Corneal opacification is the fourth most common cause of blindness globally behind cataracts, glaucoma, and age-related macular degeneration. The standard treatment of serious corneal scarring is corneal transplantation. Though it is effective for restoring vision, the treatment outcome is not optimal, due to limitations such as long-term graft survival, lifelong use of immunosuppressants, and a loss of corneal strength. Regulation of corneal stromal wound healing, along with inhibition or downregulation of corneal scarring is a promising approach to prevent corneal opacification. Pharmacological approaches have been suggested, however these are fraught with side effects. Tissue healing is an intricate process that involves cell death, proliferation, differentiation, and remodeling of the extracellular matrix. Current research on stromal wound healing is focused on corneal characteristics such as the immune response, angiogenesis, and cell signaling. Indeed, promising new technologies with the potential to modulate wound healing are under development. In this review, we provide an overview of cell-free strategies and some approaches under development that have the potential to control stromal fibrosis and scarring, especially in the context of early intervention. The cornea is the clear window of the eyes. The rays of light pass through the cornea before reaching the retina. Its main functions include 1) mechanical protection of inner eye tissues from physical, chemical, and UV damages, 2) an optically clear structure for the undisturbed passage of light, 3) a highly refractive medium (providing two-thirds of the eye’s refractive power) to converge the incident light rays through the pupil and focus on the fovea, and 4) an immune barrier  from external agents. Anatomically, the cornea has three cellular layers  in a sandwich with two acellular layers: the Bowman’s layer and Descemet’s membrane. The corneal stroma forms about 90% of the overall corneal thickness, giving the strength and structural integrity of the cornea. The cornea’s transparency is maintained by the regular alignment of collagen fibers and the relative dehydration status inside the stroma. Besides, corneal transparency is associated with corneal avascularity and the self-renewal of corneal epithelium.Corneal transparency is critical for optimal vision. Damage to the cornea by trauma or infection can cause irreversible reduction of transparency, leading to distorted vision and eventually blindness. Corneal blindness is a leading cause of vision loss, with 4.2 million people worldwide affected by corneal opacities . Affected individuals lose their independence and have a poor quality of life. Treatment for corneal injury is determined by several factors that include etiology, location, and severity of the injury. First-line treatments for corneal abrasions are antibiotics and lubricants, while mild to moderate pain can be controlled with nonsteroidal anti-inflammatory drugs. When corneal pathologies cause severe and irreversible scarring, a corneal transplant often is the best solution. In a global survey, more than 180,000 cases of penetrating keratoplasty (PK) were conducted in 116 countries and 284,000 corneas were procured in 2012 . An AustAfter more than a century of full-thickness corneal transplantation on patients (the first surgery by Dr. Eduard Zirm in 1905 ), the clhttps://clinicaltrials.gov/ct2/show/NCT02948023). Another clinical trial showed that the stromal implantation of autologous adipose-derived mesenchymal stem cells improved the pachymetric and visual parameters in patients with advanced keratoconus  presents a potentially new approach to treat corneal stromal disorders . An inteatoconus . Differeatoconus .In this review, we provide an overview of conventional cell-free medications and new strategies that can be developed to control corneal fibrosis and scarring. Some of these could be applied as an early intervention to stimulate native tissue regeneration or work in combination with other treatments to enhance vision recovery.The corneal stroma is composed of specialized extracellular matrix (ECM) components and collagen fibrils. These fibrils are mainly type I and V collagens and are organized in parallel bundles that are aligned into layers, or lamellae. About 300 to 500 collagen lamellae are positioned in perpendicular orientations to comprise the stroma, establishing the mechanical strength and optical properties of the cornea . KeratocFibrosis occurs when myofibroblasts appear and produce excessive and disorganized ECM, which interferes or blocks the light passage. Transient haze from corneal abrasion or ultraviolet (UV)-mediated riboflavin crosslinking can be reversed by the regeneration of damaged epithelium and basement membrane, which usually occurs within days . The heaPathological conditions in the cornea also affect innervation. Corneal nerves produce neurotrophic factors such as substance P, calcitonin gene-related peptide, epidermal growth factor, nerve growth factor, brain-derived neurotrophic factor, and neurotrophins , that maTopical corticosteroids, such as prednisone acetate ophthalmic eye drops, minimize fibrosis through its downregulating TGFβ-mediated inflammatory and fibrotic events . The scaMMC is a classical DNA damaging agent, that forms covalent linkage with DNA, inhibiting DNA replication, transcription, and protein synthesis, causing irreversible senescence of cells . For yeaTGFβ isoforms (β1 and β2) regulate fibrosis and scarring through the differential activation of canonical TGFβ/Smad signaling . Ligand Vitamin C (sodium ascorbate) has been known to facilitate collagen synthesis and deposition by keratocytes and to produce parallel arrays of ECM fibrils . Its antThe cornea is an ideal tissue for gene and protein therapies, as it is easily accessible by topical application or injection via subconjunctival, intracameral, or intrastromal routes. Regarding particular mechanisms of action, protein-based pharmaceuticals can address corneal surface integrity, tear composition, wound healing, inflammation, haze and scarring, nerve regeneration, and neovascularization. The topical administration of drug usually has a lower risk of systemic side effects due to the limited absorption into the bloodstream, as opposed to intravenous or intramuscular injections. In a rabbit keratectomy model, topical application of recombinant human bone morphogenetic protein (BMP)-7 suppressed TGFβ-related fibrosis, as observed by the reduced differentiation of αSMA-positive myofibroblasts . As a meIn a murine model of microbial keratitis, topical decorin reduced corneal opacities, with the downregulation of αSMA, fibronectin, and laminin, and the treatment promoted the healing of damaged epithelium . DecorinImmunoglobulin CD147, also known as ECM-MMP inducer (EMMPRIN) or basigin, promotes TGFβ-mediated myofibroblast differentiation . This moBlocking myofibroblast proliferation could be a potential therapeutic strategy to prevent corneal fibrosis. Intermediate-conductance calmodulin/calcium-activated potassium channel 3.1 (KCa3.1) regulates cell cycle progression and proliferation. Its expression has been reported in the development of fibrosis of the cornea and different organs . TreatmeEarly re-routing of the scar-forming process could be beneficial for scar-free healing and should occur a short time after injury, ideally before the scarring process initiates. The process of wound healing can be described as changes of the microenvironment that involve a multitude of dynamic and interactive molecular and phenotypic events initiated after injury . This prIn vitro stromal keratocyte culture studies have shown that the human amnion matrix and its soluble protein extract reduced TGFβ1 and β2-related signaling  is an anti-fibrotic molecule that reduces fibrosis in various organs . In a muhttps://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019169380).MicroRNA therapeutics is becoming an important field in medical research, due to the pleiotropic nature of microRNAs, which make them particularly attractive as candidate targets for diseases with a multifactorial origin and no effective treatment. A growing number of reports support the use of miRNAs as biomarkers for diagnosis, and drug intervention . MicroRNin vitro and in vivo models. Target gene search for each miRNA is available using online databases, such as TargetScan (http://www.targetscan.org/vert_72/) and miRDB (http://mirdb.org/). Since each platform uses proprietary algorithms in calculating the binding score to a target sequence, the use of more than 1 platform can increase the confidence in hits identified independently while compensating for the limitations of each approach. The top list of genes shared in the output from both platforms can then be selected for enriched pathway analyses. Since one or several miRNAs could mediate gene regulatory effects, the gene lists from different miRNA candidates should be combined and imported for enriched gene ontology terms and pathway analysis using tools, such as DAVID  (https://david.ncifcrf.gov/), KEGG (Kyoto Encyclopedia of Genes and Genomes) (https://www.genome.jp/kegg/pathway.html) and IMPALA  . After the removal of non-coding genes, the remaining gene candidates will be examined for over-representation in specific signaling pathways, biological processes, and phenotypes.We anticipate that not every miRNA identified in CSSC-EV will influence fibrosis and scar reduction. Some miRNAs can be redundant, while miRNAs with relevant activity may work in groups or combinations. This makes the identification of specific miRNAs difficult and complicated, as well as costly in setting up experiments for validation. Bioinformatics approaches, such as target gene search and enriched pathway prediction, should be employed to identify single or groups of miRNAs that regulate inflammation, fibrosis, and/or immune responses to subsequently validate using Due to the vast number of miRNAs upregulated in CSSC EV that reduce corneal scarring  and trigger an innate immune response . TLR sigTet1 and Tet2 are Oct4-regulated enzymes that sustain 5hmC in mouse embryonic stem cells (ESC) and are induced concomitantly with 5hmC during the reprogramming of fibroblasts to induced pluripotent stem cells. ESC depleted of Tet1 by RNAi treatment had reduced expression of Nodal antagonist Lefty1, and displayed hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro  to 5-hydroxymethylcytosine in DNA. in vitro . Their ein vitro . The prein vitro . Human CMany reports have illustrated the changes or dysregulation of miRNAs in different types of disorders, including cancers, tissue injuries, and age-related diseases . There hThough miRNAs have features that can be developed for drug design and efficacy, there are challenges that need to be overcome before considering this strategy for clinical uses. A major obstacle is that miRNAs exhibit pleiotropy in multiple senses (one miRNA can regulate multiple genes and a given gene can be regulated by more than one miRNA), making their therapeutic use potentially very complex . ComplexThe current standard for treating severe and irreversible corneal scarring in the visual axis often entails a corneal transplant, which carries risks of rejection, infection, and graft failure. Complications also include prolonged rehabilitation, lifelong use of immunosuppressants, and loss of corneal strength, with a considerable human and financial cost. In addition, the worldwide shortage of donor materials has greatly hampered the applicability of corneal transplantations, in particular in under-developed and developing countries. If the density of corneal opacities can be reduced, then other non-surgical therapies (such as the use of contact lenses) can correct corneal astigmatism and provide visual rehabilitation. This will lessen the demand for transplantable donor grafts, which can be reserved for treating serious and dense scars. Early intervention would thus be a valuable approach to delay visual deterioration, and reduce complications, ideally decreasing the need for corneal transplantation. A desirable approach to delay scarring would be to stabilize the wound soon after injury and redirect the healing process to reduce inflammation and fibrosis. The use of regenerative cytokines as a first-line treatment would elevate the expression of anti-fibrotic cytokines and suppress cytokines, reducing tissue inflammation and fibrosis, hence potentially stimulating regeneration of clear stroma. If successful, this would also improve the effectiveness of subsequent treatments to recover visual acuity. On the other hand, different microRNAs with anti-fibrotic activity have been identified, however, their use to reverse or reduce tissue fibrosis is still under investigation. Their specific actions and potential side-effects or contraindications need to be clarified before any of these molecules can be developed for miRNA therapeutics. Besides, these miRNAs can be potentially applied for cell product screening. In cell-based therapy, the cell stocks for clinical application must be stringently selected and quality controlled. Only cells that are carefully screened for correct phenotypes and functions can be released for patient uses. If a panel of CSSC-EV-derived miRNAs with anti-inflammatory and anti-fibrotic properties were characterized, they could be used to define the release criteria for therapeutic CSSC. Their detection in the secretome will have minimal influence on the cell manufacturing process and does not require extra cells for the assays. This will assist medical professionals to justify the use of high-quality cell products for patients.


Mapping the neuronal response during cognitive processing is of crucial importance to gain new insights into human brain function. BOLD imaging and ASL are established MRI methods in this endeavor. Recently, the novel approach of functional PET (fPET) was introduced, enabling absolute quantification of glucose metabolism at rest and during task execution in a single measurement. Here, we report test-retest reliability of fPET in direct comparison to BOLD imaging and ASL. Twenty healthy subjects underwent two PET/MRI measurements, providing estimates of glucose metabolism, cerebral blood flow (CBF) and blood oxygenation. A cognitive task was employed with different levels of difficulty requiring visual-motor coordination. Task-specific neuronal activation was robustly detected with all three imaging approaches. The highest reliability was obtained for glucose metabolism at rest. Although this dropped during task performance it was still comparable to that of CBF. In contrast, BOLD imaging yielded high performance only for qualitative spatial overlap of task effects but not for quantitative comparison. Hence, the combined assessment of fPET and ASL offers reliable and simultaneous absolute quantification of glucose metabolism and CBF at rest and task. Human brain function has been subject to research for centuries and is yet not fully understood due to its vast complexity. Thus, characterizing the neuronal response during cognitive processing is of pivotal importance and can be achieved with various approaches. and consequently, only an indirect, non-specific proxy for neuronal activation. Other drawbacks are the instability of the BOLD signal for longer task durations, spurious effects such as scanner drifts and heating, field inhomogeneity, heart rate and respiratory influences, or draining veins. These are non-trivial issues and to a certain extent reasons that absolute quantification of BOLD signal changes require substantial effort.The most common imaging method to investigate task-induced changes in the brain is functional magnetic resonance imaging (fMRI) based on the blood oxygen level-dependent (BOLD) signal. Advantages are high sensitivity to changes in blood oxygenation, readily accessible MRI sequences, established tasks and high temporal and spatial resolution. However, the BOLD signal is a composite of changes in cerebral blood flow (CBF), cerebral blood volume (CBV) and blood oxygenation8 This absolute quantification represents a major advantage of ASL, facilitating a comparison between rest and task-specific flow changes. However, the requirement to acquire images in pairs reduces the temporal resolution and also the spatial resolution is inferior compared to BOLD imaging.Another fMRI-based approach is arterial spin labeling (ASL) which enables quantification of CBF. Here, arterial blood is magnetically labeled to use it as endogenous contrast agent. Perfusion-weighted images are then computed from a labeled and an unlabeled image, enabling estimation of CBF.18F]FDG is a suitable tool to map neuronal activation. Indeed, task-specific changes in glucose metabolism were already assessed before BOLD imaging and ASL were introduced. A crucial drawback of this method is that measurements have to be conducted in at least two separate sessions, mostly carried out even on separate days, one at rest and another one during task execution. Changes in daily performance or resting activity between these measurements will influence the results and subjects are exposed to ionizing radiation twice. Recently, these issues were resolved with the novel approach of functional PET (fPET). The method enables the quantification of resting and task-specific glucose metabolism within a single measurement.13 The protocol was further optimized by the administration of [18F]FDG as bolus plus constant infusion. This increases the signal-to-noise ratio and the amount of freely available radiotracer to track even subtle task-related changes with a temporal resolution of minutes (instead of hours or days as previously required). Disadvantages include the necessity for arterial cannulation to enable absolute quantification and the radiation exposure of participants.Task-induced changes are also reflected in altered glucose metabolism. Hence, positron emission tomography (PET) using the glucose analogue FDG was administered as bolus followed by constant infusion while a complex cognitive paradigm was employed. After 8 minutes of rest, 4 task conditions were carried out with varying difficulty . Each task was followed by a rest condition (5 min) where subjects were instructed to look at a black crosshair on grey background and to let their thoughts wander. Simultaneously with the task blocks, ASL was acquired during one easy and one hard condition. During the remaining task blocks, BOLD imaging was acquired for functional connectivity as described elsewhere. Immediately after fPET and ASL acquisitions, the same task was carried out in a conventional block design and BOLD data was acquired , again separated by rest blocks (10 s each). Although the duration and number of task blocks differed across imaging modalities, we aimed to employ an optimal acquisition protocol for each parameter. The entire study design is depicted in Forty healthy subjects  underwent a single PET/MRI scan on a fully-integrated PET/MRI system . To assess the test-retest reliability of functional imaging parameters, a subgroup of 20 subjects  also underwent a second measurement (4.2 ± 0.7 weeks apart). The scans of the remaining 20 subjects  solely served for an independent region of interest definition. Measurements were initiated with the acquisition of a structural T1-weighted image followed by an ASL sequence during rest. Subsequently, FDG was freshly synthesized every morning using FASTlab FDG cassettes with phosphate buffer formulation and a FASTlab platform . The substance was administered via a cubital vein as bolus for 1 minute followed by constant infusion for 51 minutes with an infusion pump , which was placed in an MR-shield. PET data was acquired in list-mode, enabling the retrospective definition of frame lengths during reconstruction.The radioactive glucose analogue deoxyglucose measurements within the same day but also with [18F]FDG and intervals of one to twelve weeks between measurements. Similar reliability was achieved in this study with excellent ICC and small CoV. In a comprehensive evaluation of CBF, test-retest reliability was compared within session and 1 hour and 1 week apart. As expected, the CoV increased with the time between measurements, which could be based on technical challenges and varying resting activity. Although our measurements were separated by 4 weeks, we could achieve similar CoV. With the caveat of longer measurement time, Ki exhibited a higher reliability than CBF in terms of CoV and ICC, suggesting fPET as a robust quantitative tool to map resting activity. Other than Ki and CBF, estimation of BOLD changes during rest is theoretically possible by calibrating the signal but is usually not performed due to technical challenges. Consequently, a comparison to Ki and CBF was omitted in this work.Measurements at resting state reflect resting activity, which plays a key role in research and clinical applications, for example, to investigate altered metabolism or blood flow in patients. A high test-retest reliability of glucose metabolism at rest was already demonstrated decades ago with 2-FDG. Astrocytes metabolize glucose to supply neurons with energy in form of lactate. Also, in neurons themselves, glucose is transformed into ATP for action potentials and synaptic transmission. The radiotracer is irreversibly bound in cells and thus, task-specific increase in energy consumption is proportional to [18F]FDG uptake. Of note, also glucose metabolism influences the vasodilation by certain neurovascular signal pathways, including lactate. This complex neurophysiological interplay between the presented imaging approaches suggests to consider them as complementary and not as substitution of one another.In summary, the test-retest reliability during task performance varied across the presented imaging approaches. A possible explanation is that each modality reflects different factors that are coupled to neuronal activation to varying degrees. While a close relationship was observed between metabolic processes and CBF, a mismatch occurs upon task performance between oxygen supply and utilization.Another, more technical, reason for differences in test-retest variability might arise from different measurement durations between the three modalities. Acquisition times for ASL and BOLD imaging were shorter than for fPET. It is known that the temporal signal-to-noise ratio (tSNR) increases with the square root of the measurement time, which may also result in higher reliability. Hence, extending the measurement time of BOLD imaging and ASL to that of fPET (52 min) would theoretically increase the tSNR by approximately 150% and 70%, respectively (see Supplementary figure S1). Of note, this does not imply an increase in test-retest reliability by the same amount, because external influences such as physiological effects are not considered. Thus, it is likely that the test-retest reliability of task-based fMRI reaches a plateau similar to that of resting-state fMRI.Currently, BOLD imaging is often the first choice to map task changes on the whole-brain level, although several pitfalls are known. As mentioned, the BOLD effect is rather unspecific due to its signal complexity which is often compensated for by employing a control condition. However, finding a suitable control condition is challenging as it bears the risk to remove potential active regions after contrasting the conditions and has the drawback of introducing additional variance as also shown in this work.39 Given the current improvement of PET scanners with higher sensitivity, temporal resolutions of up to 100 milliseconds and advanced reconstruction algorithms, event-related designs also seem to be highly feasible with fPET in the near future.Based on our results and aforementioned difficulties with BOLD imaging, we propose to consider fPET and ASL as a robust combined tool to tackle novel research questions using fully-integrated PET/MRI systems. This enables the simultaneous acquisition of two complementary aspects of neuronal activation. Different to BOLD imaging, fPET and ASL directly reflect glucose metabolism and CBF, respectively, making them less dependent on control conditions to achieve higher specificity. The advantage of absolute quantification further enables longitudinal comparisons during task performance and at rest. However, the acquisition requires an adaption of already established paradigms. While fPET requires longer tasks, which have to be stable for several minutes, BOLD imaging employs shorter paradigms with several repetitions. Furthermore, BOLD changes can resolve event-related task effects due to the higher temporal resolution. Differently, fPET and ASL are currently limited to block designs, although real-time and event-related designs were proposed for ASL within a specific technical setup. or a hierarchical design. For the latter, a fast on/off design is embedded in longer blocks, enabling a simultaneous acquisition of fPET and BOLD signal with respect to the paradigm requirements. Utilizing a dual-echo ASL sequence would further allow the acquisition of all three modalities at the same time with such a design. Still, this would imply tradeoffs mostly at the cost of the BOLD signal as ASL employs longer TR. Although a hierarchical design enables a direct comparison between the modalities, the short rest periods within a block might influence the fPET signal.In cases where the simultaneous acquisition of fPET and BOLD is favorable, we suggest short task blocks18F]FDG PET brain imaging suggest the acquisition of a 15-30 min static image. This may also include stimulation paradigms outside the scanner to identify brain areas involved in specific task performance e.g., before surgery. However, this omits metabolic dynamics and therefore potentially misses important functional alterations connected to a disease. With fPET, resting and task-specific glucose metabolism can be robustly determined within a single 30 min measurement, enabling an optimized therapy planning , but also potentially allowing for an enhanced monitoring of progression in neurodegenerative diseases.The assessment of task-specific glucose metabolism may also prove feasible in clinical routine. The current European Association of Nuclear Medicine (EANM) procedure guidelines for [44 Another drawback is the radiation exposure of participants (here approx. 6.4 mSv for a subject with 75 kg body weight). However, the development of PET systems with high sensitivity41 will allow further reduction of the radiation burden up to an order of magnitude. Reliability obtained with a complex visuo-spatial motor task with different task loads might be more susceptible to individual daily performance than simple tasks such as checkerboard stimulation or finger tapping. However, mapping the neuronal response to complex behavioral processes requires performance of similarly challenging tasks, which makes knowledge about the corresponding reliability an essential aspect. Another limitation is the slight improvement in task performance between the two measurements. This may be ascribed to an improved control to align the bricks, which may have resulted in altered task-specific activation in the second measurement. To diminish this effect, the measurements were separated by 4 weeks and the ICC3,1 was used describing consistency rather than absolute agreement. Furthermore, no changes in imaging parameters or correlations with task performance were observed. This indicates that the reported test-retest reliability was not affected by the change in task performance. Lastly, task-specific test-retest metrics were directly compared between modalities, although data acquisition varied across modalities in duration and the number of task blocks. Strictly speaking, only Ki and CBF were acquired simultaneously and BOLD changes immediately afterwards within the same session. These differences emerge from the different requirements of imaging modalities. fPET and ASL perform best with relatively long task blocks, whereas the BOLD signal is unstable for long continuous task durations. To meet these requirements, we employed a study design that is technically most feasible but also allows to optimize the task-specific acquisition independently for each imaging modality. Our results therefore provide test-retest data similar to commonly employed designs and it is likely that longer measurement times would increase the test-retest reliability of BOLD imaging and ASL because of a higher tSNR. However, this has to be evaluated systematically with the acquisition of more task blocks and longer measurement durations to identify a plateau of reliability.Although high test-retest reliability of fPET was demonstrated, we have to note a few limitations. fPET currently requires arterial cannulation for absolute quantification of glucose metabolism but there is great effort to substitute the AIF with an image-derived input function which will broaden the applicability of the approach.In conclusion, matching task-specific neuronal activations were robustly detected with fPET, ASL and BOLD imaging in both task conditions. Despite its recent introduction, similar or higher reliability was achieved for fPET in comparison to the optimized and well-established modalities of ASL and BOLD imaging, albeit with a longer measurement time. Indeed, BOLD changes exhibited a substantially lower reliability, especially when using a control condition. Nevertheless, BOLD effects showed the highest qualitative overlap between the measurements, suggesting to use BOLD imaging preferably for the spatial assessment of task changes. On the other hand, the combination of fPET and ASL offers a robust tool, enabling the simultaneous absolute quantification of glucose metabolism and blood flow during rest and task performance. Considering that fPET will benefit from numerous further advancements such as progressive modeling strategies, more sensitive scanners and increased temporal resolution, an even higher reliability of this technique is to be expected in the near future. This enables the application of fPET not only in research, but also in clinical routine.Click here for additional data file.Supplemental material, sj-pdf-1-jcb-10.1177_0271678X211020589 for Reliability of task-specific neuronal activation assessed with functional PET, ASL and BOLD imaging by Lucas Rischka, Godber M Godbersen, Verena Pichler, Paul Michenthaler, Sebastian Klug, Manfred Klöbl, Vera Ritter, Wolfgang Wadsak, Marcus Hacker, Siegfried Kasper, Rupert Lanzenberger and Andreas Hahn in Journal of Cerebral Blood Flow & Metabolism


In neonates, a variety of postnatal diseases affect organ perfusion and oxygenation during the first weeks of life. During this critical period, preterm infants are highly susceptible to impaired organ blood flow and oxygen delivery, potentially resulting in end-organ injury. In addition, infants with intrauterine growth restriction, perinatal hypoxia, congenital heart disease, or requiring cardiothoracic interventions are similarly at risk for cerebral and/or peripheral organ hypoxic-ischemic injury. Such perturbations during the earliest stages of life are associated with significant neonatal mortality risks and an elevated likelihood of neurodevelopmental impairment among survivors .2, and lactate, aid neonatologists in assessment of systemic hemodynamics in these infants. However, these indirect monitoring techniques provide information generated after organ injury has already begun, at which point reactive therapeutic maneuvers may be conducted , and pulse oximetry, are monitored to guide the clinical management of sick neonates. In addition, biochemical measurements, including pH, pOThe ability to assess real-time individual tissue oxygenation, perfusion, and oxygen extraction may help clinicians recognize infants at risk for hypoxic-ischemic organ injury. Indeed, such monitoring may result in earlier management of varied neonatal disease states, including hypoxic-ischemic encephalopathy, clinically significant anemia, and necrotizing enterocolitis, among others. Furthermore, tissue oxygenation monitoring could also provide information concerning regulation of tissue perfusion and/or tissue metabolic demand, thus providing enhanced insight into the pathogenesis of various neonatal disease states.Over the last decades, there has been increasing interest in various non-invasive modalities to assess macro- and micro-perfusion among hemodynamically unstable newborn infants . Near-inWe present a series of articles on this topic concerning the use of NIRS and non-invasive cardiac output measurements to assess organ perfusion and oxygenation in the sick infant.Pazandak et al., who demonstrated in neonates with hypoxic-ischemic encephalopathy that BP-based requirements for dopamine and severity of brain injury were not associated. The authors suggest that BP measures alone provide an inadequate assessment of cerebral perfusion.Ongoing research into neonatal cerebral oxygenation, either as a factor preceding the development of intraventricular hemorrhage or neurodevelopmental impairment, or as an outcome measure of various clinical parameters, has resulted in several papers. The need for cerebral measurements is supported by Kalteren et al. provided a systematic review of the literature which revealed that both anemia and red blood cell (RBC) transfusions are associated with cerebral oxygenation changes, brain injury, and neurodevelopmental impairment in preterm infants. The authors suggest to further investigate the role of cerebral oxygen saturation as a potential contributor to anemia-related neurodevelopmental outcomes in the individualized treatment of neonatal anemia.Pritišanac et al. concluded from their literature review that, although from limited data, it seems that lowering the fraction of fetal Hb decreases peripheral muscle oxygen extraction, suggesting improvement in oxygen availability after administering adult packed red blood cells.Mattersberger et al. studied the literature on glucose and lactate levels in relation to cerebral oxygen saturation and confirmed a negative correlation between blood glucose level and cerebral oxygenation but found only one study with a negative correlation between blood lactate and cerebral oxygen saturation.Besides hemoglobin, other factors may affect cerebral oxygenation in infants. Wolfsberger et al. demonstrated that fetal inflammation defined as elevated umbilical cord blood interleukin-6 values was associated with higher cerebral oxygen extraction shortly after birth. The simultaneous lower SpO2 and heart rate suggested potentially compromised cerebral oxygen delivery in cases of fetal inflammation.In a small cohort of preterm infants, Cimatti et al. demonstrated that changes in cerebral oxygen saturation, possibly resulting from impaired cerebrovascular autoregulation during the transitional period, preceded the development of IVH in preterm infants. This finding supports previously published reports on the relation between impaired autoregulation measured using NIRS and cerebral hemorrhage in preterm infants . The authors speculate that pulmonary venous return is reduced using the lateral approach, resulting in reduced cardiac output with subsequent cerebral perfusion pressures below the lower autoregulatory limit. infants , 5. CereMichel-Macías et al. noticed from two cases and associated literature review that a drop in somatic NIRS values may precede clinical signs of hypoperfusion. They suggest that adding somatic NIRS monitoring to the assessment of cerebral oxygenation after ductal ligation may enhance early identification of post-ligation cardiac syndrome and guide interventions at earlier stages. Further prospective investigation is required to explore this further.Dix et al. demonstrated that fetal growth restricted (FGR) infants are less likely to demonstrate cerebral vasoconstrictive responses following noxious stimuli, possibly as a result from brain-sparing mechanisms. FGR infants appeared to have lower stroke volume and cerebral oxygenation during the second and third postnatal weeks. Richter et al. found that brain-sparing was associated with higher cerebral oxygenation after birth and enhanced behavior and executive function at 2 years of age. However, brain-sparing was negatively associated with performance IQ. Combining both observational studies by Dix and Richter, we speculate that cardiac function affected by FGR overrules postnatal brain-sparing with increasing postnatal age  in sick neonates. A review of the literature indeed suggests significant promise for renal oxygenation trending to prevent or decrease severity of AKI.Terstappen et al. analyzed multi-site oxygenation data in a subgroup of infants born in the Dutch Strider Trial, which investigated the effect of prenatal sildenafil in FGR. The data suggested that prenatal sildenafil lowered renal but not cerebral oxygenation during the first 72 postnatal hours. The observed changes in renal oxygenation could reflect a vasoconstrictive rebound from sildenafil, as the authors hypothesized. Similar changes observed in accompanying vital parameters supported this hypothesis.Alternatively, the assessment of both cerebral and renal oxygenation allows one to measure systemic hemodynamic effects of interventions. Dotinga et al. concluded from the literature. They found that the preterm intestine possesses a decreased “reserve” in cases of impaired intestinal oxygen delivery, as intestinal oxygen extraction may already be exhausted under stable conditions. A better understanding of the balance of intestinal oxygen supply and demand, perhaps assessed using NIRS, may help in guiding clinical management to prevent intestinal tissue hypoxia, as is suggested by the authors.NIRS could also aid in detecting attenuated intestinal oxygenation in preterm infants, as O'Neill et al., the authors evaluated the current utility of NICOM. They conclude that regardless of structural differences with echocardiographic derived cardiac output (CO), this approach is increasingly being used in various research settings. Its non-invasive nature informs the clinician continuously on CO trends, and could potentially impacts management and patient outcomes. However, before implementation in clinical practice, the effect of its use on clinical outcomes needs to be further addressed in future research.Another tool to assess neonatal circulation is non-invasive cardiac output monitoring (NICOM). In the review by This series of cohort observations and literature reviews on cerebral and somatic oxygenation and perfusion assessment using NIRS and NICOM adds to the increasing evidence and knowledge base on the value of these tools to augment current neonatal hemodynamic monitoring practices. Moreover, this series contributes to our understanding of the role of hypoxia-ischemia in several neonatal diseases. A major advantage of NIRS and NICOM are their non-invasive nature allowing for the continuous assessment of trends in neonatal hemodynamics. Lack of gold standards for end-organ perfusion and oxygenation assessment, however, hampers validation of both tools and the absolute values these devices present need to be interpreted with caution. However, the clinical application of the more reliable trend-observation in end-organ perfusion and oxygenation, in addition to conventional hemodynamic monitoring, will hopefully result in improved care for the sick newborn. Further investigation is clearly warranted.EK, AB, and JM edited the papers discussed in this editorial. EK wrote the first draft of the editorial. AB and JM edited this version into the current version. All authors approved submission of this paper.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.


Similar to environmental factors, EDCs  can influence gene expression without modifying the DNA sequence. It is commonly accepted that the transgenerational inheritance of parentally acquired traits is conveyed by epigenetic alterations also known as “epimutations”. DNA methylation, acetylation, histone modification, RNA-mediated effects and extracellular vesicle effects are the mechanisms that have been described so far to be responsible for these epimutations. They may lead to the transgenerational inheritance of diverse phenotypes in the progeny when they occur in the germ cells of an affected individual. While EDC-induced health effects have dramatically increased over the past decade, limited effects on sperm epigenetics have been described. However, there has been a gain of interest in this issue in recent years. The gametes (sperm and oocyte) represent targets for EDCs and thus a route for environmentally induced changes over several generations. This review aims at providing an overview of the epigenetic mechanisms that might be implicated in this transgenerational inheritance. EDCs are environmental stressors able to activate or block hormone receptors. Their consequences can be deleterious, leading to cancer, malformations, or autistic disorders  pyrene (B[a]P) is one of the most important types of PAHs. PAHs are extensively metabolized by cytochrome P450 enzymes in humans and animals. The major metabolites of PAHs are monohydroxy-phenols (hydroxylated metabolites of polycyclic aromatic hydrocarbons (OH-PAHs)) and dihydrodiols, which are urinary biomarkers useful for the characterization of PAH exposure. Paternal preconceptional occupational exposure to PAHs was associated with increased risks of all childhood brain tumors . In testEpigenetic inheritance refers to the transmission of epigenetic marks to offspring . It is iEpigenetic information can be disrupted by environmental factors and could be inherited, leading to long-term adverse consequences affecting offspring health. For example, diet is an environmental factor modifying gene expression. Evidence of the epigenetic transgenerational inheritance of disease in humans is observed in Dutch and Swedish cohorts exposed to prenatal famine . ExposurIt is difficult for human studies to highlight a clear connection from exposure in an ancestor leading to molecular changes in germ cells, driving a specific phenotype in descendants. However, associations have been made between several aspects involving epidemiological, epigenetic, and genetic approaches . An examA transgenerational transmission of increased incidence of disease is described through the male germline after exposure with vinclozolin  or throuEDCs are an important part of our environment. Exposure to EDCs results in epigenetic modifications in cells. Germ cells are particularly vulnerable to EDC exposure. The epigenetic markers most studied are DNA methylation, the modification of histones, ncRNA and EVs .Sperm cells have long been considered as delivering the paternal haploid genome and then the genetic information to the oocyte. However, spermatozoa also harbor epigenetic information that plays a remarkable role during offspring early development and long-term health. These multiple epigenetic marks make up the epigenome . The bloThe DNA methylation profile of male germ cells is significantly different from that of somatic cells and is stable throughout spermatogenesis and in mature sperm. Germ cell DNA demethylation and remethylation in a sex-specific manner occurs during fetal development and is maintained throughout the individual’s entire life . EnvironIn adulthood, during spermiogenesis, chromatin is reorganized in the male gamete. Most of the histones are replaced by protamines, allowing supercoiling and chromatin compaction, but up to 10% of histones are preserved in the sperm . This prLarge (mRNAs and lncRNAs) and small (sncRNAs) sperm-borne RNAs are delivered to the oocyte during main fertilization. The sperm of transgenerational males that were ancestrally exposed to DDT have differentially expressed lncRNAs. Paternally acquired characteristics may be transmitted to the offspring via sperm RNAs in the first post-fertilization mitotic divisions of the zygote . miRNA aSpecific post-testicular sperm maturation occurs through the epididymal secretion of EVs . ReproduOocytes are subjected to prolonged periods of arrest. There are critical periods wherein the oocyte undergoes nuclear and cytoplasmic maturation, where the translation of stored mRNAs, accumulated during the growth period, is crucial for meiotic maturation and subsequent embryogenesis. The maintenance of protein homeostasis is needed to achieve successful fertilization despite proteostasis being “reset” during embryogenesis . EnvironIn utero exposure to a plastics mixture during the period of fetal gonadal sex determination promotes the epigenetic transgenerational inheritance of adult-onset disease resembling primary ovarian insufficiency and polycystic ovarian syndrome (PCOS) . PlasticVinclozolin-lineage granulosa cells displayed significant transgenerational alteration in both the transcriptome and epigenome, bringing up a new paradigm for the etiology of ovarian disease . MoreoveSignificantly, more DMRs are found when gestating female rats are exposed to vinclozin, in F3 compared with F1 sperm, knowing that these animals were exposed during a period of PGCs deprogramming and subsequent reprogramming. Methylation patterns between generations depend on the period during which the F1 animals are exposed .The transmission of environmentally induced epigenetic changes through the female germline appears to be stable, but a combination of both paternal and maternal alleles is needed to transmit testis disease to the male offspring . DespiteIn the oogenesis and early development of mammals, the functional activity of chromatin is regulated by the unique epigenetic landscape created by post-replication DNA modifications, post-translational modifications of DNA-associated proteins and ATP-dependent nucleosome remodeling. In the oocyte, chromatin undergoes chromosome condensation, withstands double-stranded breaks during meiotic recombination, and survives a long meiotic arrest in mammals . HoweverAmong lncRNAs, Xist (X-inactive specific transcript) acts in the silencing of the X chromosome by modifying the structure of the chromatin and the factors interacting in chromosome X of mammalian females during development. Dicer1 is an important RNase III enzyme that processes pre-miRNA into the shorter miRNA duplex. Conditional knockout of Dicer in mice increases the degeneration of follicles and decreases ovulation rates. miRNAs are involved in ovarian folliculogenesis and granulosa cell physiology . piRNAs Concerning EVs’ implication in oocyte maturation, EVs derived from plasma promote cumulus expansion and oocyte maturation by enhancing Has2 and Ptgs2 mRNA expression in the cumulus–oocyte complex .Blimp1, Prdm14, and Tcfap2c, Lin28 and the microRNA let-7 [Lin28/let-7/Blimp1 pathway in three successive generations of males mice [Exposure during pregnancy to environmental factors increases the risk for disease development later in life . TransgeNA let-7 . Blimp1 les mice . Followiles mice . These tles mice . This obles mice . Somaticles mice .Today, a growing number of studies indicate that environmental factors and particularly EDCs are transduced by both paternal and maternal gametes over the course of a pregnancy.


The role of the microbiome in immunology is a rapidly burgeoning topic of study. Given the increasing use of immune checkpoint inhibitor (ICI) therapy in cancers, along with the recognition that carcinogenesis has been linked to dysregulations of the immune system, much attention is now directed at potentiation of ICI efficacy, as well as minimizing the incidence of treatment-associated immune-related adverse events (irAEs). We provide an overview of the major research establishing links between the microbiome to tumorigenesis, chemotherapy and radiation potentiation, and ICI efficacy and irAE development. As indications for immune checkpoint inhibitors (ICI) for treatment of malignancies have broadened, outcomes remain variable and difficult to predict. Save for specific tumor traits such as genomic microsatellite instability (MSI-H), high programmed cell death ligand-1 (PD-L1) expression, or other genetically hypermutated (POLε) states, it appears that only a minority of patients respond to ICI therapy. Despite low response rates, patients whose cancers regress under ICI therapy may be more likely to enjoy longer progression-free survival. Unfortunately, this suboptimal chance of benefit is accompanied by increased risk of immune-related adverse effects (irAEs). These phenomena are associated with ICI activation of T cells throughout the body, potentially leading to a multitude of occasionally life-threatening autoinflammatory organopathies. In short, the current state of immunotherapy in oncology leaves much room for improvement.The microbiome has emerged as a possible therapeutic avenue as well as conduit for understanding mechanisms by which ICI effects are modulated: why do some patients develop crippling toxicities from treatment, while others virtually none? How can we explain the variance in outcomes between patients with similar cancers and tumor PD-L1 expression following treatment with the same drug? Is the oft-noted association between development of irAEs with response to ICI therapy in actuality describing two phenomena that can be decoupled?The microbiome has already been recognized for its significant role in human metabolism. Specific bacteria in the gut are responsible for digestion and metabolism in a symbiotic manner. There is increasing study of the microbiome’s role in gluconeogenesis and lipid metabolism ,2. The gOn a daily basis, our microbiome—and the metabolomic networks formed within it—are instrumental in mitigating the body’s inflammatory state ,9, but iIt has been long theorized that the microbiome plays a pivotal role in carcinogenesis, although the nature of its exact contribution is yet to be confirmed. Multiple viruses, including hepatitis B and C, HTLV-1, and HPV , have beFirmicutes has been associated with CRC [Bacteroides, Coprococcus, Corynebacterium, Enterococcus, and Neisseria enrichment with higher likelihood of CRC development, although the causative mechanisms remain unclear [The breadth of findings tying microbiome composition to carcinogenesis vary widely. They can be very narrow, attributing one or two bacterial genera to tumor development; other studies implicate alterations in the abundance of an entire phylum or even the overall number of detectable taxonomic species in a microbiome. Studies of colorectal cancer (CRC) patients have associated decreased α-diversity, or overall species enrichment, of the gut microbiome with tumorigenesis ,15. At twith CRC . More gr unclear ,17. It iActinobacteria in the gut microbiome of lung cancer patients [Proteobacteria and decrease in Firmicutes was identified [Fusobacteria and Bacteroidetes in the gut microbiota of lung cancer patients [In our and others’ research, lung cancer patients had different gut and respiratory microbiome compositions compared to their healthy controls ,19,20. Zpatients . In a mupatients , have bepatients ,22,23 buFusobacterium has been associated with development of CRC but has also been identified inhabiting hepatic metastatic lesions [Fusobacterium can facilitate tumor growth and metastasis, either by generating a tumor microenvironment tolerant of its development or by directly assisting with tumor cell adhesion [Multiple mechanisms have been posited to explain the significance of observed dysbiosis.  lesions ,24. Thisadhesion ,26.Proteobacterium Helicobacter pylori has been shown to lead to gastric activation of NOD1-like receptor, which in turn stimulates an IL-33 dependent response, leading to generation of inflammatory macrophages and T-cell phenotypes [Salmonella typhi and other Enterococcus genera have also been associated with development of biliary cancer [Significant preclinical evidence has identified bacteria-induced localized inflammation as a potential common thread. The colonization of the stomach with the enotypes ,28,29. Sy cancer ,31.G12DKras mutation and Tp53 deletion, leading to tumorigenesis. Local respiratory bacteria induced MYD88-dependent lung inflammation and led to activation of IL-17-producing T cells, facilitating cancer development. Notably, mice whose commensal respiratory bacterial loads were ablated exhibited slowed progression of disease. This further reinforces the theory that alterations in the local/respiratory microbiome influences the manner of carcinogenesis [A major 2019 study by Jin et al. described a novel mechanism in the development and growth of lung cancer. Intratracheal transfection of cre recombinase encoded adenovirus in mice caused activation of parenchymal cell ogenesis . Consistogenesis .H. pylori colonization in the mouse intestinal tract has been shown to increase production of c-myc, interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF), but reduced interferon-gamma (IFN-γ), with subsequent development of colorectal cancer [Bacteroides fragilis generates enterotoxins causing colitis and subsequent production of IL-17A via recruitment of Th17 and γδ T cells, among others [Multiple other studies corroborate adjustments in the tumor microenvironment to induce an inflammatory state. In addition to its effects in the gastric body, l cancer ,35. Bactg others . The infg others ,37.The contribution of the microbiome in generating or dampening inflammatory immune responses, predominantly via T cell population shifts, has drawn interest for both pathophysiologic and therapeutic reasons. The bedrock principle of ICI efficacy is to harness cytotoxic and effector T cells to generate an anti-tumor effect—it is this modulatory aspect of the microbiome, carcinogenic it may be, that may also provide a way to improve its treatment.Less nebulous than its role in carcinogenesis is the clear fact that specific members of the microbiome are responsible for metabolizing—thus potentiating or weakening—particular drugs or therapies. This is valuable for demonstrating how the microbiome can be used to affect the immune system, even if the intervention was not originally intended to induce an immune response.Mycoplasma hyorhinis—an abrogation repotentiated by depleting intratumoral bacteria [Parabacteroides disastonis in the gut can also decrease the efficacy of doxorubicin [An example of the direct activity bacteria can have on chemotherapy was demonstrated by Geller et al., showing the intracellular effects of gemcitabine can be abrogated by a deamination reaction initiated by bacteria . Similarorubicin ,40.The adverse effects of chemotherapies can also be mitigated by bacteria. A well-known adverse effect of irinotecan, mucositis, and diarrhea is one major example. The active metabolite of irinotecan, SN-38, is glucuronidated (inactivated) in the liver, and then excreted fecally. However, upon coming into contact with gut bacteria, the glucuronidate moiety is removed and the drug is reactivated in the gut, leading to enterospecific toxicities .Enterococcus hirae, Lactobacillus johnsonii, and Barnesiella intestihominis) in the mesenteric lymph nodes, with resultant stimulation of an effector immune response [Interestingly, the interactions between microbiota and medications are not always dependent on enzymes: effects can also be secondary to the intended therapeutic mechanism. In mice, cyclophosphamide was shown to induce interferon-γ and IL-17, a phenomenon occurring to a lesser degree in germ-free counterparts . Furtherresponse ,42 that Fusobacterium in their gut microbiota were found to be less likely to respond to 5-fluorouracil and oxaliplatin, the principal components of first-line therapy for metastatic colorectal cancer. This was theorized to occur by F. nucleatum-specific activation of TLR4 and MYD88 pathways, leading to tumor cell autophagy in response to chemotherapy rather than apoptosis [In addition to its previously mentioned contribution to colorectal cancer metastasis, colon cancer patients with higher abundances of poptosis ,44.Proteobacteria exhibited increased abundance, and Firmicutes decreased abundance, in mouse models that developed radiation proctitis. That inflammation was tied to postradiation dysbiosis, whose metabolic functions generated inflammatory cytokine IL-1β [Alterations in the microbiome have also been seen after treatment with radiation therapy. In a similar fashion to cyclophosphamide, researchers found that total body irradiation (TBI) was able to improve the antitumor efficacy of CD8+ T cells in mice by upregulating influx of circulating inflammatory cytokines via increased enteric lumen permeability . In anotne IL-1β .It is clear that the gut microbiome is instrumental in mediating the effects and toxicities associated with multiple treatment modalities, and thus begs the question of its contribution to the same in immune checkpoint blockade.To date, several studies regarding the gut microbiome and immunotherapy efficacy have been performed ,50,51,52At a superficial level, the microbiome has already been associated with immunotherapy efficacy ,54. HighBacteroides species were associated with poor clinical benefit; higher abundances of Faecalibacterium, Holdemania, and other Firmicutes bacteria in the gut were associated with higher odds of long-term benefit [Proteobacteria phylum [Immunotherapy efficacy has also been attributed to the relative enrichment (or lack thereof) of specific species in the gut microbiome. In melanoma patients, increased abundance of  benefit ,50,58. OFirmicutes and ICI response in our 16S evaluation of lung cancer patients [Akkermansia, a member of the phylum Verrucomicrobia, was enriched in NSCLC patients who responded to ICI. When broadening that cohort to include renal cell carcinoma patients—for which ICI typically comprises first-line therapy with good propensity for response—Akkermansia muciniphila was still associated with best response to therapy, as well as increased duration of response [Other studies have linked ICI outcomes in nonmelanomatous cancers to gut microbiome composition. We found a similar association between patients ; these fpatients . Routy eresponse . This isRuminococcaceae and Lachnospiraceae in patients with prolonged progression-free survival, and high levels of Bacteroides species in those with shorter progression-free survival [Firmicutes phylum bacteria in association with improved outcomes. The DELIVER trial evaluated differences in the gut microbiome of advanced gastric cancer patients treated with nivolumab immunotherapy. With extremely stringent (Bonferroni corrected) analysis, changes in Odoribacter (phylum Bacteroidetes) and Veillonella (phylum Firmicutes) were found to be associated with improved responses to immunotherapy [Firmicutes and Verrucomicrobia phyla almost universally had better response from ICIs, whereas those who were enriched in Proteobacteria universally presented with unfavorable outcome [More recently, the microbiome’s contribution to ICI efficacy in gastrointestinal cancers has come into focus. A study of 89 patients with advanced gastrointestinal cancer  treated with ICI whose fecal samples underwent 16S sequencing were found to have higher abundances of survival . These aotherapy . In conc outcome .Bifidobacterium has been demonstrated to be associated with increased likelihood of ICI response [Bifidobacterium breve genome (SVY) whose homology to an SIY model tumor antigen led to development of SVY-specific T cells. In vivo, these SVY cells targeted SIY-positive melanoma cells and subsequently prevented tumor growth [Bifidobacterium, into mice with faster-growing melanoma led to a similarly decreased rate of tumor growth and increased antigen-specific T cell tumor infiltration in the latter population [What could be the mechanism(s) linking the microbiome to immunotherapy response? Multiple possible mechanisms are outlined in response . A strikr growth . The trapulation .As mentioned in the microbiome and cancer section, antigenic homology is not the only way bacteria can elicit a T cell response. Bacterial toxins can also stimulate recruitment of T cells, which in turn release inflammatory cytokines such as IL-17 , and couBacteroidetes phylum is associated with poorer odds of ICI efficacy, but for reasons that remain unclear, two of its members, Bacteroidetes thetaiotaomicron and Bacteroides fragilis, mark possible significant exceptions to the rule [B. fragilis has been shown to produce polysaccharide A (PsA), which also stimulates regulatory T cell production [Another theory involves specific metabolic functions of the bacterial genera implicated. In general, the the rule ,58. Thesthe rule . B. fragoduction . This suBacteroidales and increase in small bowel concentrations of B. thetaiotaomicron. However, feeding germ-free mice mixtures of the same Bacteroides species reconstituted the anti-CTLA-4 effect, rendering the answer to this question murky at best [A common question is whether these differences in composition actually influence immunotherapy efficacy, as opposed to reflecting its byproduct. The latter possibility is suggested by the observation that injection of mice with anti-CTLA-4 therapy induces a fecal decrease in  at best ,67. ThisFinegoldia (of phylum Firmicutes) in nasal and Megasphaera  in buccal swabs correlated with better response in NSCLC patients treated with chemoimmunotherapy [As previously seen, much attention has been paid to changes in the gut microbiome, but given the finding by Jin et al. with respect to local bacterial factors in pulmonary carcinogenesis , it seemotherapy .One of the most complex issues facing the use of immunotherapy is that while ICIs are largely better tolerated than chemotherapy, they also carry the possibility of developing an immune-related adverse event, with clinical severities ranging from asymptomatic to fatal. As immunotherapy mobilizes immune cells across the body, the possible adverse effects a patient may develop can include colitis, dermatitis, nephritis, hepatitis, neuritis, pneumonitis, myocarditis, and even endocrinopathies . With exFirmicutes at onset of colitis, but enriched Firmicutes at baseline, whereas increased baseline Bacteroidetes was seen in patients who did not develop colitis [Proteobacteria are similarly associated with decreased likelihood of irAE development [The fecal microbiota of melanoma patients who developed colitis after being treated with ipilimumab, an anti-CTLA-4 immune checkpoint inhibitor, were found to have decreased abundance of  colitis . Our sys colitis . In addielopment .Bacteroidetes species, B. fragilis has been demonstrated to suppress proinflammatory cytokine production through secretion of polysaccharide A [Firmicutes bacteria are responsible for production of short-chain fatty acids (SCFAs), metabolites that exert regulatory effects on inflammation and T cell differentiation. While a recent clinical study demonstrated that elevated fecal SCFA concentration is significantly correlated with better clinical outcomes with anti-PD-1 treatment [Firmicutes with the development of irAEs, and a thorough understanding of their role in various immunologic scenarios are needed. Nevertheless, these observations beg the question whether the therapeutic response and irAEs from ICIs can be decoupled.These phenomena may be due to the metabolic products of these bacteria. When considering the negative association of irAEs with haride A . Firmicureatment , prior sreatment ,72. Thesreatment . Howeverreatment ,75. In areatment ,75. ThisB. fragilis and B. cepacia improved CTLA-4 ICI efficacy in germ-free animal models and simultaneously reduced the risk of developing ICI-induced colitis [Bifidobacterium has also been reported to hamper development of ICI-mediated colitis [Firmicutes and Bacteroidetes to enhance immunotherapy response yet mitigate irAEs were also found to have promising therapeutic implications [The 2015 Vetizou study found that repletion of  colitis . Bifidob colitis . Attemptications . All theFirmicutes bacteria with increased likelihood of ICI response and propensity for irAEs, and increased enrichment of Bacteroidetes and Proteobacteria in ICI nonresponders as well as those less likely to suffer irAEs.The increased interest in this field has uncovered a number of additional questions. Although overall trends in alpha-diversity with respect to progression-free and overall survival appear to be concordant, the specific genera implicated in each study are not always congruent. Further, the 16S and metagenomics tools/algorithms used to study these populations are not standardized. This disparity in cross-study comparisons has led to the concern that the lack of replicability may indicate the findings in these studies are more noise than signal. It is important to note, then, that the composition of the microbiome can vary widely from geographic environ to environ ,79. FurtBacteroidetes species in the gut microbiome of ICI responders bucks the trend exhibited by the rest of the phylum [It is also important to stress that a bacteria’s taxonomic branch does not necessarily dictate its function: the observed enrichment of specific e phylum . Thus, tThis review has outlined outcomes in studies involving skin, gastric, lung, kidney, and colon cancer patients. It can be argued that specific associations between microbiome composition and the metabolism of immunotherapy are likely to be independent of disease subtype, couched by the possibility there may be some cancers that will never respond to immunotherapy regardless of its potentiation.However, the implications of these microbiome studies are important on several levels. In the most rudimentary sense, the identification of a microbiome composition favorable to immunotherapy efficacy or tolerability could be leveraged simply by using fecal transplantation to achieve the desired makeup. Yet the possible benefits extend far beyond that: they beget a better understanding of the mechanisms, proteomic and epigenetic, that may be involved in modulating immunotherapy responses in vivo. This field remains in its infancy, but its value is beginning to make itself apparent in preclinical studies for other treatments. For example, mouse models treated with a glucuronidase inhibitor have been shown to dampen irinotecan enterotoxicity , providiThe true value of microbiome study likely lies in the use of pathway and network analysis to elucidate complex mechanisms leading to tumor growth and immunotherapy potentiation. If the microbiome truly represents an independent factor in immunotherapy outcomes, it would comprise another tumor-agnostic therapeutic avenue in an ever-expanding repertoire, a major development for oncology patients who have not yet benefited much from ICI use. This is why continued work in this vein remains vital to the whole of clinical oncology.


Current standard of care for glioblastoma is surgical resection followed by temozolomide chemotherapy and radiation. Recent studies have demonstrated that >95% extent of resection is associated with better outcomes, including prolonged progression-free and overall survival. The diffusely infiltrative pattern of growth in gliomas results in microscopic extension of tumor cells into surrounding brain parenchyma that makes complete resection unattainable. The historical goal of surgical management has therefore been maximal safe resection, traditionally guided by MRI and defined as removal of all contrast-enhancing tumor. Optimization of surgical resection has led to the concept of supramarginal resection, or removal beyond the contrast-enhancing region on MRI. This strategy of extending the cytoreductive goal targets a tumor region thought to be important in the recurrence or progression of disease as well as resistance to systemic and local treatment. This approach must be balanced against the risk of impacting eloquent regions of brain and causing permanent neurologic deficit, an important factor affecting overall survival. Over the years, fluorescent agents such as fluorescein sodium have been explored as a means of more reliably delineating the boundary between tumor core, tumor-infiltrated brain, and surrounding cortex. Here we examine the rationale behind extending resection into the infiltrative tumor margins, review the current literature surrounding the use of fluorescein in supramarginal resection of gliomas, discuss the experience of our own institution in utilizing fluorescein to maximize glioma extent of resection, and assess the clinical implications of this treatment strategy. Glioblastoma is the most common and aggressive primary brain cancer, with over 12,000 new cases diagnosed each year . DespiteSurgical resection remains the mainstay of treatment. The only modifiable prognostic indicator of outcome in glioblastoma is extent of resection, which has been shown to correlate closely with improved survival. The current standard of surgical treatment is maximal safe resection , 5. HoweDespite advances in surgical management, all patients with glioblastoma eventually experience disease recurrence. Importantly, this recurrence almost always arises within 2 cm of the original lesion, thought to be related to the residual microscopic disease , 10. MorIn pursuit of increasing the safety and accuracy of brain tumor resection, various technologies have been developed with the goal of providing more reliable intraoperative guidance. Frameless stereotactic neuronavigation is the method most typically used in operating rooms today but is not without its weaknesses. This technology relies heavily upon accurate registration prior to surgery, and even with initial accuracy, as tumor resection progresses brain shift inevitably develops and compromises the precision of localization. Compounding this issue is the fact that deeper areas of tumor, which are often the more dangerous areas with the greatest need for reliable guidance due to their proximity to white matter tracts and vascular structures, are most affected by this phenomenon. Intraoperative MRI has been explored as an alternative means of providing more up-to-date navigational information but is disruptive to the operating room workflow; it also fails to provide true real-time information and represents a significant capital investment . IntraopFluorescent agents that selectively localize to pathologic tissue and can be viewed intraoperatively, such as 5-aminolevulinic acid (5-ALA), do provide genuine real-time feedback. 5-ALA has been extensively studied in glioma neurosurgery and is proven to significantly increase extent of resection and improve patient survival . FluoresFluorescein has been shown to reliably correlate with areas of contrast-enhancement on MRI . Most inIn its most basic sense, supramarginal resection refers to the removal of tumor exceeding “gross total resection”, or, more specifically, the resection of tissue beyond the contrast-enhancing tumor border . HoweverDespite these differences, almost all clinical studies investigating supramarginal resection for glioblastoma have shown that compared to gross total resection, supramarginal resection correlates with prolonged survival, with reported increases in median overall survival ranging from 5 to 25 months, and increases in progression-free survival as high as 19 months , 21, 25.Even with complete resection of the contrast-enhancing tumor, glioblastoma invariably recurs, and recurrence is almost always within centimeters of the original lesion, suggesting that the peritumoral region—the region marked by T2/FLAIR signal and lacking contrast enhancement, hence traditionally left behind following surgical resection—plays an important role in this process , 27.The peritumoral region of diffusely infiltrating gliomas contains many different cell populations, both neoplastic and nonneoplastic, that likely contribute to tumor progression and treatment resistance . The clivia neuron-glioma synapses may also contribute to tumor progression , which has increased specificity for glioma tissue  positive gliomas of varying grade who underwent surgery with fluorescein guidance . Specifia tissue –51. Hista tissue . Notablya tissue , 52.While fluorescein has proven useful in detecting non-contrast-enhancing, glioma-associated tissue, especially at the infiltrative margins, there are limits to this approach. Although fluorescein can certainly contribute to supramarginal resection by identifying regions of interest beyond what is traditionally considered surgical goal, large areas of non-enhancing infiltrated brain that also do not demonstrate fluorescent signal likely remain. In other words, fluorescein does not comprehensively define the boundaries of clinically adequate supramarginal resection. As alluded to earlier, there is still little consensus about what constitutes supramarginal resection, and what definition or criteria best maximizes survival benefit while minimizing patient harm . Thus, iWhile studies have investigated different means of achieving supramarginal resection in glioblastoma, perhaps the best way to maximize extent of resection is to combine the various navigational tools at our disposal. 5-ALA and fluorescein have distinct mechanisms of action that complement each other in detecting pathologic tissue, and guidance of supramarginal resection using both fluorophores might prove more effective than using either alone , 54. AltBy localizing to areas of pathologic tissue that fail to enhance on T1-weighted MRI, fluorescein may serve as a way of safely and reliably defining targets for supramarginal resection beyond the traditional contrast-enhancing goal of resection. To maximize patient safety, this strategy must be balanced with other well-established tools for increasing the safety and efficacy of surgical resection. Although supramarginal resection for glioblastoma is a promising treatment approach, larger prospective studies are needed to establish a consistent definition of “supramarginal” and determine the extent of resection that maximizes survival benefit while optimizing patient safety. While prolonged survival is an important outcome measure, it is important that these studies also assess the longer-term effects of supramarginal resection on neurological functioning and patient quality of life.LW and MB drafted the manuscript. PC and JB supervised and finalized the manuscript. All authors contributed to the article and approved the submitted version.This research was funded in part through the NIH/NCI Cancer Center Support Grant P30CA013696.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.


In the past decade, the emergence of machine learning (ML) applications has led to significant advances towards implementation of personalised medicine approaches for improved health care, due to the exceptional performance of ML models when utilising complex big data. The immune-mediated chronic inflammatory diseases are a group of complex disorders associated with dysregulated immune responses resulting in inflammation affecting various organs and systems. The heterogeneous nature of these diseases poses great challenges for tailored disease management and addressing unmet patient needs. Applying novel ML techniques to the clinical study of chronic inflammatory diseases shows promising results and great potential for precision medicine applications in clinical research and practice. In this review, we highlight the clinical applications of various ML techniques for prediction, diagnosis and prognosis of autoimmune rheumatic diseases, inflammatory bowel disease, autoimmune chronic kidney disease, and multiple sclerosis, as well as ML applications for patient stratification and treatment selection. We highlight the use of ML in drug development, including target identification, validation and drug repurposing, as well as challenges related to data interpretation and validation, and ethical concerns related to the use of artificial intelligence in clinical research. Machine learning (ML) is one subset of artificial intelligence (AI) that aims to build analytical models by learning from existing data. The concept of AI and ML can be traced back to the mid-20th century when building a “machine that can learn from experience” was proposed by mathematician Alan Turing . After dGlossary). Supervised learning algorithms investigate relationships between predictive variables and outcome from labelled training datasets and apply the learned rule to establish a model for classifying new data   and clinical research .Data characteristics, such as universality and potential applicability for developing effective precision medicine approaches, facilitate ML-based clinical studies. Electronic medical records (EMR) data are the most systematically collected patient data with standardised format and are frequently applied in clinical ML applications because they are relatively accessible and easy-to-implement. EMRs are digital data compiled by healthcare systems, they contain longitudinal information from individuals, such as medical history, current diagnoses, medication, disease activity and other clinical measurements collected at a particular clinical visit. EHRs contain information beyond EMRs, including cumulative laboratory and imaging data available for a certain patient as well as information about their overall health from all the clinicians involved in their care. Applying ML to data from EMR/EHRs is a major area of interest within the field of personalised diagnosis and treatment .Medical imaging including magnetic resonance imaging (MRI), computed tomography, nuclear imaging, x-ray, electroencephalography and ultrasound etc., are all techniques with standardised imaging acquisition protocols. These data are predominantly analysed by deep learning algorithms, which are the most suitable due to their strength and competence in analysing the complex detail present in medical images. Deep learning techniques have shown particular progress in precision oncology including early diagnosis, identifying cancer subtypes, early detection of metastasis and aiding clinical decision-making .There are various applications of ML techniques in radiology, from automatization of routine tasks usually performed by radiologists and clinicians requesting various investigations, such as assessment of imaging appropriateness, creating study protocols to improve image quality and minimise radiation, and standardisation of the way radiology studies are reported .medical image segmentation ; computer-aided detection and diagnosis  images  images  . ML methods allow a robust biomarker selection process, enabling researchers to quickly screen out and combine the most relevant markers for more comprehensive decision-making. Effective biomarker selection has been applied extensively in diseases with a strong genetic determinant such as cancer  of the total decrease in node impurity of variable, weighted by the proportion of samples reaching that node in each individual decision tree in the random forest; thus, a higher “mean decrease in Gini” implies a greater contribution of a variable to the overall model performance , a heterogeneous autoimmune disease, using immune-based ML approaches . ImmunopIn a study of patients with neuropsychiatric SLE , researcA number of studies have begun to examine the application of ML techniques to the diagnosis of complex autoimmune diseases using EHR and EMR data . In a prIn another study using EHR data to identify SLE patients , an enseSimilar studies have applied EMR data to classify patients with rheumatoid arthritis (RA)  and IBD + monocytes in SLE patients with active disease. Although models trained by gene expression data remain challenging for implementation in the clinical setting from the point of view of feasibility and cost-effectiveness, the gene expression features identified between active and inactive groups of patients may boost the understanding of SLE pathogenesis.ML classification models can also be applied in disease activity prediction of complex autoimmune diseases . This ha16 models with different variable settings for optimisation purposes, the final model including seven laboratory variables and three demographic variables identified with 88.6% accuracy whether a certain SLE patient had HDA or not. The model training used data from all visits, irrespective of their time point and this limited the possibility of using certain earlier time-points to predict later disease development status. The study shows the possibility of using a limited amount of routinely available laboratory measurements and demographics to select SLE patients with HDA, which could help the early identification of SLE patients likely to require treatment escalation after testing the model in a clinical setting.Another study attempted to identify SLE patients with high disease activity using ML algorithms without making use of the validated disease activity score usually implemented in routine practice (SLEDAI) . The lonGlossary) was employed to classify patients without chronic damage at baseline but who developed damage in the following 2 years versus those who did not develop chronic damage. Clinical data including demographics, diagnosis date, co-morbidities and medical history, and laboratory data including important markers of SLE were used as predictors for RNN model training. The RNN model uses the all the longitudinal time point (≥5 visits for each patient) of chronic damage measurement as the sequential input, then processes the network through the hidden layer (layers in between) until connecting the output layer, which generates the prediction results (see Glossary). To avoid overfitting, an early stopping technique (stop when AUC reaches 0.95) and 8-fold cross-validation were applied. The model performance was stable at AUC (0.77) for predicting a chronic damage-developing group.Another study accurately predicted chronic damage in SLE with the aim to improve disease management . 413 SLESimilar studies have also been described in patients with MS. Seccia and colleagues applied supervised ML algorithms to predict disease progression of MS and potentially provide treatment decision support . Four coGlossary) using all predictors. After variable selection from the model, a refined model with 12 predictors was employed. The refined elastic net model significantly outperformed the traditional cardiovascular risk assessment scores in predicting generalized subclinical atherosclerosis and the risk of progression in 3 years. Notably, this model improved the false-negative prediction rate meaning that fewer high-risk individuals were mis-classified in the “no disease” group.The classification and biomarker selection properties of ML algorithms can also help to predict the prognosis of diseases with a long asymptomatic phase. In a recent study of atherosclerosis, Sánchez-Cabo and colleagues applied ML to predict cardiovascular risk in asymptomatic individuals . Non-invIn a recent study of SLE , researcPersonalised treatment is a fundamental aim of precision medicine, where individuals receive tailored therapy instead of the one-size-fits-all approach. The precision of the treatment is increasingly important in heterogeneous diseases, including autoimmune inflammatory diseases, where significant disease signature differences between patients can be overlooked by the same diagnosis. An effective way of delivering personalised treatment is by performing a more precise subpopulation identification based on their distinct pathogenetic signatures. Signatures can be extracted from genomes, metabolomics, immunophenotyping and other types of data. Supervised ML is an ideal tool, specialised in the identification of unique signatures, while clustering approaches from both supervised and unsupervised ML are designed for partitioning complex high dimensional data. An increasing number of studies have applied ML models to identify subgroups of patients and show promising results toward more personalised treatment  McKinne.SLE is a chronic ARD with no cure. Due to the heterogeneous nature of SLE, predicting treatment response of SLE patients remains challenging. Figgett and colleagues  applied n = 88) and stratified by k-means clustering into two groups with distinct immune profiles. The balanced random forest model identified a signature of eight T-cell subsets that differentiated between the two groups with high performance (AUC = 0.99). The 5 year clinical trajectory analysis identified differential damage scores and disease activity between the two groups. The study suggests the potential of differentiating pSS and SLE patients based on their immunological profile and could provide the opportunity for more accurate targeted treatments across diagnostic boundaries.In the recent study of primary Sjögren’s syndrome (pSS) and SLE , researc+ T cells and methylation analysis in PBMCs  . More thDrug development is a complicated, costly, and time-consuming process, which depends on a large number of factors. The pipelines of drug development can be simply divided into two phases: the drug discovery phase and drug-testing phase . The druThe first step of drug development is target identification which often heavily depends on the extensive study of disease mechanism. Understanding disease mechanisms can be time and labour intensive; common experimental techniques ranging from using immunoprecipitation assays to identify protein-protein interactions in biological samples to genome-wide CRISPR-Cas9 screens to knock down genes of interest. Modern high-throughput techniques generate abundant molecular and biological data, which makes it difficult to screen potential drug targets using conventional methods. To speed up the drug target selection process, numerous studies have developed automated in-silico approaches for drug target identification and validation.A recent study by A Bayesian machine learning approach (BANDIT) developed by Madhukar and colleagues  integratDrug repurposing is another powerful application aiming to discover, validate and apply existing approved drugs for new application. The process of drug repurposing is much conserved by renouncing the standard drug development pipeline approach and investigating similarities between various disease processes potentially targeted by the same therapeutic interventions, so that new effective treatments can be delivered faster to patients. This is a more cost-effective approach which led in recent years to the testing and licensing of similar classes of therapeutic agents across many immune-mediated chronic inflammatory diseases . In a repro) as a potential drug target because it was highly conserved and encoded by a distinct gene. Due to the 96.08% similarity between SARS-CoV-2 and SARS-CoV . One of the studies selected the SARS-CoV-2 main protease  to explore potential treatment options for COVID-19 using existing anti-cytokine therapies which enabled large-scale clinical trials to be rapidly conducted . ResearcDespite the promise of ML research in the field of precision medicine, many challenges still need to be addressed to ensure the further development and acceptance of ML approaches  – a rare ARD. In one study, researchers applied a ML model to stratify JSLE patients based on their immune profile . Only 67Another challenge in the development of ML models is access to high quality and well-defined datasets, needed for algorithm training and evaluation. In recent years there has been a big push to make research data FAIR  . DatasetIssues with multiple testing and p-hacking has contributed largely to the reproducibility “crisis” in science. The 2016 Nature survey pointed out that more that 70% of scientists have failed to reproduce other scientists reported results . P-hackiTo achieve the highest model performance, many clinical studies tend to avoid data splitting for model development. Resampling methods such as bootstrapping and k-fold cross-validation are economical internal validation, therefore, they are often applied to prevent model overfitting. On the other hand, external validation using an independent cohort is not often performed, potentially due to limited access to similar cohorts, despite being the most straightforward way to evaluate the generalizability of the model. Less than 10% of autoimmune studies combine cross-validation with an independent test dataset for validating model performance . HoweverGlossary) are applied to represent the predictive efficacy of ML models in clinical studies. However, most of the metrics do not directly affirm the clinical applicability and can be difficult to evaluate with limited interdisciplinary knowledge (Transforming a well-performed model into an actual clinical application associated with improvement in patient outcomes can be challenging; the term “AI Chasm” describes the discrepancy between the model development and translation of models to real-world applications . The clinowledge . Anothernowledge . To helpnowledge . TRIPOD-nowledge , which tThe upsurge of ML applications in personalised medicine has raised potential ethical concerns regarding data privacy, as a wide range of big datasets including personal information from genetics data, demographic data and medication history are stored and used in various studies. Anonymisation is the most straightforward and common way for privacy protection of medical datasets by removing personal data for de-identification purposes. However, advanced re-identification techniques were developed and used to target the vulnerability of the anonymisation system by data mining companies, and data were then exploited by health insurance companies . Thus, mThe phenomenon of ML algorithm-driven discriminating decisions has been well-observed in other areas of research using AI, such as racial discrimination in criminal charge facial recognition technology  and gendWith such challenges evident at every possible step during the application of ML approaches, the ambition of personalised medicine to ensure that every individual receives an optimal treatment decision guided by their disease particularities and individual risk becomes uncertain. To warrant a future for ML applications in the clinical field, it is crucial to have universal procedure guidelines from data collection, data processing to model training, validation, and implementation . By ensuThe versatility of ML applications allows researchers to tackle divergent unmet clinical needs of immune-mediated inflammatory disease with the most effective tools . Predict


Dog park members initially join and attend dog parks for the wellbeing of their dogs, but often experience their own biopsychosocial benefits. This mixed methodology  ) pilot study utilized qualitative heuristic interviewing  and the Satisfaction with Life Survey . Data gathered from interviews and surveys administered to participants of a members-only dog-park indicate a high satisfaction with life. Members 60 years and older reported feelings of life satisfaction almost 7 points over the total respondent average, placing them in the “highly satisfied” range. All members experience the dog-park as a supportive social environment that benefits their physical health, mental health and the well-being of their canine companions. Five qualitative themes were identified: Canine Well-being, Community, Mental Health Benefits, Physical Health Benefits and Fights, Falls & Frustrations. These findings demonstrate the need for more research into the impact and importance of pet ownership, community dog parks and outdoor green spaces on older adults and life satisfaction.