Disorders of consciousness (DOC) has been an object of numbers of research regarding the diagnosis, treatment and prognosis in last few decades. We believe that the DOC could be considered as a disconnection syndrome, although the exact mechanisms are not entirely understood. Moreover, different conceptual frameworks highly influence results interpretation. The aim of this systematic review is to assess the current knowledge regarding neurophysiological mechanisms of DOC and to establish possible influence on future clinical implications and usage.We have conducted a systematic review according to PRISMA guidelines through PubMed and Cochrane databases, with studies being selected for inclusion via a set inclusion and exclusion criteria.Eighty-nine studies were included in this systematic review according to the selected criteria. This includes case studies, randomized controlled trials, controlled clinical trials, and observational studies with no control arms. The total number of DOC patients encompassed in the studies cited in this review is 1,533.Connectomics and network neuroscience offer quantitative frameworks for analysing dynamic brain connectivity. Functional MRI studies show evidence of abnormal connectivity patterns and whole-brain topological reorganization, primarily affecting sensory-related resting state networks (RSNs), confirmed by EEG studies. As previously described, DOC patients are identified by diminished global information processing, i.e., network integration and increased local information processing, i.e., network segregation. Further studies using effective connectivity measurement tools instead of functional connectivity as well as the standardization of the study process are needed. CRS-R tAlthough effective treatment for this group of patients is not yet available, some progress has been made by introducing neuromodulation techniques, in the first-place deep brain stimulation (DBS). Historically, Morruzi and Magoun first showed brainstem reticular formation and thalamus stimulation of the anesthetized animals leading to desynchronization of low-frequency disorganized electroencephalograph (EEG) activity and background activity comparable to the patterns in wakeful states . MentionIn mentioned primary, as well as in later studies, the vast majority of patients manifest eye-opening and incomplete movements when receiving stimulation consistent with an arousal effect. Nevertheless, arousal effect occurrence did not predict any improvement. Furthermore, arousal effect, including eye-opening, autonomic function changes and EEG desynchronization characterize a fundamental and wide activation of the forebrain, brainstem, and spinal cord systems. It seems that apparent wakefulness and incomplete movements do not demonstrate higher integrative brain function recovery – it just gives evidence that DBS electrodes hit the target .Several research groups are continuously making efforts to improve patient selection criteria for DBS as well as neurostimulation protocols. The inclusion criteria for such procedures traditionally require the presence of multimodal evoked potentials , which are believed to be gross neurophysiological markers of cortical functional integrity.Today, however, with the rapid advances in computing and neuroimaging techniques to test brain connectivity, we are faced with enormous amounts of data and novel mathematical modeling techniques. All this information might improve our understanding of underlying pathophysiological processes and lead us to better decision-making, but the careful interpretation is crucial.11 and there are roughly 1014 synaptic connections, it is impossible (and even pointless) to investigate disrupted connectivity patterns at a single neuron level  studies with patients diagnosed with DOC; (b) studies published up to January of 2023; (c) published in the English language; (d) published in indexed and peer-reviewed journals; and (e) evaluated consciousness using validated scales and scoring systems .Exclusion criteria include: (a) studies published in regional languages other than English, (b) examination in an acute state or during sedation/anaesthesia, and (c) no clear methodology or testing parameters described. Additionally, we excluded papers on basic research, brain-computer interfaces, machine learning, or pharmacological treatment response.Studies were checked for quality, and finally, 89 studies were included .We find increasing evidence linking DOC with interference in brain connectivity both locally and connecting remote brain areas. Disconnection generally indicates brain dysfunction following lesions to white matter connections . TheoretSo, despite severe and irreversible damage of RT, cortical neurons and their connections with the thalamus may still be preserved. These pathological findings explain the fact that in some patients with severe DOC (VS and MCS), cortical somatosensory evoked potentials can still be elicited. RAAS bind together different cortical regions, and ischemic injury selectively lesson RAAS while leaving some cortical neurons intact, resulting in loss of consciousness of various degrees. On the other hand, awareness is mostly related to frontoparietal associative cortices. It is later subdivided into an internal awareness network (mostly midline regions), and an external awareness network .In the last decades, an extraordinary flourishing of structural and functional imaging techniques, combined with mathematical models, offered us new ways for further advances in lesion mapping. Network theory is an umbrella term due to mathematical theory for the networks description. Therefore, a number of non-invasive novel techniques and methods analysis have provided whole-brain connectivity patterns inspection using electrical and magnetic brain activity , as well as cerebral blood flow changes as quantification of neural activity (fMRI) for network construction . Thus, tTo describe the relationship between pairs of network nodes, the term functional connectivity (FC) has been introduced. However, it is essential to point out that functional connectivity does not necessarily correspond to structural  connections. Understanding the relationship between structural and functional organization represents one of the most critical challenges in neuroscience. In contrast to functional connectivity, effective connectivity includes information about the direction of the connection. Even so, present-day fMRI resting-state methods for causal connectivity are limited .The network model is convenient for describing static brain properties, but another mathematical model is introduced, the so-called neuronal oscillations model, to evaluate dynamic interactions. Neural oscillations are widespread phenomena ranging from the microscopic level of individual neuron electric state oscillations to large neuronal ensembles macroscopic oscillations.The recuring presynaptic neurons firing generates oscillatory activation, while the synchronized activity of several neurons generate macroscopic oscillations. Mentioned was first observed by Hans Berger in 1924 in EEG, leading to the brain rhythms classification into frequency bands. Rather than using single-neuron models, it is helpful to emerge a low-dimensional models to imitate the assembly of number of near-identical interconnected neurons with a preference to operate in synchrony. These neural mass models are composed out of state variables that track coarse-grained measures of the average membrane potential, firing rates, or synaptic activity .What fires together, wires together" is a well-known (but simplified) Hebb's principle and, while mnemonic, could be very misleading: if neurons activations occur at the same time, the activation of one neuron cannot cause activation of the other. The actual ' 'Hebb's quote was: “when an axon of cell A is near enough to excite a cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one of the cells firing B, is 'increased”. An attentive reveals the principle of causality and consistency  . DiffusiThe signals obtained from PET, fMRI, and fNIRS rely on the detection of localized alterations in cerebral blood flow that are linked to neural activity. Functional neuroimaging investigations in DOC can be categorized into two types: resting-state studies and stimulus-based studies . RestingThe introduction of fMRI has brought significant advancements, with the majority of studies on patients with DOC relying on fMRI. While fMRI has limited temporal resolution (within a few seconds), it offers excellent spatial resolution (within millimeters). PET, despite being a more robust technique than fMRI, seems to be more sensitive than active fMRI in aiding the clinical diagnosis of DOC patients, although it may have led to a higher rate of false positives .Historically, the analysis of brain networks has focused on their anatomical organization. However, there is a recent emerging trend that highlights the importance of examining the topological aspects of these networks. Topological metrics allow us to understand the relationships between elements within a system, irrespective of their physical placement. In this section, we will first discuss the patterns of dysfunctional connectivity observed in the anatomical organization of brain networks, followed by an exploration of the subsequent topological reorganization. PET has been used to assess cerebral metabolic activity in DOC patients and has shown modified connectivity between intralaminar nuclei of the thalamus and prefrontal and anterior cingulate cortices in the VS, while not after consciousness recovery . HypermeLacking cortico-thalamic and reduced cortico-cortical connectivity patterns were also described in fMRI studies , as wellDepending on the analysis method, there are several strongly connected resting state neural networks (RSNs) of sensory and cognitive relevance including the default mode network (DMN), somatomotor network, dorsal attention network, ventral attention network, limbic system network, fronto-parietal and visual network, that are widely accepted . These nHowever, there is evidence of globally impaired functional connectivity within multiple RSNs . PatholoMoreover, intrinsic functional connectivity strength in many brain regions significantly correlates with consciousness level and recovery outcome . Among tAnother approach to global network analysis is connectome-based. Brain networks have a topological organization, consisting of nodes that are grouped into modules, while extensively connected nodes are called the “hubs” of the connectome. The hubs play a critical part in coordinating communication between distant parts of the brain . The higConsidering both local (intrinsic) and global network alterations, recent studies proposed that regions sensorimotor integration of high arrangement play a crucial part in supporting consciousness. The view is supported by evidence of a significantly reduced number of connections in the sensorimotor cortex and their correlation with levels of consciousness . FurtherFrequency band analysis provides valuable insights into assessing consciousness. Studies have revealed decreased alpha power and increased delta power in patients with the UWS compared to those in the MCS . FurtherThe absence of consciousness in patients in the VS is paralleled by impairment in the overall EEG operational architecture . NeuronaBrain networks have traditionally been analysed in anatomical space, but recent research has highlighted the importance of considering the topological aspects of brain networks. Further studies explored even more detailed frameworks to evaluate functional connectivity, for example using multiplex and multilayer network analyses of frequency-specific and area-specific networks . AlteratNeuromodulation protocols could be used for diagnostic or therapeutic purposes. In this review, we will primarily discuss their diagnostic value. Several methods have been used to access functional connectivity after stimulation protocols. We can divide them into two categories: (a) non-invasive – which is transcranial magnetic stimulation (TMS), and (b) invasive: spinal cord stimulation (SCS) , vagus nThe invasive techniques are used primarily for therapeutic reasons. Since it is the most common, non-invasive, and painless technique, we will discuss TMS in more detail. The main advantage of TMS-evoked EEG responses compared to resting-state FC is “active probing” of effective connectivity. In contrast to resting state EEG measurements, TMS-EEG measures and maps cortical excitability and reactivity. Three types of TMS can be distinguished: single-pulse activated once every few seconds, paired-pulse, where two pulses are activated out of phase to inhibit or excite neurons of one hemisphere or to inhibit in one while exciting in the other hemisphere, and repetitive TMS (rTMS), where pulses are sent in fast sequence . It has The first observation a significantly different effect of TMS treatment comparing VS and MCS patients; there was an improvement in EEG functional connectivity and increases in power spectra in the majority of MCS patients but modest or no effect in VS patients . TherefoDBS has been shown to have significant effects on functional connectivity in patients with MCS . A studyHowever, these results must be interpreted cautiously because of the small patient samples . Among oFunctional connectivity studies in DOC have predominantly relied on neuroimaging techniques, with a particular focus on resting-state fMRI . RestingInitial fMRI studies revealed disruptions in functional connectivity in regions such as the basal ganglia, thalamus, and frontal cortex, shedding light on the structural basis of functional disconnection underlying these conditions. Specifically, using RSNs, studies have shown local hypoconnectivity within multiple RSNs, particularly in the DMN and frontoparietal associative networks, suggesting a breakdown in the coordination and communication between different brain regions critical for normal cognitive functioning .In addition to hypoconnectivity, DOC patients also exhibit local hyperconnectivity in certain regions, including limbic structures, brainstem areas, and the cerebellum. This abnormal increase in connectivity may reflect compensatory mechanisms or maladaptive processes in the brain. Abnormal interactions between different RSNs have also been observed, and these abnormal connectivity patterns have been correlated with levels of consciousness, providing valuable information about the severity of the condition and potential prognosis. Furthermore, fMRI studies have demonstrated whole-brain topological reorganization in DoC patients, particularly affecting sensory-related RSNs. This reorganization is characterized by diminished global information processing (network integration) and increased local information processing (network segregation).In addition to fMRI, electrophysiological techniques (particularly EEG) have also been used to estimate functional connectivity in DoC . AlthougThese findings emphasize the importance of frequency-specific connectivity patterns, EEG organizational changes, and network dynamics in assessing consciousness and differentiating between DoC states. Future research aims to determine levels of functional connectivity as biomarkers for responsiveness to potential neuromodulatory interventions.Connectomics and network neuroscience provide valuable quantitative frameworks for analysing dynamic brain connectivity, offering new insights into the pathophysiology of disorders of consciousness. By integrating neuroimaging and electrophysiological techniques, functional connectivity studies hold promise for enhancing diagnostic accuracy, guiding treatment approaches, and assessing prognosis in DOC patients. Functional connectivity studies, particularly those conducted using resting-state paradigms, have significantly contributed to our understanding of the underlying neural mechanisms in DOC. These studies have revealed disruptions in functional connectivity, abnormal interactions between networks, and whole-brain topological reorganization.However, several challenges need to be addressed, such as accurately extracting real signals from artifacts and irrelevant data arising from complex mathematical algorithms. To mitigate these challenges, it is essential to maintain a grounded understanding of basic neuroanatomy and neurophysiology to avoid misinterpretation of the data.Considering that a significant number of patients exhibit a higher degree of preserved consciousness than clinically classified, functional connectivity techniques can aid clinicians in avoiding misdiagnosis. Expanding research on EEG functional connectivity, given its low-cost and routine implementation, has the potential to become a new gold standard for evaluating cortical integrity, particularly due to its strong correlation with clinical CRS-R testing. However, it should be noted that improved functional connectivity observed after various neuromodulatory interventions does not necessarily imply the full restoration of consciousness levels.In DOC both awakenees and awareness are significantly impaired. Thalamocortical as well as cortico-cortical dysconnectivity play important role in the pathogenesis of DOC. Furthermore, there is critical role of high-order sensorimotor integration in supporting consciousness. The evidence of preserved sensorimotor integration in the higher-order cortex may hint at the potential for brain recovery. Additional research is required to validate these assumptions.GP designed the study and wrote the first version of the manuscript. GP, MR, VD, and DC conducted the literature research, contributed to the data analysis, study concept, and design. VD and DC interpreted the results and revised the manuscript. All authors read and approved the final version of the manuscript as submitted.This research was supported by the Croatian Science Foundation project CSF-IP-2020-02-4308, DC.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The COVID-19 pandemic caused many palliative care services to rapidly implement telehealth services to reduce face-to-face human contact to limit virus transmission. Examples of palliative care telehealth services implemented during COVID-19 include clinical monitoring, patient assessment, psychological support, spiritual care and day therapy services.614 The World Health Organisation has recently announced that COVID-19 is no longer a global health emergency, however, its impact in increasing palliative care telehealth will likely remain, partly due to increasing global need for palliative care, meaning technological innovation is needed to provide care.Palliative care telehealth  and telemedicine (describing use of technology for care delivery) is increasingly being used in the delivery of palliative care. For example, telehealth could facilitate more efficient time management for the multidisciplinary team. Improved management of resources could streamline (and reduce) the number of face-to-face clinical appointments for patients. Telehealth could improve access to palliative care services for people in areas without access specialist palliative care services , and support the identification of people requiring specialist evaluation and treatment . From a service level, telehealth could help organisations to support staff coordination, education and pastoral care. Finally, novel data technologies provide new possibilities to innovate care through its incorporation with telehealth, for example artificial intelligence , audio  and visual , which could potentially provide the clinician with data that adds value to the consultation.Palliative care telehealth offers potential to improve access and choice for patients.18 Governments commonly cite cost reduction, improved self-management of illness and homecare delivery as reasons to adopt telehealth systems. However, this premise is potentially troublesome when considering palliative care needs, as telehealth may lead to increased intensity of face-to-face support as physical function worsens. In this situation, telehealth may (appropriately) identify those who require more in-person support, following which telehealth may no longer be possible as the individual may no longer be able to use telehealth systems due to their declining health. Without long-term economic analysis, there is risk that some telehealth programmes (implemented rapidly during COVID-19) were not costed appropriately, meaning details of long-term running costs  are unknown. Telehealth systems may increase the risk of bias towards some users if  technology is developed with data unrepresentative of certain communities . A failure to adequately design telehealth systems for the changing requirements of people with palliative care needs may cause them to be deemed expensive, ineffective and inefficient.Many palliative care telehealth interventions lack detailed evaluation in real-world settings, meaning their usefulness (and transferability) is uncertain.Evidence to identify how telehealth services should be used (including appropriate models of care and economic considerations) are needed for this technology to achieve its full potential in palliative care. Interdisciplinary research partnerships  should be conducted, to adequately research the areas needed to determine how telehealth can be used, safely and effectively, to improve palliative care for patients and caregivers.


Intraocular inflammation is common after anterior or posterior segment surgery. They typically manifest either as non-infectious inflammation of the anterior or posterior segment, known as toxic anterior or posterior segment syndrome (TPSS), or as sterile or infective endophthalmitis. In this report, we describe a rare case of TPSS following vitreoretinal surgery, presenting as hemorrhagic retinal vasculitis.A 58-year-old male diagnosed with a left eye acute rhegmatogenous retinal detachment underwent an uneventful primary pars plana vitrectomy with silicone oil endotamponade on the same day of presentation. At presentation, there were no signs of intraocular inflammation, and his visual acuity in the affected eye was 20/200.The retina was well-attached with silicone oil in place on the first post-operative day. Along the inferior retinal periphery, a hemorrhagic occlusive vasculitis was observed. Clinical examination revealed retained intraocular cotton fiber along the inferotemporal quadrant over the retinal surface. In addition to the standard post-operative medications, a course of systemic steroids (40 mg per day of Prednisolone tablets) was started. At the end of the first post-operative week, clinical signs of hemorrhagic retinal vasculitis were beginning to resolve, and by the end of the fourth post-operative week, they had completely resolved.This report describes an unusual diagnosis of TPSS after vitreoretinal surgery, most likely due to the presence of an intraocular cotton fiber. This excessive inflammation of the posterior segment usually responds to a course of topical and systemic steroids. Following an anterior or posterior segment surgery, intraocular inflammation is not uncommon . TypicalIn this report, we describe a case of TPSS presenting with a picture resembling hemorrhagic occlusive retinal vasculitis, which was successfully treated with systemic and topical steroids, possibly due to an accidently introduced intraocular cotton fibre.A 58-year-old healthy man was referred to the retina department by the general ophthalmologist with a diagnosis of rhegmatogenous retinal detachment in the left eye. In the past four days, he was complaining of a sudden, painless decrease in vision in his left eye. His right eye visual acuity was light perception, while his left eye visual acuity was 20/200. His poor right eye vision was the result of a failed retinal reattachment procedure performed five years ago. His anterior segment examination and intraocular pressure examination were normal upon ocular examination. Both eyes exhibited pseudophakia with posterior intraocular lenses. The right eye fundus was vitrectomised and filled with silicone oil, with a thin, detached, atrophic retina and severe macular scarring. The fundus of his left eye revealed a total rhegmatogenous retinal detachment with a large superior retinal break extending posterior to the equator as well as an associated vitreous hemorrhage Fig. . ThroughThe intraocular pressure was normal and there were no signs of anterior segment inflammation on the first postoperative day. The left eye’s fundus examination revealed a well-attached retina, a healthy optic disc, and silicone-oil in situ. There were sclerosed retinal vessels emerging from the equator and extending anteriorly along the inferior retinal periphery. Multiple blot-deep retinal hemorrhages were observed in the same region. A small number of these hemorrhages had white centres. In addition, a white lesion located preretinal along the inferotemporal arcade that resembled cotton fibres was observed upon clinical examination Fig. A. An optTo summarize, we present a case of exaggerated, non-infective posterior segment inflammation following retinal reattachment surgery that manifested as occlusive haemorrhagic retinal vasculitis on the first post-operative day. The presence of non-infective posterior segment inflammation, most likely caused by the toxicity induced by the retained intraocular cotton fibre, led to the diagnosis of TPSS.The term TPSS refers to posterior segment inflammation of the eye caused by a reaction or toxicity to any substance used in the surgical procedure. Toxic posterior ocular reactions are usually caused by the use of intracameral adjuvants, such as those used in cataract surgery, or by the use of saline irrigating solution, silicone oil or gas tamponades, or perfluorocarbon liquid during vitreoretinal surgery , 4, 6, 7Multiple factors, including the re-use of a previously cleaned vitrectomy set or instruments, saline infusion, intraocular air during fluid-air exchange, silicone oil endotamponade, and the presence of intraocular cotton fibre, may have contributed to the development of hypersensitivity in this case. The remaining cases, which were operated on the same day with the same vitrectomy set and instruments, as well as the same batch of silicone oil and saline infusion, showed no toxic intraocular reaction. As a result, we believe the hypersensitivity reaction was caused by an accidentally retained intraocular cotton fibre. In addition, the patient recovered from TPSS with a favourable visual outcome following the disappearance and disintegration of the intraocular cotton fibre and additional treatment with topical and systemic oral corticosteroids. This reinforces our belief that the toxic intraocular reaction was most likely caused by the retained intraocular fibre. However, a contaminated silicone oil vial or saline infusion solution cannot be completely ruled out as the likely source of TPSS in this case.In conclusion, the appearance of a picture resembling occlusive retinal vasculitis following vitreoretinal surgery indicates exaggerated posterior segment ocular inflammation, which can be potentially vision-threatening in eyes with macular involvement. In such cases, identifying the trigger agent(s), particularly in TPSS clusters, is critical. Topical and systemic steroid treatment is usually effective.


Pulmonary hypertension is a debilitating condition that frequently develops in the setting of interstitial lung disease, likely related to chronic alveolar hypoxemia and pulmonary vascular remodeling. This disease process is likely to be identified more frequently by providers given recent advancements in definitions and diagnostic modalities, and provides practitioners with emerging opportunities to improve patient outcomes and quality of life. Despite years of data suggesting against the efficacy of pulmonary vasodilator therapy in patients with pulmonary hypertension due to interstitial lung disease, new data have emerged identifying promising advancements in therapeutics. The authors present to you a comprehensive review of pulmonary hypertension in interstitial lung disease, reviewing our current understanding of pathophysiology, updates in diagnostic approaches, and highlights of recent clinical trials which provide an effective approach for medical management. Pulmonary hypertension (PH) is an often progressive and debilitating condition comprising a diverse collection of disease processes, characteristically due to elevated pulmonary artery pressures from either precapillary, postcapillary, or mixed etiologies. This framework allows for the stratification of a patient’s underlying etiology of PH into one of five groups based on pathophysiology utilizing the World Health Organization (WHO) classification system. WHO Groups 1, 3, and 4 represent precapillary causes, Group 2 represents postcapillary causes, and Group 5 represents diseases with multifactorial etiologies [The recently gathered 6th World Symposium of Pulmonary Hypertension defined PH utilizing a mean pulmonary artery pressure (mPAP) of greater than 20 mmHg, which was reduced from the prior cut-off threshold of 25 mmHg to improve diagnostic sensitivity ,5. More The development of PH-ILD can be quite common in patients with ILD. Prevalence is thought be variable among different subsets of ILD and increases with disease severity, with the bulk of our understanding deriving from patients with idiopathic pulmonary fibrosis (IPF). Patients with IPF have been cited as having anywhere from 8% to 86% prevalence of PH-ILD, with this wide range likely being related to prior variability in the definition of PH and the methods used for diagnosis. General consensus among PH experts places overall prevalence somewhere between 30 and 50% . At diagAmong patients with ILD, the identification of PH typically is a finding of grave consequence. PH-ILD portends increased morbidity with decreased functional capacity, more frequent hospitalizations, higher supplemental oxygen requirements, and reduced quality of life when compared to patients with ILD without PH ,12. ThisThe development of PH-ILD is complex and not fully understood, though chronic alveolar hypoxia is likely a key mediator. Unlike systemic vascular structures which dilate in response to decreased blood oxygen tension, pulmonary vascular tone increases in response to low oxygen tension in a process called hypoxic pulmonary vasoconstriction (HPVC). Hypercapnia or acidemia can additionally augment the response of the pulmonary vascular bed to hypoxemia. Healthy pulmonary tissue performs HPVC in an effort to maintain systemic arterial oxygenation by diverting blood flow from regions of relative hypoxia and proportionately towards regions of normal oxygenation, improving ventilation/perfusion matching. This adaptive mechanism is intended to correct oxygen saturation, so patients may initially demonstrate near-normal oxygen saturation despite large regions of fibrosis and alveolar hypoxia if they are appropriately compensated .The effects of HPVC can typically be reversed with the implementation of supplemental oxygen, though repetitive exposure to hypoxia can lead to vascular remodeling of peripheral vessels and increased smooth muscle in the pulmonary vascular tissues. Vascular remodeling occurs through various mechanisms in the pulmonary vasculature, notably with muscularization of the media and thickening of the intima of small-to-medium pre-capillary pulmonary arterioles and arteries . Though HPVC and damage to the pulmonary vascular bed are unlikely to solely account for the explanation of PH-ILD development . The lacIn response to increasing PVR, patients with PH-ILD commonly demonstrate progressive right ventricular (RV) dysfunction. Relative to the left ventricle, the thin-walled RV is highly sensitive to hemodynamic changes and maintaining function is exquisitely important to support systemic cardiac output due to ventricular interdependence. With the progression of PH-ILD, RV afterload significantly increases due to increasing PVR, promoting progressive RV dysfunction. Chronically increased afterload leads to RV hypertrophy through neurohormonal activation with increased adrenergic tone . This isAlthough it is clear that advanced PH-ILD leads to RV dysfunction and this drives worsening outcomes, why relatively modest increases in pulmonary artery pressures (PAPs) that do not manifest in overt RV dysfunction are associated with such poor outcomes is less clear. It is possible that these patients rapidly progress to have more severe PH-ILD and studies fail to capture this progression over time. Another possibility is that these patients have exercise-induced PH that causes RV strain with activity. A final possibility is that these patients suffer from a relatively low cardiac output state that accounts for an elevated PVR despite relatively low mean PAPs. These conditions could limit oxygen delivery to tissues with exertion. Further study of patients with mild PH-ILD to determine the response to exercise and natural history is essential in answering these questions. Furthermore, whether treatment with pulmonary vasodilators will halt the progression of mild PH-ILD and improve outcomes requires formal evaluation in clinical trials.Unfortunately, there is limited information on which patients should be screened for PH-ILD and a paucity of consensus guidelines exist to direct a clinician’s diagnostic workup. The symptoms of PH-ILD have significant overlap with those of fibrotic lung disease and only a portion of patients with ILD may go on to develop PH-ILD, further complicating the diagnostic process . AdditioGiven the challenges of identifying PH-ILD from history or exam, clinicians must maintain a high index of suspicion. Careful review of non-invasive parameters may be suggestive of the diagnosis of PH-ILD. Chest CT can be a useful tool for both monitoring ILD progression and identifying PH-ILD. One notable CT finding is a pulmonary artery to aorta diameter ratio of >0.9, which has been suggested to predict a mPAP of >20 mmHg and reduced survival . IdentifPulmonary function testing (PFT) has limited utility in the identification of PH-ILD, though diffusion capacity of carbon monoxide (DLCO) appears to have both diagnostic and prognostic utility. Severely reduced (<30% of predicted) DLCO has been shown to increase the likelihood of PH-ILD twofold . Basic sIn general, we recommend the regular use of transthoracic echocardiography (TTE) to screen for PH-ILD in patients thought to be at risk based off of history, exam, and diagnostic findings, in concert with a careful review of PFTs, cross sectional imaging, and exercise data. We also recommend to repeat TTE annually in patients with an established diagnosis. Screening should be serial in nature given the progressive nature of ILDs, as the incidence of PH-ILD may as much as double over five years of follow up . HistoriGiven the inadequacies of noninvasive diagnostic strategies, the gold standard for confirming a diagnosis of PH-ILD remains right heart catheterization (RHC) . We recoOnce PH is confirmed in a patient with ILD using RHC, it is imperative to rule out other possible contributors to PH and comorbid conditions prior to initiation of therapy. Serologic testing and historical assessment for disorders which cause pulmonary arterial hypertension (Group 1) should be undertaken if not already carried out, including HIV, thyroid and liver disease, stimulant medications, and autoimmune diseases . ImagingPH-ILD presents a potentially unique physiologic challenge with the introduction of pulmonary vasodilators, different from other PH physiologies. Use of these agents poses a theoretical risk of worsening hypoxemia through uncoupling of the ventilation/perfusion (V/Q) ratio in areas with significant fibrosis . ConcernOne potential explanation for the varied responses to therapy described may be the presence of two distinct phenotypes of PH-ILD. In some cases, PH-ILD is likely representative of an adaptive process. The PH in these patients reflects the severity of the underlying fibrotic disease and its resulting alveolar hypoxemia, and PH develops through an attempt to preferentially shunt blood flow to better ventilated regions . These aThere was initial interest in employing endothelin receptor antagonists (ERAs) for management of PH-ILD. These trials unfortunately resulted in disappointing outcomes. One study on the ERA, bosentan, demonstrated no improvement in pulmonary hemodynamics, respiratory symptoms, or functional status after 16 weeks of therapy . AnotherCommonly used to manage Group 4 PH, the guanylate cyclase stimulator riociguat has also garnered interest in PH-ILD. This was initially spurred by a 2013 open-label, uncontrolled pilot trial of 22 patients with PH-ILD which showed improvements in cardiac output and PVR after 12 weeks of therapy . A largeNo prospective trial to date has demonstrated positive outcomes from the administration of phosphodiesterase-5 (PDE-5) inhibitors for the management of PH-ILD, though several trials and related registries suggest a potential benefit. Within the Sildenafil Trial of Exercise Performance in Idiopathic Pulmonary Fibrosis (STEP-IPF), sildenafil failed to significantly improve 6MWD, though it was found to improve numerous secondary outcomes, including oxygen saturation and DLCO . DespiteInvestigation of all of these agents has been fraught with challenges including wide variability in how PH is defined, small sample sizes, and the abstraction of data from patients with ILD without PH. Given these issues, recommendations for vasodilator therapy for PH-ILD have been limited and without strong evidence until recently.After years of trials without reassuring data, the Inhaled Treprostinil in Pulmonary Hypertension Due to ILD (INCREASE) trial brought forward a novel therapeutic with proven benefit for PH-ILD . The INCStudies have noted that even modest elevations in mPAP (>17 mmHg) can lead to increases in mortality by as much as 45.5% . Given tSome experts may choose to reserve treatment for patients with severe disease (PVR > 4 WU), as subgroup analysis of the INCREASE trial showed that inhaled treprostinil only demonstrated significant improvement in 6MWD with these patients . As thisThough monotherapy with inhaled treprostinil may be adequate to manage some individuals, patients with more advanced disease with severely increased PVR may require additional pharmacotherapy. Other patients may prove to be intolerant to inhaled treprostinil due to side effects, so alternative therapies may be considered. Recommendations for second line and adjunctive therapy are limited. The use of sildenafil appears to have the potential for benefit and safe use in some patients based upon the results of the COMPERA registry and subgroup analysis from the STEP-IPF trial ,50. The 2 < 60 mmHg or SpO2 < 92%, should be considered for continuous supplemental oxygen to reduce hypoxic vasoconstriction and improve PVR [Beyond pulmonary vasodilators, management of PH-ILD continues to be anchored in the treatment of the patient’s underlying lung disease and management of contributors to hypoxemia . Patientrove PVR ,57. Addirove PVR ,59. A strove PVR ,60. Vaccrove PVR ,61. Addirove PVR ,63.In general, all patients with PH-ILD should be considered for early referral to centers of excellence in the management of pulmonary hypertension and advanced lung diseases . SeveralPH-ILD is a debilitating disease with significant associated morbidity and mortality. Identifying cases of PH-ILD can be challenging given the overlap of symptoms with their underlying ILD and limitations of current diagnostic strategies. Given these challenges, providers should have a high index of suspicion for PH in patients with ILD and a low threshold to screen these individuals. This is especially important in light of the recent developments in pharmacology for PH-ILD, as inhaled treprostinil appears to be the first safe and effective pharmacologic intervention that can modify disease severity.


Pathologic hypertrophy of the cardiac muscle is a commonly encountered phenotype in clinical practice, associated with a variety of structural and non-structural diseases. Coronary microvascular disease is considered to play an important role in the natural history of this pathological phenotype. Non-invasive imaging modalities, most prominently positron emission tomography and cardiac magnetic resonance, have provided insights into the pathophysiological mechanisms of the interplay between hypertrophy and the coronary microvasculature. This article summarizes the current knowledge on coronary microvascular dysfunction in the most frequently encountered forms of pathologic hypertrophy.The online version contains supplementary material available at 10.1007/s12350-022-03040-2. LVH can be primary or secondary and characterizes a wide variety of cardiac structural and non-structural disease-entities such as ischemic heart disease, arterial hypertension, heart failure, valvular disease, and a range of genetical cardiomyopathies. The estimation of LV wall thickness and mass in routine clinical practice is derived from measurements obtained by echocardiography or cardiac magnetic resonance (CMR).Cardiac hypertrophy refers to an increase in the mass of the left or the right ventricle or both as an adaptive mechanism to pressure and/or volume stress, genetic mutations of several proteins, and/or loss of contractile mass as a result of prior infarction.2 This remodeling process commonly affects the entirety of the left ventricle, indicating a more diffuse nature of the disease.3 CMD in LVH is considered to confer important clinical and prognostic implications. Non-invasive imaging modalities allowing for assessment of myocardial blood flow (MBF) and myocardial flow reserve /rest MBF) have provided valuable insights on CMD in pathological LVH. Myocardial perfusion positron emission tomography (PET) which is considered the gold standard for routine clinical MBF assessment, but also CMR and contrast-enhanced transthoracic echocardiography (contrast-TTE) have been shown to be valuable tools in both clinical and research settings.Coronary microvascular disease (CMD), although not the hallmark and the primary mechanism for the pathogenesis of pathological hypertrophy, is considered to hold a critical role in the natural history of this pathological phenotype. Several pathophysiological mechanisms have been proposed and although our understanding remains incomplete, in most cases, CMD in LVH is multifactorial Table . PatientThis review aims to summarize the current knowledge on CMD in the most commonly encountered LVH-phenotypes in clinical practice.4 HCM is commonly morphologically characterized by asymmetrical hypertrophy of the left ventricle, with a predilection for the interventricular septum and histologically by hypertrophy of cardiac myocytes, myocyte disarray, interstitial fibrosis, and thickening and narrowing of the intramural coronary arteries.6 Given the several secondary etiologies of LVH, diagnosis of HCM should be established in the absence of other cardiac, systemic, or metabolic factors or diseases resulting in a hypertrophic phenocopy while at the same time, genetical testing if available is essential in the diagnostic process.7 Impaired MBF during stress and its sequelae, myocardial ischemia is considered an essential feature of HCM patients, and altered microvascular function is associated with poor outcomes.9 Compared to patients without pathological hypertrophy, HCM patients have significantly reduced hyperemic MBF and MFR while resting MBF is preserved.12 Clinically, a significant association has been reported between pathological MFR and symptoms (primarily angina), risk of sustained ventricular arrhythmia, progression to end-stage heart failure, and cardiovascular death.16Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiovascular disease, inherited through an autosomal dominant pattern.18 The latter contributes to endothelial dysfunction, which in turn leads to inadequate hyperemic MBF response predisposing for myocardial ischemia.10 Remodeling of intramural coronary arterioles increases coronary vascular resistance, and it seems to affect the entirety of the myocardium, even remote regions that are not hypertrophied.19 Increased loading conditions and wall stress in these left ventricles with severely impaired diastolic dysfunction as well as the increased metabolic demand of the hypertrophied myocardium further contribute to the presence and extent of CMD, particularly in the subendocardium.9 Several studies have shown that MBF is more blunted in the subendocardium and in the more hypertrophied areas, but without always sparing of the non-hypertrophied segments.19 , at CMR) has been shown to be present without CMD in some patients.21 In the majority of patients, myocardial segments without fibrosis have normal global MFR without regional heterogeneity of flow, whereas in the presence of LGE, the majority of symptomatic HCM patients exhibit impaired global MFR with significantly lower hyperemic MBF within fibrotic versus non-fibrotic segments.22The prevalence of CAD is no different in patients with versus without HCM.23 Genetical factors might also play a role, in view of the results from a 13N-ammonia PET and CMR study showing significantly lower peak MBF in patients with sarcomere myofilament mutations compared to matched genotype-negative patients.24Similar to the clinical nature of the disease and the phenotypical heterogeneity  a substantial heterogeneity in MFR reduction and CMD is observed in HCM patients, potentially owning also to the time-point of the diagnosis and the time of assessment of the microvasculature. While some patients present with normal MFR, others exhibit severely reduced MFR (consistent with significant CMD).25 Studies assessing the coronary microsiculation in patients with LVOT obstruction have shown contradictory results. A 13N-ammonia PET study comparing 11 non-obstructive patients, 12 obstructive patients, and 10 with latent-obstruction (defined as a provoked-LVOT gradient of > 30 mmHg), found no significant correlation between LVOT gradients and global parameters of ischemia or hyperemic MBF and MFR on PET, when comparing symptomatic patients with obstructive versus non-obstructive HCM.23 Other groups using 15O-water PET showed an inverse correlation between LVOT gradients and hyperemic MBF.26 These discrepant findings could potentially be explained by the labile nature of LVOT obstruction in HCM; a highly variable echocardiographic finding heavily dependent on a myriad of factors, including volume status, autonomic nervous system, physical position, and others.7HCM patients with obstructive physiology and elevated left ventricular outflow tract (LVOT) gradients appear to have a worse prognosis compared to those with non-obstructive disease.23 Petersen et al. demonstrated that hyperemic MBF decreased by .011 mL⋅min−1⋅g−1 for each millimeter increase of end-diastolic wall thickness.27 Similarly, pathology studies in HCM patients have demonstrated that the percentage of luminal narrowing of intramural coronary small arteries inversely correlated with heart weight and myocyte size and also that there was an inverse relationship between normalized coronary arteriolar lumen and degree of LV hypertrophy.28 CMR studies in HCM patients, utilizing cardiac diffusion tensor imaging for in vivo characterization of myocardial microstructure, demonstrated that even in myocardial segments with normal wall thickness, normal perfusion, and without scar, diffusion is more isotropic than in controls, suggesting the presence of underlying cardiomyocyte disarray.29 In the same population, hyperemic MBF and MFR were reduced, particularly within the subendocardium, not only in hypertrophied and scarred segments but also in segments with normal wall thickness. Transmural perfusion gradient (TPG) is a relative novel quantitative PET metric defined as the ratio of endocardial-to-epicardial MBF. In hypertrophic left ventricles, TPG is decreased during vasodilator-induced hyperemia, and in HCM patients, a reduction of TPG under stress indicates that the impairment of MBF affects primarily the endocardium.31 In HCM, impaired TPG is related to pathological LVEF stress response, potentially suggesting that the stress-induced subendocardial ischemia may also cause transient LV dysfunction.32It has further been shown that myocardial wall thickness is the strongest predictor for reduced global hyperemic MBF and MFR, while MBF decreased in proportion to the increase in end-diastolic wall thickness.33 The benefit of implementing an assessment of the microvasculature in the individual management of HCM patients remains to be elucidated by future studies.Being the most frequently encountered form of pathological LVH, with an estimated prevalence of 1 out of every 200 adults (0.5%), HCM represents a very heterogeneous population whereby CMD is present in several stages of the disease with important clinical and prognostic implications.34 The estimated prevalence of AFD in patients with unexplained LVH in adults is around .5% to 1%.37 Cardiac deposition of globotriaosylceramide in myocytes, vascular endothelium, and smooth muscle cells leads to myocardial ischemia, myocardial wall thickening, and progressively to interstitial replacement fibrosis.40 These alterations increase coronary vascular resistance and myocardial oxygen demand, which along with direct endothelial cell dysfunction due to globotriaosylceramide storage, nitric oxide pathway dysregulation, and microvascular remodeling are considered to mediate CMD in patients with AFD.42 In fact, CMD is considered to play an important role in AFD patients, in some representing the first manifestation of cardiac involvement, in others it is responsible for anginal symptoms.44  is caused by mutations in the gene encoding α-galactosidase A, resulting in inability to break down glycosphingolipids, which in turn leads to accumulation in multiple organs with subsequent renal, cardiac, and cerebrovascular injury.45 Compared to healthy controls, AFD patients seem to have reduced MFR and hyperemic MBF, irrespective of LVH or gender.42 Although AFD is an X-linked disease with males primarily being affected, most heterozygous females also express clinical manifestations, including cardiac involvement, but usually at a later age.47 CMD might be the earliest manifestation of cardiac involvement, seen even prior to any signs of LVH.42 Similarly, reduced hyperemic MBF is inversely related to the degree of LV wall thickness and to age in males, potentially corroborating the hypothesis that CMD precedes the development of LVH. Multiparametric CMR imaging has been used to describe the several stages of AFD, including the initial globotriaosylceramide storage phase characterized by low native T1 mapping values, and followed by the LVH stage with focal and global inflammation leading to fibrosis.51 At CMR, a reduction in hyperemic MBF has also been shown to be present early in the disease course and was demonstrated to be related with the severity of LVH, as well with regional inflammation and fibrosis.52 A CMR study in 114 AFD patients revealed that compared to matched, healthy controls, the patients had significantly lower MBF even prior to the occurrence of LVH and reduction of native T1 mapping values.53 Supporting the findings from PET studies, CMD was shown to be present not only prior to hypertrophic phenotype but is also preceding the storage phase of cardiac involvement.CMD in AFD has been investigated primarily in PET studies.54 This concept was assessed in PET-based studies with discrepant results, potentially owing to sample sizes too small to assess any ERT response effect.55 Technological advancements and increased availability of imaging modalities could allow for better evaluation of the effect of therapeutic options on the microvascular function and the potential long-term benefit.Assessment of the microvasculature function could have implications for the management of AFD patients: Early recognition of cardiac involvement could potentially prompt early initiation of disease-specific treatment that includes either intravenous enzyme replacement therapy  or oral chaperon therapy , or even cardiac supportive therapies.56 Primarily affecting the lungs and intrathoracic lymph nodes, cardiac involvement is present in a considerable amount of sarcoidosis patients, ranging from 5% to 10% of individuals with systemic manifestations, albeit the exact prevalence is most probably underestimated.58 Myocardial involvement in cardiac sarcoidosis (CS), phenotypically described as a chameleon-disease, is usually focal or scattered and commonly affects the free lateral LV wall or the basal intraventricular septum, that appears thinned, while in some cases it can result in LVH mimicking HCM.61 Atrioventricular conductance disturbances, ventricular arrhythmias, and progressive heart failure are the most common clinical manifestations and CS patients may present with angina symptoms even in the absence of significant epicardial CAD. The latter has been attributed to CMD, although this hypothesis up till recently was based on qualitative assessment of myocardial perfusion (with single-photon emission computed tomography) and not on quantification of MBF.65Sarcoidosis is a systemic inflammatory disease characterized by the formation of non-caseating granulomas upon an unknown trigger in genetically predisposed individuals.13N-ammonia and 18F-fluorodeoxyglucose (FDG) PET study has provided insights on the relationship between cardiac inflammation and MBF in CS patients.66 Sarcoid-mediated inflammation was shown to be associated with regional impairment of MFR. Although resting MBF were found to be within normal limits, irrespective of the presence of inflammation or not, hyperemic MBF and MFR were significantly lower in inflamed myocardium compared to non-inflamed regions. In a small portion of the study participants, assessment of treatment-response on the microcirculation was also evaluated, whereby those that did not respond to immunosuppressive treatment  had a further decline in hyperemic MBF and MFR. In contrast, response to steroid therapy was associated with preserved coronary circulatory function.67A retrospective, combined 68 Work utilizing contrast-TTE reported that in the absence of traditional risk factors for CAD, sarcoidosis patients without known cardiac involvement had a lower MFR as compared to healthy controls.69 The mechanism of CMD in CS patients is undoubtedly multifactorial. An early systematic inflammatory state affects the microvascular endothelium. It is considered to result in decreased nitric oxide production, which in turn impairs vasodilation response, particularly in actively inflamed myocardial regions.71 In advanced stages, a more global and diffuse impairment of MBF might also be present and potentially precedes the phase of structural myocardial changes, including fibrosis and scarring that leads to overt heart failure.66 Whether CMD in CS is a by-stander and to what extent it confers prognostic or therapeutic implications remains to be elucidated.Active inflammation in CS patients has also been associated with enhanced coronary vasoconstrictive reactivity at the epicardial and microvascular level, while medical treatment  was shown to reduce epicardial spasm effectively.72 Cardiac amyloidosis (CA) is caused by cardiac amyloid fibril deposition and the main forms clinically encountered (termed after the precursor protein of the amyloid deposit) include light-chain amyloidosis (AL) or transthyretin-types amyloidosis .74 Amyloid can be deposited in almost all cardiac structures from pericardium, epi-/endocardium to cardiac valves, myocardium and cardiac vasculature. Both left and right ventricular myocardium can be infiltrated, resulting in biventricular hypertrophy  that progresses to a restrictive form of cardiomyopathy.74 Deposition of amyloid leads to structural alterations predominantly within the subendocardial and midwall regions ultimately resulting in replacement fibrosis.75 Pathological hypertrophy of the ventricles may impede subendocardial perfusion due to capillary rarefaction and compression.76 Although significant luminal obstruction of the epicardial coronary arteries rarely occurs, direct vascular deposition of fibrils can further precipitate microvascular obstruction. Functional abnormalities, including autonomic dysfunction and endothelial dysfunction, that characterize amyloidosis are also considered to play a significant role in CMD in CA patients.Amyloidosis is a collection of systemic disorders characterized by the extracellular deposition of insoluble fibrils composed of misfolded proteins in multiple organs.80 Although most studies have focused on the non-bioptic identification of cardiac amyloid depositions, a few smaller studies have also assessed the microvasculature in CA using all available imaging modalities.85 In patients with both AL-CA and ATTR-CA and in the absence of epicardial CAD that were assessed with 13N-ammonia PET, rest MBF, hyperemic MBF and MFR were significantly lower when compared to a matched hypertensive patients’ group with LVH.76 , defined as the blood circulation time from the orifice of the coronary arteries to the pooling in the coronary sinus. This surrogate marker of CMD was significantly prolonged in CA patients compared with the HCM and the healthy controls.87 Real-time contrast TTE with flash bubble destruction has also been used for quantification of MBF in AL CA patients, whereby significant reduction of hyperemic MBF was observed.85 Even in patients with rare renal amyloid A (AA) amyloidosis, MFR was significantly lower at contrast-TTE compared to a group of individuals with non-amyloid chronic inflammatory disease and healthy controls.81 As cardiac involvement in AA amyloidosis usually occurs at later stages of the disease, assessment of microvascular function could potentially enable earlier identification of patients at risk.The last decade has heralded a paradigm shift in the field of CA, given the advent of disease-modifying therapeutic options (primarily for ATTR) such as stabilizing molecules (tafamidis) and genetic silencers (patisiran and inotersen) which have been shown to improve quality of life.Current knowledge indicates a close association and interplay between the coronary microvasculature and pathologic myocardial hypertrophy while the prevalence of patients with a hypertrophic phenotype is increasing in clinical practice. Multimodality imaging approaches for non-invasive quantification of MBF provide a distinctive opportunity to evaluate the function of the coronary microcirculation. Technical refinements and wider availability of said diagnostic tools hold the promise to allow for a better understanding of the pathophysiological mechanism and the related clinical and prognostic implications. Finally, integration of patient- and disease status-specific functional assessment of the microvasculature in the diagnostic approach may pave the way for better risk-stratification and patient-tailored therapeutic options.Supplementary file1 (PPTX 905 kb)Below is the link to the electronic supplementary material.


Functional motor skills are crucial to keep a good quality of life in older adults. While motor learning/relearning may be relatively preserved with aging, it typically requires repetitive practice, which can be challenging. This challenge becomes even more important when considering rehabilitation protocols for older individuals recovering from stroke, falls, or hip surgery, as they often experience physical fatigue. Therefore, there is a need to promote reliable interventions, simple to implement, and less physically demanding, with the aim of preserving or even improving motor functions in the elderly population.Among different options, there has been a growing interest in motor imagery (MI) over the past two decades. MI refers to the mental simulation of a motor action without actual physical output. It is often performed from a first-person perspective, where individuals mentally perceive  what they would normally perceive during the actual execution of the movement. Numerous investigations have provided evidence that MI positively influences motor learning and recovery across various populations. While inter-individual variability is high, overall MI ability remains preserved until around 80 years, given intact working memory . In factRegardless of the studies on MI in the elderly, it has been shown that anodal transcranial direct current stimulation (a-tDCS), involving the application of a weak current (1–2 mA) between an anode and a cathode placed on the scalp, may enhance MSL in older adults. For example, the combination of physical practice with a-tDCS applied to the primary motor cortex over five consecutive days of training resulted in greater online effects (compared to sham stimulation) on implicit MSL, in the second, third and fourth days . Also, tWe thus conducted a randomized controlled study aimed to investigate the cumulative effects of three consecutive daily sessions of MI in combination with either a-tDCS (active group) or sham tDCS (sham group) over the primary motor cortex, on learning a SFTT, in healthy older adults . Both grTo sum up, we showed that three consecutive daily sessions of MI led to performance improvements in a SFTT, primarily driven by online effects observed during the first two training sessions. Additionally, we observed a stabilization of performance between training sessions (offline consolidation) and long-term retention one week after training. This work emphasizes the importance of repeatedly practicing MI to maximize its effect in older adults. However, as already demonstrated in young adults, we recently confirmed that combining MI to physical practice led to similar outcomes than physical practice alone in the elderly (unpublished data). If an older individual is capable of making a movement but finds it taxing, the combination of physical and MI practice appears the best way to use MI. Finally, how combining anodal tDCS with MI in the elderly, including questions about where, how, and when stimulation should be applied, is one of the next challenges in this field of research.


Cell-to-cell communication between the immune system and tumors is of the utmost importance; it influences the development of tumors, their growth, and how they respond to treatments. In this article, we provide an overview of why understanding the interactions between immune and tumor cells is so significant for developing anti-cancer therapeutics, particularly cancer immunotherapy. We delve into the methods and tools used to decipher these interactions and discuss the potential impact on the future of cancer treatment. Moreover, we emphasize the power of unraveling these interactions in advancing cancer immunotherapy. We also explore the challenges that can be tackled by gaining insights into these interactions.The tumor and tumor microenvironment (TME) consist of a complex network of cells, including malignant, immune, fibroblast, and vascular cells, which communicate with each other. Disruptions in cell–cell communication within the TME, caused by a multitude of extrinsic and intrinsic factors, can contribute to tumorigenesis, hinder the host immune system, and enable tumor evasion. Understanding and addressing intercellular miscommunications in the TME are vital for combating these processes. The effectiveness of immunotherapy and the heterogeneous response observed among patients can be attributed to the intricate cellular communication between immune cells and cancer cells. To unravel these interactions, various experimental, statistical, and computational techniques have been developed. These include ligand–receptor analysis, intercellular proximity labeling approaches, and imaging-based methods, which provide insights into the distorted cell–cell interactions within the TME. By characterizing these interactions, we can enhance the design of cancer immunotherapy strategies. In this review, we present recent advancements in the field of mapping intercellular communication, with a particular focus on immune–tumor cellular interactions. By modeling these interactions, we can identify critical factors and develop strategies to improve immunotherapy response and overcome treatment resistance. Intercellular interactions play crucial roles in organism function and development. Cells can interact with each other either directly  or indirectly . These interactions are the basic building blocks of physiological communication and are essential for tissue formation, immune response, homeostasis, and regeneration. In direct cellular communications, contact between cell surfaces can occur via gap junctions, cell adhesion, tunnel nanotubes, and ligand receptor signaling. When cells interact indirectly, cellular information is shared through signaling from extracellular vesicles, cytokines, chemokines, growth factors, metabolites, and exosomes. These intercellular interacting mechanisms contribute to tissue development and physiological functions . A healthy immune system can precisely identify and eliminate precancerous cells, and it eliminates them before they cause any harm, by a process referred to as tumor immune surveillance. Numerous extrinsic and intrinsic factors impair immune–precancerous cell interactions, contributing to tumorigenesis. Once developed, tumor cells evade and disrupt the host immune system, leading to an immune-suppressive tumor microenvironment (TME). The TME is a complex ecosystem comprising tumor cells, immune cells, fibroblasts, extracellular matrices, and signaling molecules. The interaction between the immune cells and cancer cells within the TME evolves and can result in either pro- or anti-tumorigeneses . RestoriCancer immunotherapy began in the 1980s with an interferon-α2 inhibitor as the first immunotherapeutic agent approved by the FDA in 1986 . Since t+ T cell abundance is often associated with favorable overall survival, whereas increased regulatory T cells are associated with poor overall survival [+ abundance is not always associated with a better prognosis, as the spatial cellular assemblies are also crucial [Studies over the years have showcased the cell types present in the TMEs associated with positive and negative outcomes of immunotherapy. High CD8survival ,7. HowevMethods that can characterize the deformed intercellular interactions and identify the cell (sub-) populations that are involved in the interactions are extremely critical for prognosis. Identifying which cell population at which cellular state is associated with positive or negative outcomes in immunotherapy is extremely valuable to patient stratification, prognostic biomarkers, and resistance mechanisms. This review aims to explore the diverse manifestations of cell–cell interactions in the context of cancer, providing an overview of quantitative approaches to assess these interactions. Moreover, it seeks to investigate the potential of leveraging the influence of cell–cell interactions to enhance the efficacy of cancer immunotherapies.The immune system consists of two compartments: innate cells  and adaptive cells (B cells and T cells). Innate cells rapidly respond to foreign pathogens, presenting antigens to adaptive cells to initiate specific immunological responses. In the context of cancer, antigen-presenting cells detect tumor antigens and present them to naïve lymphocytes. This communication primes and activates lymphocytes, which then migrate to the tumor site. The activated T cells recognize and eliminate tumor cells. The innate and adaptive immune cells collaborate in a process called cancer immunoediting to eliminate tumors. However, if this process is unsuccessful or suppressed, the tumor microenvironment forms. The progression of the TME towards malignancy can be understood within the framework of cancer immunoediting. Cancer immunoediting refers to the dynamic interplay between the host immune system and the tumor cells, whereby immune mechanisms either restrain or promote tumor development. This process can be divided into three distinct phases: elimination, equilibrium, and escape .During the elimination phase, innate and adaptive immune responses collaborate to recognize and eliminate malignant cells. The innate immune system, including dendritic cells and antigen-presenting cells, primes and activates T cells by presenting tumor antigens. These activated T cells are then mobilized to directly interact with cancer cells, leading to their destruction . HoweverTumors that successfully evade elimination enter the equilibrium phase, during which they remain dormant but develop resistance mechanisms against immune surveillance . This reEffective immunotherapy can revert tumors to the elimination phase, where the suppressive mechanisms are counteracted, leading to the elimination of the tumor . HoweverUnderstanding the intricate dynamics of cancer immunoediting and intercellular interactions between tumor and immune cells is crucial for the development of effective therapeutic approaches aimed at restoring immune control over tumors and achieving durable clinical responses.Almost all types of immunotherapies involve direct cell–cell interactions for anti-tumor effect. One of the key cell–cell interactions during immunotherapy is the interaction between T cells and tumor cells. T cells can recognize and target tumor cells through the recognition of specific antigens presented by the tumor cells. However, tumors can evade T cell recognition by downregulating antigen presentation or by producing immune-suppressive molecules. Cell–cell interactions are the pharmacological basis of immunotherapy . The mosAnother mechanism in which immune cells can have a cytotoxic effect on cancer cells is the process of antibody-dependent cellular cytotoxicity (ADCC) ,16. In AAnother therapeutic approach that utilizes antibodies is bispecific T cell engagers (BiTEs) . BiTEs aChimeric antigen receptor (CAR) cell therapies represent a novel immunotherapeutic approach that signifies a significant advancement in personalized cancer treatment . This ap+ T cells to the tumor site. This process results in the reduction in regulatory T cells (Tregs) and facilitates an effective immune response against the cancer cells [Oncolytic virus therapy holds promise as an immunotherapy approach that involves T cell activation and cell–cell interactions. This therapy utilizes either genetically engineered or naturally occurring viruses that can selectively replicate within cancer cells and kill them while sparing non-cancerous cells ,21. Uponer cells ,21.In summary, intercellular interactions between effector cells (such as cytotoxic T lymphocytes and NK cells) and tumor cells have emerged as crucial steps for the efficacy of immunotherapies. Therapeutic approaches such as BiTEs, CAR cell therapies, and oncolytic virus therapies exploit these interactions to enhance the immune response against cancer cells, and they hold promise for the improvement of cancer treatment outcomes.The intricate interplay between immune cells and structural components within the TME significantly influences patient outcomes, therapeutic response, and disease progression . While eTo investigate and elucidate the intricate cellular interactions within the TME, numerous methodologies and systems have been developed . These aThe study of cell–cell interactions employs various in vitro experimental systems, including two-dimensional (2D) cell culture and three-dimensional (3D) methods such as spheroids and organoids, as well as tissue samples . TraditiNevertheless, in vitro systems lack the host tissue contexture and immune system, which are critical components for studying cell–cell interactions. Cell–cell interactions rely heavily on the tissue contexture, and techniques that support in vivo investigations have the potential to unveil novel modes of cell–cell interactions and their impact on tumor response to therapies. In vivo systems with an intact host immune system, such as syngeneic mouse models, are useful for studying the TME since the host immune system can interact with the TME ,29. HoweFurthermore, tumor xenograft models using cell lines often undergo substantial genetic changes, fail to recapitulate the natural tumor structure, and may lead to mouse-specific tumor evolution . While mIntercellular proximity labeling approaches have revolutionized the study of cell–cell interactions by providing spatially resolved information. These methods involve the tagging or labeling of proteins or other molecules that are in proximity to a specific cell type or surface marker  Table 2Table 2. Unlike the IHC or fluorescent staining approaches, proximity labeling techniques can go beyond a time-frozen snapshot, helping to identify and track cells and providing a more dynamic approach to cell–cell interactions. This becomes important when studying where immune cells go after interacting with cancer cells and how that cellular movement affects the composition of the TME. To ensure the applicability of proximity labeling approaches in both in vivo and in vitro environments, it is essential that these methods are non-disruptive and non-toxic to cells. This consideration ensures that the labeled cells maintain their physiological properties and behave naturally during the experimental process. By utilizing labeling techniques that are minimally invasive and compatible with live cell imaging, researchers can gain a comprehensive understanding of cell–cell interactions in the TME.Two prominent intercellular proximity labeling methods, EXCELL and LIPSTIC, employ the Staphylococcus aureus enzyme Sortase A (SrtA) to measure cell–cell interactions. LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts) enables the identification of ligand–receptor interactions between immune cells and their target cells ,35. CellGFP-based Touching Nexus or G-baToN harnesses the trogocytosis communication of cells to transfer GFP from a donor cell to an acceptor cell . HoweverPafA or bait [A subsequent method that is able to detect membrane proteins through proximity labeling is population-based interaction tagging (PUP-IT) . In this or bait . PUP-IT  or bait . However or bait . This hi+ T cell was used to target a specific antigen (NY-ESO-1) that can mediate tumor size in melanoma patients. The 2CT method was used to control the selection pressure and killing effects shown by the T cell as well as to modulate the effector to target ratio. Furthermore, the 2CT method was used in combination with a CRISPR-Cas9 library that held over 100,000 single-guide RNAs which impaired effector function in T cells. The 2CT method allowed for the analysis of genes necessary for immunotherapy, specifically those that target effector T cell function. The 2CT method has exciting translation and clinical opportunities to uncover genes in immunotherapy-resistant patients [2CT-CRISPR assay is a novel and interesting approach for identifying genes that are essential for effector T cell function in tumors. In the 2CT-CRISPR assay, human T cells were represented as effectors, and melanoma cells were represented as targets . The purpatients . Other proximity-based methods for studying cell–cell interactions include TRACC  and SynNotch-activated MRI (magnetic resonance imaging), both of which exploit specific receptor–ligand interactions between two interacting cells to facilitate labeling and detection ,43. TRACMost proximity-based methods are dependent on cell–cell contact and interaction. Cherry-niche, caged luciferins, and CLIP (cre-induced intercellular labeling protein) are three approaches that do not solely rely on direct cell–cell contact for labeling ,42,44. IIn summary, intercellular proximity labeling or imaging approaches offer significant potential for elucidating the intricate cellular interactions and communication networks within the TME. These methods have been successfully employed in various research areas, including the investigation of tumor metastasis ,41, T ceEnzyme-based intercellular proximity labeling approaches are predominantly employed in experimental systems. However, with the growing availability of large clinical datasets, bioinformatic methods have gained significance in the study of cell–cell interactions and the identification of novel interactions. In clinical settings, bioinformatics methods play a crucial role in inferring intercellular interactions or communication by examining the coordinated expression patterns of ligand–receptor pairs’ cognate genes. Ligand–receptor analysis has emerged as a valuable approach for investigating intercellular communication, particularly in the context of cancer immunotherapy. This approach enables the identification of the specific ligand–receptor pairs involved in immune cell interactions with cancer cells or the TME . This approach proves to be especially valuable in deducing intercellular interactions that are not solely reliant on cell-to-cell contact. This is evident when immune cells and cancer cells release diverse cytokines, chemokines, and growth factors that govern immune reactions and inflammation. These signaling molecules and their corresponding receptors may be modulated based on environmental cues. Through these bioinformatics methods, we can deduce the likelihood of intercellular interactions based on their ligand–receptor profiles; this is potentially pivotal in forecasting patient prognosis and treatment outcomes.The analysis of ligand–receptor interactions primarily relies on single-cell RNA sequencing (scRNA-seq) or bulk RNA-seq data. The procedure typically involves the following steps:Data preprocessing: This step involves normalizing, quality controlling, and filtering of gene expression data to ensure data integrity and reliability.Gene set selection: A specific set of ligand and receptor genes is chosen based on prior knowledge or by utilizing databases such as CellPhoneDB or Interactome INSIDER.Calculation of ligand–receptor expression: The expression levels of ligand and receptor genes are calculated for each cell or cell type within the dataset, regardless of whether the data are scRNA-seq or bulk RNA-seq. Ligand–receptor interaction analysis: Interactions between ligands and receptors are predicted by assessing the co-expression patterns of ligand and receptor genes across different cells or cell types. Several methods, including CellPhoneDB, scRNA-seq-based ligand–receptor pair analysis (sLRPA), and LigandNet, are available for performing this analysis. Visualization and interpretation: The results of the analysis are visualized using heatmaps, networks, or other visualization techniques. These results can be interpreted to identify the specific ligand–receptor pairs that may be involved in intercellular communication and to gain insights into the underlying biological processes.Bioinformatic methods offer valuable tools for inferring intercellular interactions and communication based on transcriptome data. These approaches provide valuable insights into the intricate network of cell–cell interactions in the context of cancer immunotherapy. Notably, ligand–receptor analysis holds promise for the identification of predictive biomarkers for immunotherapy response and the monitoring of treatment efficacy.Nevertheless, it is important to acknowledge the limitations of these bioinformatic methods in inferring intercellular interactions. Firstly, their predictions solely rely on gene expression data and overlook additional factors like post-translational modifications or protein localization that can influence interactions. Secondly, these methods hinge upon existing knowledge of ligand–receptor pairs, which may be incomplete or imprecise. Thirdly, the biological relevance of the predictions is not guaranteed, necessitating experimental validation. Lastly, technical artifacts such as batch effects, sequencing depth, and normalization methods can influence the accuracy and reproducibility of the results. Considering these limitations, it is crucial to exercise caution and combine bioinformatic predictions with experimental validation to ensure the reliability and significance of the findings. Continued advancements in bioinformatic techniques and complementary experimental approaches will enhance our understanding of intercellular interactions and their role in cancer immunotherapy. A wide array of scientific questions concerning cancer immunotherapy could be answered by utilizing these cell-cell interactions techniques. These questions include:What are the subpopulations of immune cells interacting with the tumor cell during immunotherapy?What are the molecular features of these interacting immune cells, and how are the molecular features related in response to immunotherapy? Do the effector cells that have interacted with the tumor cells migrate across tumor metastatic lesions? Ultimately, the answers to these questions can uncover the pharmacological actions of cancer immunotherapy and reveal the underlying molecular mechanism of resistance. The TME represents an intricate network comprising diverse cell types engaged in communication; it plays a pivotal role in shaping the tumor landscape. Effective communication between immune cells and cancer cells holds great significance in the determination of the patients’ responses to immunotherapy, and it contributes to treatment resistance and interpatient variability in the responses. The investigation of cell–cell communication in immune cells under normal and pathological conditions provides crucial insights into the mechanisms of cancer immunotherapy, patient responses, disease progression, and TME status. Various experimental and computational approaches exist for elucidating pathological intercellular interactions, both directly and indirectly, with the aim of identifying the communicating cell populations. Comprehensive understanding, modeling, and the discovery of cell–cell interactions within the TME hold immense potential for the identification of the critical factors and strategies influencing immunotherapy response, treatment resistance, and TME status.In recent times, advances in imaging, microscopy, cellular engineering, and bioinformatics have emerged as powerful tools for the unraveling of novel mechanisms and cellular relationships, thereby paving the way for improved immunotherapy options for patients. By leveraging these methodologies synergistically, it becomes possible to bridge existing knowledge gaps and gain a comprehensive understanding of treatment resistance and to design more potent cancer immunotherapies.


Vesicle trafficking is an essential cellular process upon which many physiological processes of eukaryotic cells rely. It is usually the ‘language’ of communication among the components of the endomembrane system within a cell, between cells and between a cell and its external environment. Generally, cells have the potential to internalize membrane-bound vesicles from external sources by endocytosis. Plants constantly interact with both mutualistic and pathogenic microbes. A large part of this interaction involves the exchange of transport vesicles between the plant cells and the microbes. Usually, in a pathogenic interaction, the pathogen releases vesicles containing bioactive molecules that can modulate the host immunity when absorbed by the host cells. In response to this attack, the host cells similarly mobilize some vesicles containing pathogenesis-related compounds to the pathogen infection site to destroy the pathogen, prevent it from penetrating the host cell or annul its influence. In fact, vesicle trafficking is involved in nearly all the strategies of phytopathogen attack subsequent plant immune responses. However, this field of plant-pathogen interaction is still at its infancy when narrowed down to plant-fungal pathogen interaction in relation to exchange of transport vesicles. Herein, we summarized some recent and novel findings unveiling the involvement of transport vesicles as a crosstalk in plant-fungal phytopathogen interaction, discussed their significance and identified some knowledge gaps to direct future research in the field. The roles of vesicles trafficking in the development of both organisms are also established. Rhizobia is achieved through symbiosome as an interfacial surface, surrounded by peribacteriod membrane (PBM). Periarbuscular membrane (PAM) however forms the interfacial surface for interaction with mutualistic fungi   and effector-triggered immunity (ETI). In PTI, when a pathogen comes in contact with the host plant, a given molecular signature on the pathogen known as pathogen associated molecular pattern (PAMP) is identified by the host plasma membrane-localized receptor proteins called pattern recognition receptors (PRRs) via interaction between the two , Golgi complex, trans-Golgi network (TGN), endosomes and vacuoles. Effective communication between these organelles and with the plasma membrane (PM) is very necessary for the continuity of cellular activities in both healthy and diseased conditions, and this is achieved through vesicle trafficking. This trafficking occurs for biosynthetic, recycling, degradative and/or defense purposes. It is central in maintaining physiologic homeostasis, signaling, growth and development of eukaryotic cells. It is also very important for the continuity of secretory and endocytic pathways. In plant cells, there are different vesicles trafficking routes including ER-TGN, ER-vacuole, TGN-endosomes, endosomes-vacuoles, TGN-PM, and many more. When a plant cell recognizes the presence of a microbial species  around it, it reprograms its molecule homeostasis and endomembrane system in preparation for that encounter. This brings about changes in molecular architecture of the cell, including changes in lipid composition and the symmetry of the plasma membrane. Intracellular vesicle trafficking is also reshaped for appropriate responses. In fact, the entire process of host cell reprogramming heavily depends on vesicle trafficking. A number of previous studies demonstrated the significance of vesicle trafficking in plants during plant–microbe interaction. However, the link between vesicle trafficking in plants and that in phytopathogens during plant-pathogen interaction is not clearly documented. In the present review, we bring together some important findings to demonstrate how phytopathogens make use of their endogenous transport vesicles to promote virulence and manipulate host immunity and how the host plants use same to counteract, disarm and incapacitate the invading pathogens.Transport vesicles are membrane-bound, small sphere-like intracellular structures containing some selected cargo molecules to be delivered to a particular destination within or outside a eukaryotic cell for a given purpose. Such vesicles could be as small as 40 nm and as large as 10 µm in diameter  of wheat and some cereals, further suggests a link between vesicle trafficking and virulence/development of phytopathogens , the SNARE proteins VdSso1 and VdSec22 regulate vesicle-mediated cargo delivery at the target membrane  production -mediated vesicle trafficking  product was also shown to localize to the EHM; some host proteins are however selectively excluded on this defense barrier ( Inada et al. M. oryzae infection sites, suggesting a role in blast resistance (Sugano et al. Oomycetes and fungal phytopathogens form haustoria through which they release effector substances to subvert the plant immune responses whereas bacterial pathogens use their secretion systems to release the virulence factors Fig. . To restPlants interact with different microorganisms and the interactions can be mutualistic or pathogenic. This review focuses on the exchange of cargo-containing vesicles between plants and phytopathogenic fungi. Findings from previous studies clearly demonstrated that both plants and their invading fungal pathogens utilize vesicle trafficking for different purposes. Both organisms need vesicle trafficking for their normal physiologic activities. Additionally, the pathogenesis of fungal pathogens largely depends on the delivery of pathogenesis-related molecules packed in membrane-bound vesicles. Most of these pathogens lose their pathogenicity when their vesicle trafficking pathways are perturbed. In other words, the pathogens package virulence factors in transport vesicles and release them to the hosts for successful colonization. Similarly, hosts also use their vesicle trafficking pathways to defend themselves against the pathogen attacks. They do not only neutralize the virulence substances from the pathogens but also block the pathogens’ vesicle trafficking pathways. However, a number of knowledge gaps in our understanding of the use of transport vesicles in plant-fungal pathogen interactions still exists. What are the factors that determine cargo selection for a kind of vesicle? What determines the specific pathway for MVB transportation? Can EVs be released for plants/pathogenic fungi detection or are they only released post detection? These and many related questions await future investigations. Understanding these will provide new horizon in plant-fungus warfare and plant disease management.