to examine a broader range of illness severity. Participants in this part of the study are referred to as cohort enrollees (year one).This report describes the results of our recruitment efforts and the subsequent participation of pregnant women in study activities, including a 6-month post-delivery follow-up interview. The main project consisted of three sub-studies. In both years, we recruited pregnant women following medical encounters for acute respiratory illness (ARI) during influenza season; the women in these sub-studies are referred to as Because our study took place within two integrated healthcare systems with longitudinal electronic medical record (EMR) data, we had the unique opportunity to identify the demographic and health characteristics of women who were eligible for our study  but could not be reached for recruitment, as well as women who were contacted but refused participation. Specifically, we examined possible indications of selection bias, such as systematic differences in age, study site, race, ethnicity, underlying health status, and medical visits. Given the focus of the original study on IVE, we were particularly interested in whether receipt of influenza vaccination differed between participants and non-participants and between the cohort vs. medical enrollee sub-studies. We also examined the associations between these factors and participation in weekly surveillance (for cohort enrollees in year one) and completion of a follow-up interview .We have previously published detailed methods of the study , 6. In bThe year one cohort recruitment methods differed slightly by site. Both sites distributed flyers about the study to women following their prenatal visits via postal and electronic mail (at site one) or directly at prenatal and laboratory appointments (at site two). Information on the flyer gave women the opportunity to accept or decline participation immediately by mail or telephone. For all other women who did not immediately accept or decline participation, both sites then called potential participants on the telephone; this was done by local staff at site one and by an external call center at site two. Verbal orientation to the study and completion of verbal consent were completed during the telephone contact, and signed written consent forms were returned by postal mail or during the home visit when enrollment interviews were completed by study staff.ARI-negative controls. Medical enrollees and ARI-negative controls provided verbal consent when recruited by telephone and then written consent by postal mail or when they met with study staff.During both years one and two, we recruited pregnant women during the influenza season following medical encounters for ARI (defined below); these medical enrollees were recruited by telephone at both sites. In year one, medical enrollees could only be recruited from women not already participating in the cohort. Completion of the enrollment interview and collection of respiratory specimens were done during a home visit by study staff. In year two only, we also recruited two pregnant women controls for each laboratory-confirmed influenza positive pregnant woman enrolled. The control participants were recruited by telephone within 2 weeks of each influenza positive case, were in the same trimester of pregnancy, and had no record of a medical encounter for ARI and no self-report of an illness with fever and cough since the start of the local influenza season. These women are referred to as Ten variables were available from the EMR for all potential participants. Most indicators describe the women’s characteristics or medical history at the time of (or prior to) recruitment: (a) socio-demographic characteristics ; (b) presence of a high-risk chronic medical condition  during the year prior to conception; (c) medical encounters for an ARI during the year prior to conception . Other indicators describe women’s medical history during the entire pregnancy or study period and thus enable us to examine how women who differed in participation may have differed in their health trajectories: (d) ARI medical encounters during the 6-month study period; (e) influenza vaccination ; (f) any pregnancy complications during the index pregnancy  who completed interviews at enrollment: (a) expanded race categories; (b) marital status; (c) education; (d) subjective social status (SSS) rated on a 9-point ladder from low (1) to high (9) and examined in tertiles see –13); (e); (e)13])refused to participate in the study when invited. Finally, we compared the characteristics of women we called to invite to join the study but who were never reached with women who we reached (including those who consented or refused). Because ethnicity data was incomplete for non-participants, the available data is presented for refusers and never reached women, but statistical comparisons are not made.The comparisons we were able to make differed for the two years of our study. For year one  when all variables were considered simultaneously.For comparisons involving women who were never reached or refused participation, local IRBs approved analyses of frequency tables rather than individual-level data; thus, we used Chi-square tests to compare counts of their socio-demographic and health characteristics Tables  and 2. FOf 13,655 potentially eligible pregnant women that we attempted to recruit across sites for the prospective cohort during year one, we reached half by telephone (51%); a substantially higher proportion of women were reached at site one  where local staff made invitation calls, than at site two  where an outside contractor made the calls. Similarly, successful completion of informed consent and enrollment in the cohort among invited women was also higher at site one  than site two ; the combined enrollment rate was 20% (1374/6938)  compared to the cohort enrollees in Year 1  was similar to the percentage of vaccine uptake among participant refusers (54%) and those never reached (53%), but were considerably lower than among cohort enrollees (76%). After adjusting for all the bivariate differences between the cohort and medical enrollees Table , the oddWe did not record the number we attempted to contact in year two. Nonetheless, of the women reached, site two enrolled a higher proportion of women  than site one 70/299, 23%); the cross-site enrollment rate was 33%  of the pregnant women who joined the cohort completed at least one weekly surveillance report; on average, these surveillance participants completed reports during 55% (95% CI = 53–56%) of the surveillance weeks (interquartile range = 38–71%). As shown in Table 74 of theNearly half (49%) of the surveillance participants used two or more methods to complete surveillance reports, and this was associated with a higher percentage of weeks with completed reports compared to using a single method  , we completed 6-month post-delivery follow-up interviews with 1595 (92%) participants Table . CompletStudy site and race were significantly associated with all cohort participation indicators ; non-Hispanic ethnicity and not having a child aged < 13 years were associated with all indicators where these factors could be examined. Women with additional health issues  were more likely to be reached for recruitment and more likely to enroll in the cohort. However, among women who were enrolled in the cohort, these health issues were not consistently associated with surveillance participation or study retention. Similarly, pregnant women who received influenza vaccination were more likely to be reached and more likely to enroll in the cohort; yet, among cohort enrollees, vaccination status was not consistently associated with surveillance participation or retention.We had greater difficulty enrolling cohort participants and conducting active surveillance than we expected , 16. We Race was a consistent predictor in all of our analyses. Non-white women were less likely to be reached and enroll in our cohort; once in the cohort, non-white women were less likely to start active surveillance, completed fewer weekly reports, and were less likely to complete the 6-month follow-up interview. This finding is consistent with previous observations of lower rates of completed contacts and research consent by non-white adults –19. HoweAlthough the differences we noted were statistically significant, many were of relatively small magnitude. Certainly, statistical differences in participation among subgroups do not necessarily imply that the study findings are biased. In our IVE study, we were most concerned about potential confounders: characteristics associated with both the exposure of interest (vaccination) and the outcome (laboratory-confirmed influenza illness). We previously reported that only gestational age at enrollment was associated with both the likelihood of vaccination and influenza outcomes in our final study sample . HoweverEnrolling women following medical encounters for ARI proved to be an efficient way to increase the total number of influenza positive cases in our study. The IVE literature typically assumes that ARI patients differ from their full local population in their propensity for medical utilization and vaccination , 25. IndEnrolling women following medical encounters also proved to be an effective way in year one of our study to reach and enroll women who were non-white, Hispanic, in poorer health, and had children at home. In year two, non-white women were also more represented among the medical enrollees. This may have been due in part to the fact that we were able to reach a more diverse population of women when many were at home sick. Although we asked the cohort and medical enrollees to perform similar activities , the commitment asked of medical enrollees was more immediate and clear, since they often completed the bulk of study tasks on the same day. In retrospect, we recognize that the scope of tasks involved in our cohort study, the uncertainty of what would be required (depending on if they became ill or not), and the extended time commitment, may have biased participation toward women who were more educated, had higher subjective social status, had fewer stressful events in the past year, already had health concerns, and were more likely to have future pregnancy and ARI issues.Among the strengths of our study is the detailed report of participation rates which start with the denominator of all presumed eligible potential participants, as recommended by the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines , but rarOur study also has at least five limitations. First, we do not have information on the specific reasons why some women refused participation or did not complete surveillance tasks. Second, the extent to which our findings will generalize to other study settings and designs may be limited by the features of our study. For example, our study benefited from being able to offer small financial incentives for participation, from conducting home visits rather than requiring special trips to research facilities, and from having regularly updated contact and medical care information within the integrated healthcare systems. Third, we did not have access to complete information on Hispanic ethnicity or racial categories beyond white vs. non-white for all potential participants, which limited our ability to describe and understand how these factors influenced participation. Fourth, we noted differences in participation between study sites, which may have been due to different recruitment and staffing methods, population differences in race and ethnicity, or other reasons. In year one, study site one used local staff  for recruitment and had higher enrollment and surveillance participation rates than site two which relied on an external contractor. Interestingly, in year two, when both sites used local staff for recruitment, the percentage of contacted women who enrolled was higher at site two. Finally, our surveillance methods did not include the use of short message service (SMS) texts, which have recently been demonstrated to be an effective way to conduct health monitoring during pregnancy e.g., , 28)..28]).In conclusion, we observed systematic differences in socio-demographic and health characteristics across different levels of participant engagement and in comparing cohort vs. medical enrollees. The indications of selection bias we observed are a reminder that having a representative sample and a high participation rate remains critically important for ensuring valid conclusions in observational studies. Faced with a growing need to conduct observational studies of pregnant women due to new disease threats , 30 and Additional file 1:Figure S1. Recruitment in Year 1 as Cohort or Medical Enrollees. Figure S2. Recruitment in Year 2 as Medical Enrollees or Acute Respiratory Illness (ARI) Negative Controls. (PPTX 40 kb)Additional file 2:Table S1. Multivariable Analyses Including Covariates with Significant Bivariate Associations. Presents full results of logistic regressions models described in the manuscript. Table S2. The surveillance methods used and the percentage of weekly reports completed by method or combination of methods. Presented the percentage who completed surveillance reports. Table S3. Characteristics of Cohort Participants who Completed Combinations of Computer Automated Telephone Interview (CATI) and Website Methods of Active Surveillance for Acute Respiratory Illness. Presents descriptive characteristics by types of surveillance methods used. (PDF 73 kb)


Vaccination against influenza is the most effective approach for reducing influenza morbidity and mortality. However, influenza vaccines are unique among all licensed vaccines as they are updated and administered annually to antigenically match the vaccine strains and currently circulating influenza strains. Vaccine efficacy of each selected influenza virus vaccine varies depending on the antigenic match between circulating strains and vaccine strains, as well as the age and health status of the vaccine recipient. Low vaccine effectiveness of seasonal influenza vaccines in recent years provides an impetus to improve current seasonal influenza vaccines, and for development of next-generation influenza vaccines that can provide broader, long-lasting protection against both matching and antigenically diverse influenza strains. This review discusses a perspective on some of the issues and formidable challenges facing the development and regulation of the next-generation influenza vaccines. The Food and Drug Administration (FDA) is responsible for regulating vaccines in the United States. In 1945, the first inactivated influenza vaccine (IIV) was licensed in the United States (US). A live attenuated influenza vaccine (LAIV) was licensed in 2003. Since 2010, US Centers for Disease Control and Prevention (CDC) has recommended annual immunization with current licensed influenza vaccines for all individuals 6 month and older. The seasonal influenza vaccines licensed in the US contain two Influenza A strains (one H1 and one H3) and either one or two Influenza B antigens in trivalent or quadrivalent formulations, respectively. The current inactivated influenza vaccines are licensed and released based on hemagglutinin (HA) content alone since HA is considered the key protective antigen for the current inactivated influenza vaccines. Conventional influenza vaccine production relies on an egg-based manufacturing process that yields inconsistent amounts of hemagglutinin (HA). The adaptation of vaccine strains to growth in eggs to increase HA yields, can result in accumulation of mutations that could impact important antigenic sites and may reduce the protective immunity against the circulating influenza strains. In addition, there is a significant time lag between virus strain selection by the WHO and FDA Vaccine Advisory Committee (VRBPAC) and the availability of vaccine for mass vaccination. The current production of egg-derived influenza virus vaccines is a slow, inefficient process and has the potential of bottlenecks at various steps during the production process and lot release. To overcome some of these limitations, cell-based and recombinant production technologies were developed for more rapid production of seasonal influenza vaccines that could also be used in response to a pandemic threat. The first influenza vaccine produced in mammalian cell culture received licensure in November 2012 in the United States. The first recombinant influenza vaccine based exclusively on hemagglutinin protein was licensed in 2013 by FDA. To improve vaccine immunogenicity and protection in the elderly population, the first adjuvanted seasonal influenza vaccine combined with adjuvant, MF59C.1., an oil in water emulsion of squalene, was licensed in November 2015 by FDA for adults 65 years of age and older.Hemagglutinin (HA) and Neuraminidase (NA) surface glycoproteins of influenza virus evolve and re-assort, leading to antigenic drift and antigenic shift. For the currently licensed influenza vaccine to be effective in protection, the vaccine’s HA and NA antigens needs to be derived from a strain that is well-matched to a circulating influenza strain. This will ensure that the antibodies elicited will protect against infection from that strain. Because multiple influenza strains are in constant circulation, seasonal influenza vaccine is produced and administered annually to protect against the three/four influenza strains expected to be most prevalent that year . Several seasonal quadrivalent influenza vaccine  and trivalent influenza vaccine  formulations are licensed for immunizations of different age groups in the US  antibodies as the primary immunological endpoint. HAI antibody is a surrogate for blocking of influenza virus binding to sialic acid receptor on the surface of red blood cells. The selection of red blood cells used for the HAI assay may be critical for accurate measurements of antibodies blocking virus-receptor interactions due to differential preference of sialic acid receptors present on various type of red blood cells by diverse influenza strains. Other virus neutralization assays have been developed, but thus far have not been used as the primary immunological endpoint for influenza vaccine licensure.In addition to the currently licensed seasonal influenza inactivated vaccines (split or subunit vaccines), several influenza vaccine candidates are currently in preclinical or early clinical development. Some target conserved influenza proteins, such as NP, M1, M2e as well as surface glycoproteins NA and conserved regions within hemagglutinin stem (HA2) and HA1 receptor binding domain with the hope of eliciting broadly cross-protective antibody and/or T cell responses ,6,7. OthAdjuvants can act as immune potentiators and reduce the vaccine dosage needed to stimulate an immune response. Several adjuvants are being evaluated for the next-generation influenza vaccines ,7. AdjuvManufacturers should provide a rationale for the use of adjuvant in their vaccine formulation, with supportive data that may be derived from preclinical studies . Mode of action (MOA) studies in vivo and in vitro are important, but not absolutely required prior to entering clinical trials. Safety and immunogenicity data from use of the same adjuvant with related vaccine antigens, if available, can be supportive. Early clinical immunogenicity trials should be performed to compare adjuvanted vs. unadjuvanted vaccines to provide evidence of enhanced immune response, antigen sparing effects, or other advantages. Since these studies are designed to provide safety information, data can be used to select the optimal dose of both antigen and adjuvant that are both immunogenic and tolerable (least frequency of serious adverse reactions in vaccine recipients). Because adjuvants are not considered active ingredients from a regulatory perspective, manufacturers are not required to demonstrate the “added benefit” of an adjuvant in comparative phase 3 efficacy trials, e.g., studies comparing vaccine antigen with and without adjuvant, but it is necessary for sponsors to demonstrate the added benefit of the adjuvant in early phase studies or in a well-defined animal model. Preclinical safety should be evaluated as per 21 CFR 312.23(a)8) and conducted prior to clinical trials to identify and characterize potential local and systemic adverse effects (SAEs). These studies should include repeat dose toxicity studies and histopathology of full tissue list based on WHO guidance for novel adjuvants with special consideration of reproductive toxicity testing [ and condThe clinical safety of the adjuvanted vaccine must be demonstrated in pre-licensure studies as per Safety requirement for vaccine licensure (21 CFR 600.3). There are special considerations for safety evaluation of adjuvanted vaccines including suggested comparisons between adjuvanted vaccine vs. unadjuvanted antigen vs. saline placebo, and specific clinical follow up regarding any identified symptoms consistent with autoimmune and neuroinflammatory diseases. A longer post-vaccination follow-up, typically 12 months following last vaccination is recommended to capture delayed vaccine associated serious adverse events (SAE) and new onset of medical conditions. However, some potential adverse events beginning after vaccination may not be recognized or diagnosed until much later. Adverse events of “special interest” (AESI) with focus on autoimmune/autoinflammatory diseases such as neuroinflammatory disorders , musculoskeletal and connective tissue diseases , and gastrointestinal disorders  should be followed long-term. This follow up may be performed as part of post-licensure phase 4 studies agreed upon by the manufacturer during the licensure process. There is no regulatory requirement to compare the safety of the adjuvanted to the unadjuvanted vaccine formulation in comparative phase 3 safety studies. Safety information submitted to the Biologic License Application (BLA) may include the safety experience obtained from domestic or foreign trials. Safety experience with the same adjuvant formulated with other vaccine antigens may also contribute to the adjuvant’s safety evaluation.Animal models that predict safety and efficacy of a vaccine antigen, vaccine platform or adjuvant/antigen combination are often used for proof-of-concept studies prior to initiation of phase 1 trials in humans. The animal model may also assist in the initial selection of antigen dose range. However, the predictive value of animal studies for evaluation of next-generation influenza vaccines may be limited by the fact that animals are naïve, while most humans have been exposed (infected/vaccinated) to influenza since childhood and have pre-existing immune memory, which may influence their response to vaccination. Moreover, the outcome of animal challenge studies post-vaccination will vary by their susceptibility to un-adapted human and avian influenza strains, and sialic acid receptor distribution in the upper and lower respiratory tract. In addition, differential pattern recognition receptor (PRR) repertoire (targeted by various adjuvants), husbandry, microbiome, inoculation routes, immune effector functions, and the interval between vaccination and influenza virus challenge may contribute to significant differences in immune responses and protection from viral challenge compared to humans. Despite these caveats, preclinical studies may be helpful to understand vaccine induced immune responses and tease out immune mechanisms that are “reasonably likely” to predict protection. They can also be useful for identifying safety signals that can inform the design of human clinical studies to mitigate vaccination induced adverse events. An animal model that recapitulates human exposure and correlates of protection will be desirable for the preclinical evaluation of next-generation influenza vaccines.FDA’s approval of Vaxchora in 2016 based on positive results from a 10 and 90-day cholera challenge trial, as well as two safety and immunogenicity trials in healthy adults that demonstrated efficacy after vaccination suggests that an appropriately designed human challenge study can provide the data to support vaccine licensure [The utility of human challenge studies for the evaluation of next-generation influenza vaccines will need careful evaluation of viral challenge dose, routes of administration, screening assays for pre-existing immunity of subjects, study age groups, and the time interval between vaccination and viral challenge, among other factors. Availability of panel of influenza virus challenge strains that differ from the vaccine strain will help to demonstrate breadth of protection. Moreover, the careful collection of appropriate pre-and post-challenge specimens at regular time intervals that mimics natural infection can help development and evaluation of relevant immunological assays, and possibly identify correlates of protection for next-generation influenza vaccine platforms, that can be used during subsequent pivotal clinical studies.Licensure of influenza vaccines in the US is based on clinical efficacy endpoints accrued during clinical trials. The production of neutralizing antibodies is a correlate of protection for many human vaccines, including currently US licensed inactivated influenza vaccines. However, no influenza vaccine has been licensed based on any immunological measurement alone. The inactivated influenza vaccines (IIVs) contain virus hemagglutinin as the active component. These vaccines are expected to induce antibodies that block interaction of circulating viruses with the host cell receptor  as measured in a red blood cell-based hemagglutination-inhibition (HAI) assay used as the primary in vitro immunological endpoint. HAI titers were historically shown to predict in vivo protection against influenza symptoms . Most ofAn immune correlate of protection may also be suggested from other sources, including post-infection immunity. Even though influenza is a respiratory infection, there is paucity of relevant mucosal immune assays. Therefore, it may be prudent to develop and evaluate assays and methods to quantify mucosal immune responses in addition to measurement of systemic immunity.Safety of a vaccine is paramount in the licensure of any vaccine by the US FDA. As indicated above, many next-generation vaccine platforms are not designed to block virus binding to its receptor. Rather, they are likely to induce antibodies that inhibit virus replication at a post-entry stage  and therefore may have the potential to mediate not only virus neutralization but also enhancement of viral infection/disease ,20,21,22New vaccines must meet regulatory requirements for safety and efficacy and must be “applicable to the prevention, treatment or cure of diseases or injuries of man” (21 CFR 610.3). Only those vaccines that are demonstrated to be safe and effective, and that can be manufactured in a consistent manner will be licensed by the FDA . The pivVaccine licensure follows either a traditional approval pathway or an accelerated approval pathway. Both pathways require demonstration of product safety and consistency of vaccine manufacturing. However, traditional approval pathway includes pre-licensure evidence of efficacy from clinical trials in which influenza illness is assessed as the primary endpoint. The two licensed influenza vaccines against avian type A H5N1 strains were approved through the traditional pathway, since they used the same manufacturing process as previously licensed seasonal influenza vaccine, and they met the CBER requirements for % seroconversion  and % seroprotection  For pandemic influenza vaccines wherein clinical efficacy trial cannot be performed, licensure might be pursued through accelerated approval pathway. In these instances, a surrogate immune correlate of protection is used for vaccine licensure, with a commitment to perform a clinical efficacy study in the event of an influenza pandemic.The licensure for next-generation influenza vaccines will follow the same general pathway as for other influenza vaccines and requires a full quality package to demonstrate consistent manufacturing process in producing a safe and potent vaccine. The product-specific challenges may involve developing assays to measure vaccine potency and stability as well as the determination of an appropriate vaccine formulation, dosage, and schedule of vaccination. A non-clinical package to demonstrate the proof of concept  will be beneficial but not required for initiation of phase 1 trials.Pre-marketing (pre-licensure) vaccine clinical trials are typically done in three phases: Phase 1; safety and immunogenicity studies performed in a small number of closely monitored subjects, Phase 2; dose-ranging studies that may enroll hundreds of subjects, Phase 3; may enroll thousands of individuals and provide the critical documentation of effectiveness against confirmed influenza infections and influenza-like illness (ILI). In addition to efficacy information, the Biologic License Application (BLA) should contain significant safety information collected during the pivotal phase 3 trials as well as other clinical trials with the same vaccine product. The phase 2 and phase 3 clinical trials are expected to be randomized, blinded, and include a strong statistical plan. Discussion with the FDA prior to and throughout the human clinical trials is highly encouraged.For a broadly protective influenza vaccine, it is important to pre-define the breadth and longevity of protection and generate the data to support these claims. The case definition for influenza illness should be prospectively defined. Placebo-controlled efficacy studies in the US may be challenging given that licensed influenza vaccines are available and recommended for yearly vaccination of all age groups. Alternatively, a population at increased risk for influenza illness complications may be studied, to demonstrate “non-inferiority” or “superiority” of the new vaccine compared with a US licensed vaccine product over more than one season. However, there is no requirement to study the superiority of the vaccine unless the sponsor wants to claim “superiority” for their product.For vaccine effectiveness studies, surveillance systems may be needed for continuous functional surveillance depending on the proposed claims of breadth and longevity of protection against influenza. This will also require identifying drift variants escaping from the immune response induced by the next-generation vaccine. It is possible that next-generation vaccines will include multiple components required to induce immunity against diverse Influenza A (Group 1 and Group 2) and Influenza B viruses. Detailed chemistry, manufacturing and control (CMC) information with validated assays to ensure manufacturing consistency and lot release criteria should be developed prior to the pivotal phase 3 trials. One or more relevant immunological endpoint assays should be identified and validated prior to initiation of the pivotal studies. It will be important to discriminate between vaccine induced responses vs. pre-existing effector functions of antibody and T cells. It is possible that repeat vaccination with the new vaccine product will be needed to maintain long term protection. Vaccine boosts may be identical or different to the primary vaccine construct. If the vaccine boost is different from the primary vaccine, then this new vaccine licensure may generate additional challenges, as currently there is no FDA licensed vaccine that includes prime-boost with different vaccine antigens. Pivotal efficacy trials may require comparison with the US standard of care against a licensed product  with follow up across several seasons, depending on the claim in the licensure package (either “non-inferiority” or “superiority” against “matched and mismatched” influenza strains). As these are new vaccine concepts, they will likely be subjected to age de-escalation criteria to perform these studies first in healthy adults followed by other age and population groups.Regulatory pathways supporting development and approval of vaccines formulated with a novel adjuvant are the same as for unadjuvanted vaccines. Efficient planning of the development pathway for any adjuvanted vaccine requires careful attention to preclinical testing, study design, dosing decisions, and safety monitoring. Although manufacturers are not required to demonstrate the “added benefit” of adjuvanted vs. unadjuvanted vaccines in clinical comparative phase 3 studies, manufacturers should provide a justification for including an adjuvant in the vaccine. Evaluation of safety of an adjuvanted vaccine needs to include special safety considerations.In summary, next-generation influenza vaccines pose significant unique challenges in development and regulation of these influenza vaccines. Some of them include development of appropriate vaccine potency assays, validated immunological assays, identifying immune correlates of protection for different vaccine platforms, development of predictive preclinical animal model for safety and efficacy and/or human challenge studies, vaccination induced long-lasting cross-protective immune responses, and definition and evaluation of what constitute improved breadth of protection during human clinical efficacy trials. Despite these challenges to make better influenza vaccines, there has been a lot of progress made over the last few years, with several new vaccine platforms already in early clinical trials. In recent years, the successful licensure of different types of influenza vaccines including cell-derived vaccine, recombinant protein vaccine and adjuvanted influenza vaccines has provided important experience that could be harnessed in the regulatory evaluation of novel influenza vaccine platforms and next-generation vaccines. Still the approval of next-generation influenza vaccines will be challenging not only for regulatory agencies and vaccine manufacturers but also for the whole influenza community. This will require dialogue, discussion, collaboration and partnership between academic/non-academic institutions, funding agencies, vaccine manufacturers, public health officials and regulatory agencies around the globe for better influenza vaccines of the world.


Attention Deficit/Hyperactivity Disorder (ADHD) is negatively associated with a range of academic achievement measures. We use Danish administrative register data to study the impact of medical treatment of ADHD on children’s academic performance assessed by student grade point average (GPA). Using administrative register data on children, who begin medical treatment, we conduct a natural experiment and exploit plausible exogenous variation in medical nonresponse to estimate the effect of medical treatment on school-leaving GPA. We find significant effects of treatment on both exam and teacher evaluated GPAs: Compared to consistent treatment, part or full discontinuation of treatment has large significant negative effects reducing teacher evaluation and exam GPA with .18 and .22 standard deviations, respectively. The results demonstrate that medical treatment may mitigate the negative social consequences of ADHD. Placebo regressions indicate that a causal interpretation of our findings is plausible. One in five individuals experiences the onset of mental health problems before reaching adulthood . Mental Recent research has linked ADHD to delayed maturation of parts of the brain directly related to cognition –12, indiOne of the modal ways to address the behavioral problems related to ADHD is through medical treatment with psychotropic drugs. Although the use of these drugs is not uncontroversial –19, drugnonresponders. Nonresponders are unaffected by the medication or have severe side effects, in either case causing them to discontinue treatment even though they show the same symptoms as responders prior to treatment [This study tests whether efficient medical treatment of ADHD affects children’s academic performance by utilizing a natural experiment. The medical literature documents that 25 to 30 percent of treated individuals with ADHD are reatment . There dreatment –23 nor dreatment –25. Undeafter graduation . We find no significant differences in the effect of treatment with and without confounders and we find no differences in the GPA between medical responders and nonresponders in the placebo sample, indicating that the main results likely express causal effects. Our results demonstrate that medical treatment of ADHD may play a substantial role in diminishing educational disparities caused by the condition.We find that medical treatment has a significant and sizeable effect on long-term academic performance outcomes in terms of school-leaving GPA. The effect of medical treatment of ADHD is comparable in size to the gender differences in GPA. To test whether a causal interpretation of the results is plausible and to account for any social gradient in treatment patterns, we estimate the effect of treatment with and without a rich set of potential confounders and perform placebo regressions on a sample of children, who initiate medical treatment The remainder of this paper is organized as follows: Section 2 reviews relevant literature and theories on the effect of ADHD and ADHD treatment on educational outcomes. Section 3 presents the data for our empirical analysis. Section 4 presents our identification strategies and statistical method. Section 5 presents results. Section 6 discusses the findings and concludes.Untreated behavioral problems may substantially impair children’s learning and educational achievement –27. PrevThe research literature on the consequences of ADHD for children’s lives indicates two mutually reinforcing pathways through which the disorder affects educational outcomes: direct biological effects on cognitive development and indirect effects on children’s home environment that likely affect learning.Neurological research has shown that children with ADHD experience delayed maturation of the brain—more specifically delayed growth of the pre-frontal cortex, i.e. the part of the brain associated with the regulation of executive functions, such as complex cognition, decision-making processes, and the moderation of social behavior. In their seminal longitudinal study of 223 children diagnosed with ADHD and 223 typically developing controls, Shaw et al.  show subYet, delayed cognitive maturation may not be the only cause driving the relationship between ADHD and low educational achievement. Neither learning nor ADHD occur in a social vacuum. Beyond the direct neurobiological effects, ADHD also places strain on children’s life circumstances outside the educational system in ways that could deter learning. Untreated ADHD increases the risk of experiencing family instability, such as parental divorce –14 and, The strong association between ADHD and the above-mentioned host of negative outcomes has given rise to a number of treatment types, with pharmacological treatment with stimulants being the most common form ,34. OverWe use data from Danish administrative registers collected by Statistics Denmark. The primary data sources are the Danish Registry of Medicinal Product Statistics (RMPS) and the national registers on educational attainment (REA). Danish registers offer extensive individual-level information on all educational courses commenced in the public educational system and various academic performance measures. Register data has almost no sample drop out or attrition and validity and coverage is very high; e.g. in 2008, 96 percent of the Danish population aged 15–69 have non-missing education information . From 20Data from RMPS contain detailed information on all legal purchases of prescription drugs in Denmark . The prescription data are linked to the individual identification number of the patient through the Danish health insurance system . By usinDiscontinued Pharmacological Treatment (DPT), Ambiguous Pharmacological Treatment (APT), and Continuous Pharmacological Treatment (CPT). The difference between these three types of treatment allows us to estimate the effect of treatment by comparing the outcome across the three different treatment patterns with the DPT acting as a control group and the CPT as a treatment group and the APT as a partial compliance group. The definition of discontinuation (DPT) follows strict restriction rules: we define DPT as having purchased medication for maximum three months within the data window, which just allows for initial medicine trial and dose titration. Likewise, the definition of continuous treatment (CPT) is restricted to those, who have a regular and stable use of medication with purchases being no more than three months apart. The patterns of the remaining group, the APTs, are then per definition ambiguous as they cannot be convincingly placed in either the DPT or CPT category. These are children, who repeatedly enter in and out of medical treatment. All three categories are mutually exclusive. In all analyses, CPT is reference group, whereby we investigate inverse treatment effects of DPT and APT (discontinuation and ambiguous treatment) compared to CPT (continuous treatment).In order to determine the effect of medical treatment of ADHD, we use longitudinal data from RMPS and define three types of pharmacological treatment patterns of use in the analysis: treatment sample). Note that amphetamine salts were not introduced in Denmark for treatment of ADHD until 2013 due to official national restrictions and that these types of drugs are therefore not included in our sample criteria. We exclude children, who begin treatment before age twelve because scholastic impairment is likely less traceable among children at this age and because they more likely suffer from hyperactivity than attention problems . All analyses were also conducted for the total sample yielding very similar results. We define an additional sample of children, who start treatment after school-leaving exams and use this as a placebo sample (we elaborate on this in section 4 on methods).We sample all children diagnosed with and treated for ADHD from the total population of children, who completed Danish compulsory schooling from 2002 to 2011. To identify medically treated children (irrespective of their subsequent treatment pattern), we select children, who redeem at least one prescription for any type of ADHD medication  between age twelve and prior to school-leaving exams  . Exams aThird, we exclude children, who commence treatment around the time point of exams to minimize the risk that we include children, whose (expected) poor exam performance causes them to commence treatment to boost future academic performance . These rTo account for any effect of the duration of medical ADHD treatment, severity of symptoms, or potential correlations between age and treatment response , we contTo account for the event that treatment patterns (DPT and APT versus CPT) vary across child and family characteristics, we include a rich set of control variables. Individual level information includes gender, immigrant status, birth weight, and gestational age. Studies show that birth characteristics predict ADHD symptoms –47 and cWe measure parental characteristics one year prior to the birth of the child to avoid post treatment confounding. We control for parental income (including dummies for negative income generated by negative income from self-employment), unemployment, educational level (ISCED code dummies), parent age at the birth of their child, and whether either parent has an ADHD diagnosis.We estimate the effect of medical treatment on school-leaving GPAs for a sample of children diagnosed with ADHD, who initiate medical treatment. Our design has the advantage of circumventing selection into diagnosis and treatment of ADHD, as all children in the sample have been diagnosed and have initiated treatment. However, selection out of treatment may be an issue. As mentioned previously, we exploit evidence from medical research showing that individual response towards ADHD medication regarding the improvement of core symptoms is arbitrary with respect to individual characteristics of the patient –23,25,36GPAi is student GPA, Xi is a vector of individual characteristics, β is a vector of corresponding regression coefficients, DPT and APT are binary indicators of discontinued treatment and ambiguous treatment. ei is the error term. The regression coefficient γ1 measures the average difference in outcomes between those continuing medication (CPT) and those discontinuing medication (DPT) from the onset of diagnosis and until completing compulsory school, where GPA is measured.To measure the effect of medication on students’ school-leaving GPA we estimate the following regression:γ2 is less clear-cut, but captures the average difference in outcome between a continued treatment pattern and the average ambiguous treatment pattern (APT). The effect thus both depends on the effectiveness of the medication  and it depends on the distribution of the ambiguous treatment patterns in the data. If APT patterns on average are close to full compliance, children in this group should have the same outcome as the CPTs. If, however, they, on average, are close to a full discontinuation of treatment, children in this group should, on average, have the same outcome as the DPTs. At any rate, a negative estimate of γ2 indicates that medication is effective.The interpretation of and simultaneously have a direct effect on the GPA outcomes [Although random nonresponse is supported by empirical evidence, drop out from medical treatment in observed data may be nonrandom for a number of other reasons. Take-up rates of prescriptions have a social gradient due to economic constraints for poorer families to finance medication consistently, and at-risk families may be less inclined to follow medical advice or to comply with medical treatment despite negative side effects. Studies find that this applies for health care in general –51 and foutcomes .To evaluate the extent that omitted variables bias our results we estimate Eq  both witDuring the last 20 years, the number of medical products available has increased, while existing medical products have been enhanced –56. Suchafter graduation (thus after receiving their grades) and for whom we should expect no treatment effect on their GPA  have significantly higher exam GPAs compared to those, who discontinue treatment partly (APT). The effect of DPT (full discontinuation) is -0.13, yet insignificant, and the effect of APT is -0.12 and significant. Including control variables (model 2) further increases these effects to -0.22 for DPT and -0.13 for APT, albeit estimates with controls are not significantly different from the estimates without controls. Nevertheless, controlling for background characteristics we thus find more than a fifth standard deviation difference between DPT and CPT and more than a tenth of a standard deviation difference between APT and CPT. Controlling for selection into our sample of initially treated students (model 3) has virtually no impact on the estimated effects across treatment groups, hence sample heterogeneity across birth cohorts seems negligible.For the placebo sample (model 4), we find no differences across treatment groups, indicating no selection bias on unobservables for the placebo sample into DPT, APT and CPT. If we are willing to assume that this also applies for the treatment sample—supported by the fact that including controls did not change parameters in the treatment sample—the results support a causal interpretation of the effect of medication on GPA. One may argue that the placebo sample is not entirely comparable with the treatment sample, as they are diagnosed a considerable time after their exams. However, restricting the placebo sample to a narrower age range (only including students beginning treatment no later than age 24 or students beginning treatment no later than five years from graduation) does not alter our conclusions.Inspecting the effect of the covariates on the outcome, we find much the expected sizes and signs: Males have lower exam GPA. Parent educational attainment level is positively correlated with exam GPA. Children whose mother is diagnosed with ADHD have a slightly higher GPA. The reason for mothers (and partly fathers) diagnosis being positively associated with GPA could be the fact that we study a selected sample; those diagnosed with ADHD . Within this sample, parental experience with a diagnosis may prove to be beneficial. For the total population, however, the association is reversed; on average, students, whose parents have ADHD, obtain lower grades than students, whose parents do not have an ADHD diagnosis.When re-estimating regressions including an interaction between duration and treatment pattern, we find that duration (measured in months) in itself has a very small yet significant positive influence on our outcome, but the interactions are nonsignificant. Thus, we do not find evidence that our main effects of treatment patterns vary with duration.Comparing the covariate estimates for the treatment and placebo sample reveals similar sizes of the estimates for most control variables. This indicates that the selection process into the two samples is similar. Had they been different, we should have seen marked differences between the estimates because different selection processes would have yielded different selection bias in the estimated coefficients.Panel B in In sum, we find evidence of a positive effect of medical treatment on school-leaving exam and teacher evaluation GPAs for students diagnosed with ADHD, who enter treatment. Our results are robust to the inclusion of control variables and selection into the group of treated students. Moreover, there are no differences in GPA outcomes across treatment patterns for students beginning treatment after receiving their school-leaving GPA, which support our main results.In this study, we find that increased efficiency of medical treatment of ADHD can alleviate a substantial part of the GPA gap between children diagnosed with ADHD and their peers. Our empirical analyses show substantial and significant negative effects from discontinued pharmacological treatment (DPT) and ambiguous pharmacological treatment (APT) on school-leaving teacher evaluation and exam GPAs compared to consistent treatment (CPT). Treatment effects are synonymously negative, in the sense that both DPT and APT lower GPA across various model estimations and are robust to the statistical control for a large set of covariates and selection models.Teacher evaluation GPA likely reflects both academic performance and student behavior in class. In this respect, teacher evaluated GPA reflects non-cognitive skills over and above the cognitive skills reflected in exam GPA. Finding that treatment effects are similar for teacher assessed performance  and exam performance (exam GPA) supports the notion that medical treatment raises student academic performance and not merely alters student behavior in class.Our main findings support an overall beneficial effect of medical treatment on long-term individual academic performance of children diagnosed with ADHD. Relying on the fact that national guidelines only recommend medical ADHD treatment for moderate to severe cases, children in the sample likely have moderate to severe symptoms, in which case benefits may exceed negative side-effects of treatment. In addition, Danish treatment rates of ADHD are more than 80 percent lower than, for example, US treatment rates . Althougand treatment as well as for the endogeneity when studying long-term social outcomes of children diagnosed with ADHD.As medical treatment affects school-leaving GPA of individuals diagnosed with ADHD, treatment likely influences subsequent educational and vocational tracks as well; for example choice of post-secondary tracks, dropout rates, unemployment propensity, adult profession, and adult income as these are all strongly correlated with school-leaving GPA. International studies have also found evidence of such effects from ADHD diagnosis. However, due to interdisciplinary gaps between social and medical studies, research is needed that can adequately account for diagnosis This study finds that medicine exerts a profound influence on the educational outcomes of children diagnosed with ADHD. Methodologically, our analysis rests on the stable unit treatment value assumption, implying that potential individual outcomes are unrelated to the treatment status, outcomes or characteristics of other individuals. However, children with ADHD are part of social contexts—school classes, families, etc.—and the individual effects of treatment presented should ideally be construed in its context. Therefore, additional research should study the impact of ADHD treatment on peers and families. A few recent studies suggest that ADHD and medical treatment hereof affects academic outcomes of non-ADHD siblings  and clasMore than four decades of sociological research has voiced concerns about medicalization, social control, and the social construction of problem behaviors –18. Rece


Glioblastoma Multiforme (GBM) is the most common malignant primary brain tumor. Despite aggressive multimodality treatment it remains one of the most challenging and intractable cancers , 3. Ther Multiple immunotherapy clinical trials in GBM have been initiated although few have reached completion. And of those few, the results observed were limited. For example, an upfront (newly diagnosed GBM) phase 2 trial tested the efficacy of a patient specific dendritic cell vaccine termed ICT-107, by observing its ability to significantly change the median survival rate of newly diagnosed GBM patients. In the ICT-107 study, patients in the experimental arm as well as control arm underwent surgical resection followed by radiation and TMZ treatment for six weeks. Subsequently, white blood cells (WBCs) from GBM patients were extracted from both experimental and control groups and cultured with antigens found in the GBM experimental group and not in the control group. Over the period of several months, the WBCs pulsed with GBM antigen for the experimental arm and WBCs not pulsed with GBM antigen for the control arm were reintroduced as vaccines to patients in the respective groups. The results showed that when compared to patients in the control group, whose extracted WBC were not cultured with GBM antigens, the GBM cultured WBCs increased survival in ICT-107 treated patients by only less than 2 months [Table Other combination trials using immunotherapy with chemotherapy and/or radiotherapy have been performed in the hopes of attaining statistically significant results, but most have fallen short. Another upfront phase 2 immunotherapy combination trial investigated the effect of immunotherapy with radiation and chemotherapy to determine the efficacy of combination therapy as treatment for GBM with the PEP-3-KLH vaccine for newly diagnosed EGFRvIII-expressing GBM patients. The PEP-3-KLH vaccine is a synthetic peptide derived from a mutated segment of the epidermal growth factor type vIII (EGFRvIII), which is overexpressed in some patients with malignant glioma . This muA second upfront combination trial analyzed the efficacy of immunotherapy in conjunction with radiotherapy and chemotherapy. In this trial, patients who had recently undergone tumor resection for their newly diagnosed GBM were first treated with radiotherapy and concurrent chemotherapy, TMZ. After completion of both radiotherapy and chemotherapy, WBCs were collected from these patients and cultured along with dendritic cells with each individual patient's GBM tumor antigens Figure . This auThe latter two upfront GBM trials mentioned above suggest a common theme in the ability to properly treat GBM. Patient outcome is highly variable, which could possibly be due to the heterogeneity of GBM tumors. In 2010, research utilizing data from The Cancer Genome Atlas developed a catalog of genomic abnormalities correlated with GBM tumors and helped to categorize four different GBM tumor subtypes: Classical, Proneural, Mesenchymal, and Neural , which hIn comparison to upfront GBM trials for combination immunotherapy, recurrent GBM trials have even fewer results. These trials include single therapy dendritic cell vaccines in which patient WBCs are cultured and reintroduced  , treatme+ T-cells) do not highly express the PD-1 receptor and therefore, cannot bind the PD-L1 (ligand) on GBM tissue resulting in the high success rate of this combined therapy  that facilitate the molecular processes of epigenetic regulation . lncRNAsSeveral lncRNAs are differentially expressed in GBM relative to normal brain tissue . For insin vivo  epigenetic reader proteins Figure . One sucAnother study involving lncRNAs looked at lncRNA LINC00470, which like HOTAIR, is overexpressed in GBM when compared to normal brain tissue. Moreover, it was reported that patients with higher levels of LINC00470 had poorer prognoses in terms of survival time when compared with patients with lower levels of LINC00470 . ResearcResearch looking into epigenetic management of cancer has shown that epigenetic inhibitors are safe and may be effective in treating certain neoplasms. For example, Vorinostat, a histone deacetylase (HDAC) inhibitor that works by preventing the action of histone deacetylases (HDACs), has been approved for treatment of Cutaneous T-cell Lymphoma and may benefit patients suffering from prostate cancer, breast cancer, and lung cancer . 5-AzacyGBM remains an incurable disease despite increased understanding of the genetic and epigenetic pathways dysregulated in these tumors. While immunotherapy trials have shown minor improvements in overall survival, the actual increase in time for the patient is still only months and treatment is highly limited to certain subtypes of GBM. Importantly, lncRNAs can be detected in patient serum and can be used as biomarkers for efficacy of drugs in patients. Thus, it seems that the pursuit of other treatment options such as combining epigenetic pathway inhibitors along with immunotherapy treatment could bring medicine closer to treating GBM. Though it may be difficult to create a blanket treatment for all types of GBM, it is worth dedicating resources into future research of individualized patient treatment.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.


Cerebral blood vessels are innervated by sensory nerves that store several neurotransmitters among which calcitonin gene-related peptide (CGRP) is the most abundant. In primary headaches, there is a clear association between the head pain and the release of CGRP. In cluster headache there is an additional release of vasoactive intestinal peptide (VIP).In connection with administration of triptans, the headache subsides and the neuropeptide release normalises, in part via a presynaptic effect.In subarachnoid hemorrhage (SAH), CGRP is released to counterbalance the blood-induced vasospasm. In severe cases, the stored CGRP may be exhausted while infusion of CGRP may limit cerebral vasospasm. Thus, interactions with the trigeminovascular system at CGRP receptors may be a useful target for understanding of cerebrovascular disease and to design novel treatments.