How do stem cells function? Why should we, as dentists, care about stem cells? How might dental procedures be substituted by stem cells? Are stem cells capable of regenerating a tooth or temporomandibular joint (TMJ)? Although the ability to regenerate a destroyed tissue has been known for a while, research into regenerative medicine and dentistry has made significant strides in molecular biology. A paradigm shift in the therapeutic toolbox for dental and oral diseases is likely to result from a growing understanding of biological concepts in the regeneration of oral/dental tissues along with stem cell research, leading to an intense search for "biological solutions to biological problems."Among other tissues, orofacial tissues effectively separate stem cells from human tissues. Because they can self-renew and produce different cell types, stem cells offer novel techniques for regenerating damaged tissues and curing illnesses. A number of significant milestone successes have shown their practical applicability, traditional biomaterial-based treatments in regenerative dentistry as therapeutic alternatives that offer regeneration of damaged oral tissues rather than merely "filling the gaps."In order to use these innovative accomplishments for patient well-being, the ultimate goal of this ground-breaking technology, well-designed clinical studies must be implemented as a crucial next step. The review's objective is to briefly synthesize the literature on stem cells in terms of their traits, subtypes, and uses for dental stem cells. It has been highlighted that stem cell therapy has the ability to treat craniofacial abnormalities and regenerate teeth in the oral and maxillofacial regions. Because of their unique capabilities, they are essential for creating advanced technologies [Stem cells are defined as unspecialized, immature cells that have the capability to transform into multiple cells . These snologies .Two fundamental types of stem cells are multipotent and pluripotent, which can only differentiate into a specific population of cells and can differentiate into any adult body cell, respectively. Depending on the source, they could be adult or embryonic stem cells. It is possible to separate embryonic stem cells from the inner cell mass of embryonal blastocysts . Adult sCurrently, researchers have discovered significant sources of stem cells in oral tissues, as they are a distinct progenitor of stem cells. In order to develop innovative and successful dental applications, dentists and researchers must further define these cells . Tissue Various fundamental and translational experiments involve stem cells as well as many different components of tissue engineering, such as bioactive compounds and scaffolds ,8. HowevSearch methodologyThis review carefully gathered literature on the application of stem cells in dentistry, utilising databases such as PubMed, Scopus, and the Web of Science. "Periodontal ligament stem cells", "dental pulp stem cells", "dental follicle progenitor cells", "stem cells from exfoliated deciduous teeth", and "stem cells from apical papilla." Articles suitable for inclusion focused on stem cell applications in dentistry or literature reviews, but publications focusing on the application of these cells in medical and other disciplines were omitted. The inclusion criteria included relevant books, articles, and reviews. The study selection procedure included screening titles and abstracts, followed by a full-text evaluation of relevant papers. The final group of included research offers a thorough analysis of the evidence that is currently available on the use of stem cells in dentistry. The results were combined and analysed to draw meaningful conclusions. The review confirmed the inclusion of high-quality studies that addressed the study’s objectives by adhering to these selection criteria.Classification of stem cellsStem cells are primarily categorized as Embryonic stem cells (ESCs), Adult stem cells, and Induced pluripotent stem (IPS), which exist naturally in humans and have lately been created synthetically by genetic alteration of somatic cells ,11. The Embryonic Stem CellEmbryonic stem cells are produced by blastocysts, which have 50-150 cells . Cells oAdult Stem CellThese are multipotent stem cells . Mature Induced Pluripotent Stem CellPluripotency is described as the individual potential of a single cell to create all adult creature lineages in response to an embryonic stimulus . InducedSources of stem cellTreatment of the oral and maxillofacial area can be done with the help of stem cells from many sources such as bone marrow, adipose tissue, and stem cells from the oral and maxillofacial region .Bone Marrow Stromal Cells (BMSCs)BMSCs are present in adult bone marrow and are peculiar multipotent progenitor cells. The sternum or iliac crest can be used to extract BMSCs. It consists of hematopoietic, mesenchymal, and stem cells. Almost all the structures of the oro-maxillofacial region produce mesenchymal stem cells. Bone marrow has the benefit of having a greater count of stem cells, which allows the development of different types of cells. Isolation of bone marrow cells is possible under general anaesthesia, with discomfort following surgery. Akintoye et al. exhibited important features of iliac and orofacial bone marrow cells from the same person, with orofacial BMSCs having stronger proliferation and osteogenic differentiation potential than iliac crest BMSCs. Furthermore, in an in vivo mouse model, orofacial BMSCs generated more bone, but iliac crest BMSCs produced a large quantity of compact bone that included hematopoietic tissue .Adipose Tissue (ASCs)Mesenchymal stem cells are abundant in adipose tissue and are intensively researched in the domain of regenerative drugs as a form of stem cells. They can be exfoliated with aspiration after liposuction or lipectomy. ASCs are a form of pluripotent mesenchymal stem cell that may develop into a variety of lineages . ASCs diStem cells from the oral and maxillofacial regionIn oral and maxillofacial areas MSCs are predominant stem cells. Various varieties have been found as well as characterized in oro-facial areas. They are seen in Figure Histopathological representation of various cells found in oro facial areas is seen in Figure Dental Pulp Stem Cells (DPSCs)In 2000, Gronthos et al. extracted DPSCs. In immunocompromised mice, researchers were capable of demonstrating DPSC-derived odontoblast-like cells generating ectopic dentin . Dental Stem Cells from Exfoliated Deciduous Teeth (SHED)This is discovered by primary teeth that develop into many cell types, having a higher rate of procreation and more frequent population doublings. Furthermore, by attracting host cells, SHED can specifically trigger the creation of a lamellar bone ,26. ThisDental Follicle Progenitor Cells(DFPCs)Dental follicle stem cells, which produce PDL cells, cementoblasts, and osteoblasts known as DFPCs, were isolated by Morsczeck et al. . Dental Stem Cells from Apical Papilla(SCAP)Sonoyama et al.  showed tPeriodontal Ligament Stem Cells (PDLSCs)The periodontal ligament is a source of adult mesenchymal stem cells in dental tissues. Extracted teeth can also be used to retrieve it. Seo et al. extracted PDLSCs from the periodontal ligament of 25 human third molars. They noticed cementoid cells and adipocytes when they were introduced into weak animals . It may Why stem cell research?Research advancements in stem cells have examined several therapeutic approaches, and the results from pilot-proof concept trials offer a plethora of previously imagined profits. They are helpful in understanding functional genomic investigations, genomic data mining, bioinformatics, and human embryonic gene expression . These cStem cell storage and transportIn contrast to the decades-long existence of bone marrow and placental cord blood stem cell banks, those that specialise in stem cells collected from teeth are relatively recent. Stem cell-containing tissue samples are to be placed in a screw-top container with a suitable medium that nourishes them while they move. The sample must be sent to the processing storage unit within forty hours . SamplesObstacles to overcome for stem cell researchMSCs are found in large numbers in tooth tissues, according to an increasing number of studies. Stem cells, signalling molecules to drive differentiation into appropriate types, such as osteoblasts, cementoblasts, odontoblasts, fibroblasts, etc., and scaffolding material for cellular proliferation support and harvest are required for regeneration of tooth or tooth-related tissues. The difficult problems surrounding factors that affect stem-cell-based bone regeneration are then addressed by the authors. These issues must be resolved for stem-cell-based therapies to be clinically effective. Pre-clinical stem cell research employing MSCs for cell-based immunotherapy and complicated oral tissue and organ engineering are also discussed.Possible applications of stem cell therapy in the oro-maxillofacial regionStem cells suited for regenerative purposes have to be subjected to comprehensive regulation of cell control in the body. Only adult MSCs have significant clinical implications in dentistry at the moment. MSC regeneration of periodontium has been extensively researched, with several trials currently reaching the clinic. Effective stem cells must be able to grow into desired tissues or organs, be conveniently extracted, and be processed easily. It should have antimicrobial properties. The pulp, dentin, periodontal ligament, enamel, cementum, craniofacial bones, temporomandibular joint, comprising fibrocartilage, bone, ligaments, tendons, skeletal muscles, subcutaneous soft tissue, and skin, and also including the salivary gland, are all structures of importance to the dentistry profession. During native development, all of these oral, dental, and craniofacial features are generated by mesenchymal cells and/or neural crest-derived cells.Reconstruction of Dental and Craniofacial StructuresCraniofacial bone grafting therapies include devitalized allogenic and autogenous bone grafting, and osteoconductive biomaterials are also used in craniofacial bone grafting treatments. Donor site morbidity limits autologous bone transplantation, and allogenic bone is frequently lost. These cells possess the ability to help with bone repair and the correction of significant craniofacial abnormalities caused by trauma, enucleation, and tumour resection. Tissue transfer is commonly used to fix a bone defect, although it has limitations such as not being capable of restoring the specific function of the missing area and donor site morbidity, which is followed by scarring, infection, and a lack of function. In cases of severe loss of soft tissues following trauma or surgery, soft tissue repair in the oro-maxillofacial region appears critical. In their experiments, Alhadlaq et al. discovered that when human MSCs are exposed to adipogenic-inducing media, they can transform into adipose cells. Soft tissue reconstruction can be accomplished using adipose cells and an appropriately formed scaffold .Periodontal RegenerationClinical research employing material therapies shows that partial periodontal bone loss can be recovered by using bioactive materials, or substances that improve the niche's ability to support resident stem cells in tissue regeneration. Platelet-rich plasma from peripheral blood was combined with autologous mesenchymal stem cells from the iliac crest for periodontal regeneration. After one year, the bone defect had healed significantly and the attachment level had improved. It also demonstrated effective repair and regeneration of the interdental papilla .Regeneration of Damaged Coronal Dentin and PulpNovel understandings in tissue engineering, as well as molecular biology, have increased the possibility of the creation of new advanced treatments, for example, pulp capping and many other techniques for the treatment of tooth-related problems . Cells pWhole Tooth RegenerationA therapeutic alternative that seemed inconceivable now appears to be a realistic aim. Even now, there are restrictions on replacing lost teeth. Mesenchymal epithelial interactions are required for tooth development. The creation of unique terminal phenotypes, or "helping cells," as a result of signal exchange between these two naive germ layer tissues distinguishes these interactions . DespiteProven studies in oral and maxillofacial regionVarious studies were evaluated as listed in Table An entirely new age has begun in the field of dentistry where orofacial bone, parts of teeth, TMJ, and various other regenerations from stem cells extracted from the oral cavity like periodontal ligament, pulp, deciduous teeth, etc., can heal and restore functional and aesthetic harmony in patients. The enormous regenerative potential of embryonic, adult, and induced pluripotent stem cells has been examined, but their use in dental practice is still problematic and constrained by a number of factors that are still out of control, including the high risk of rejection, cell behaviour, the lengthy tooth eruption period, the appropriate crown morphology, and the right colour. Even so, one of the biggest challenges in the dental field for the foreseeable future is the development of biological methods for dental reconstruction using stem cells. Therefore, dental practitioners must be aware of the potential of the developing area of regenerative dentistry as well as the chance of acquiring stem cells while performing standard procedures, which can be saved for later use in autologous therapeutics. In dentistry, stem cell-based treatment is now undergoing intensive research in tissue engineering as well as chair-side cellular grafting techniques, both of which have the potential to produce more predicted regenerative results in the future. Challenges of the study are host-immune response, isolation and identification of appropriate stem cells, long-term stability, and being very costly. Limitations of the use of stem cells are that stem cells in the oral and maxillofacial region are specialized for their specific functions, which may not translate well to other types of tissue repair or regeneration. The number and regenerative capacity of stem cells in this region may decline with age, limiting their effectiveness in older individuals.


Introduction: In the pediatric brain tumor surgery setting, intraoperative MRI (ioMRI) provides “real-time” imaging, allowing for evaluation of the extent of resection and detection of complications. The use of advanced MRI sequences could potentially provide additional physiological information that may aid in the preservation of healthy brain regions. This review aims to determine the added value of advanced imaging in ioMRI for pediatric brain tumor surgery compared to conventional imaging.Methods: Our systematic literature search identified relevant articles on PubMed using keywords associated with pediatrics, ioMRI, and brain tumors. The literature search was extended using the snowball technique to gather more information on advanced MRI techniques, their technical background, their use in adult ioMRI, and their use in routine pediatric brain tumor care.Results: The available literature was sparse and demonstrated that advanced sequences were used to reconstruct fibers to prevent damage to important structures, provide information on relative cerebral blood flow or abnormal metabolites, or to indicate the onset of hemorrhage or ischemic infarcts. The explorative literature search revealed developments within each advanced MRI field, such as multi-shell diffusion MRI, arterial spin labeling, and amide-proton transfer-weighted imaging, that have been studied in adult ioMRI but have not yet been applied in pediatrics. These techniques could have the potential to provide more accurate fiber tractography, information on intraoperative cerebral perfusion, and to match gadolinium-based T1w images without using a contrast agent.Conclusion: The potential added value of advanced MRI in the intraoperative setting for pediatric brain tumors is to prevent damage to important structures, to provide additional physiological or metabolic information, or to indicate the onset of postoperative changes. Current developments within various advanced ioMRI sequences are promising with regard to providing in-depth tissue information. Pediatric brain tumor surgery aims for a complete resection of tumor tissue while avoiding damage to healthy functional brain regions. The extent of resection (EOR) is a key indicator of the child’s prognosis after surgery . An incrThe implementation of intraoperative magnetic resonance imaging (ioMRI) aims to sustain these goals by enabling “real-time” images of the brain, allowing for intraoperative evaluation of the extent of resection (EOR) . In 38% IoMRI may also contribute to alleviate a second challenge during surgery; avoiding damage to healthy functional brain regions and preserving the quality of life . ParticuGenerally, ioMRI sites incorporate multiparametric imaging optimized for surgical aims. Conventionally used sequences in the pediatric ioMRI context are variations of 2D or 3D T1-and T2-weighted (T1w and T2w) images to visualize residual tumor tissue and to guide continuation of the neurosurgical resection .Advanced MRI sequences, on the other hand, could potentially provide additional information on physiological aspects of the brain. For example, these sequences could assess the functional integrity of white matter tracts and blood perfusion or metabolic status of the brain tissue .Potentially this might contribute to peroperative awareness and support prevention of damage to healthy functional brain regions.In this manuscript, we systematically review the literature, aiming to answer the following question: “what is the added value of advanced imaging in ioMRI for pediatric brain tumor surgery, as compared to conventional imaging?”We conducted a Pubmed search of the literature on advanced MRI in the pediatric ioMRI setting, based on the following search terms: (pediatr* OR paediatr* OR child*) AND (ioMRI OR iMRI OR iopMRI OR “intraoperative MRI” OR “intra-operative MRI”) AND (tumor* OR tumour* OR glioma*). Papers were screened on title and abstract, and relevant articles were read in full text.- Case series, cohorts, or trials including pediatric patients (age <19 years) undergoing ioMRI for brain (tumor) surgery.- The reported MRI sequences were intraoperatively acquired.- The added value of advanced MRI sequences was reported.Articles were included based on the following criteria: in vitro studies, articles not written in English, or with no focus on surgery; articles on low-field MRI (<1.5 Tesla), no focus on ioMRI or no pediatric patients  were excluded.Literature reviews, book chapters, articles on epilepsy, Conventional MRI sequences were defined as T1w, T2w, or fluid-attenuated inversion recovery (FLAIR) sequences (with or without gadolinium contrast) that can provide structural information about the brain . AdvanceThe following MRI sequences were considered as advanced MRI sequences: arterial spin labelling (ASL), amide-proton transfer-weighted imaging (APTw), dynamic contrast-enhanced (DCE), dynamic susceptibility contrast (DSC), diffusion kurtosis imaging (DKI), intravoxel incoherent motion (IVIM), multicomponent-driven equilibrium single pulse observation of T1 and T2 (mcDESPOT), magnetic resonance spectroscopy (MRS), myelin water imaging (MWI), neurite orientation and dispersion density imaging (NODDI), quantitative magnetization transfer (qMT), quantitative susceptibility mapping (QSM), relaxometry, vascular, extracellular and restricted diffusion for cytometry in tumors (VERDICT) ; diffusiFrom each included study, the following data were extracted: study center, magnetic field strength (Tesla), number of brain tumor patients included, histopathological diagnosis, advanced MRI sequences used, and their added value as reported in the study.As this search yielded limited information, we extended our search beyond the initial research question, using the snowball technique, to gain more information on the technical background of each advanced MRI technique, their use in ioMRI in adults, and their use in routine pediatric brain tumor care.The literature search on PubMed yielded 128 articles, of which ten met our inclusion criteria ; Table 1The ten articles that reported the use of advanced ioMRI sequences for pediatric brain tumor surgery focused on diffusion MRI (DWI and DTI), perfusion MRI (DSC), and metabolic MRI (MRS and CSI). Diffusion MRI was used in 288 patients, covered by seven studies . Three oIn conclusion, the sparse literature demonstrated that advanced sequences in ioMRI for pediatric brain tumor surgery was used to reconstruct fibers to prevent damage to important structures, provide information on relative cerebral blood flow or abnormal metabolites, or to indicate the onset of hemorrhage or ischemic infarcts.The explorative literature search was confined to the same advanced MRI fields: diffusion-, perfusion-, and metabolic MRI. It focused on the technical background of the sequences, their use in the ioMRI setting for adult brain tumor surgery, and their use in routine pediatric brain tumor care.Diffusion MRI (dMRI) is based on the diffusion of water molecules and provides information on the microstructural tissue organization. In dMRI, multiple diffusion-weighted images are acquired in multiple spatial directions.Diffusion tensor imaging (DTI) leverages dMRI data acquired at a single diffusion weighting  that is therefore called single-shell acquisition . It is tDMRI metrics such as the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) can be used to disentangle tissue components  that can be useful for clinical decision-making . DKI hasIn the adult ioMRI setting, dMRI was used for fiber tracking of white matter fibers in eloquent brain areas . Due to DMRI metrics, such as the ADC and FA, were used in routine pediatric clinical practice to grade tumor tissue and differentiate it from healthy brain tissue  Ouadih . ADC wasBrain tumor perfusion characteristics can be investigated with gadolinium contrast-based perfusion MRI sequences. Examples of such imaging methods are dynamic susceptibility contrast- (DSC) and dynamic contrast-enhanced (DCE) imaging . GadolinArterial spin labelling (ASL) is a perfusion MRI sequence that can quantify absolute CBF based on endogenous blood water . The ASLASL has the advantage that it is not susceptible to blood-brain barrier leakage artifacts usually observed for gadolinium . AnotherIntraoperative ASL could be used for iatrogenic changes in CBF and to depict residual tumor tissue in adults  Lindner. LindnerSeveral studies have indicated that ASL could be a reliable method to evaluate perfusion patterns of brain tumors in pediatric populations . ASL-basSome limitations of ASL in pediatric routine brain tumor care should be considered. First, various ASL perfusion patterns existed for both high- and low-grade gliomas depending on vascular characteristics . Second, ASL hypoperfusion in children could also be caused by edema or scar tissue . Due to Proton magnetic resonance spectroscopy (MRS) is a non-invasive metabolic MRI technique that can detect metabolites in the tissues . For exaAnother metabolic MRI modality is chemical exchange saturation transfer (CEST). CEST is an MRI modality that exploits the abundance of exchangeable protons of a certain metabolite, and its chemical exchange with water protons, to image the relative concentration of a certain metabolite . In CESTIn the intraoperative setting, metabolic MRI could be used to provide biochemical information about relative metabolite concentrations of potential residual tumor tissue . Pamir eHypothetically, intraoperative APTw imaging might be an alternative to a T1w-Gd sequence . Yu et aMRS has been used in clinical practice to support diagnosing and differentiating brain tumor subtypes in children . In suchAPTw imaging could also be used in pediatric brain tumor care to grade and identify the proliferative activity of tumor tissue . The incThis study is a retrospective study of the literature on the added value of advanced MRI in the intraoperative setting of pediatric brain tumors compared to conventional MR imaging. Our systematic literature search revealed that the fields of diffusion-, perfusion-, and metabolic MRI have been reported for selective cases during surgery. The available literature was sparse and demonstrated that advanced sequences were used to prevent damage to reconstruct fibers to prevent damage to important structures, provide information on relative cerebral blood flow or abnormal metabolites, or to indicate the onset of hemorrhage or ischemic infarcts.Our explorative literature search revealed developments within each advanced MRI field that have been studied in the adult ioMRI population but have not yet been applied in pediatrics. First, multi-shell dMRI could offer “real-time” fiber tractography that was said to be more anatomically accurate than models based on single-shell data. Second, ASL could give information on intraoperative cerebral perfusion and could indicate residual tumor tissue intraoperatively without a contrast agent. Third, APTw imaging and ASL could potentially match T1w-Gd images. Despite these promising advances, the technical and practical limitations of each of these advanced MRI sequences should be carefully considered before implementing them in standard pediatric ioMRI protocols.Advanced MR images acquired during surgery could gain insight into the effect of mechanical manipulation of a child’s brain. Data from these sequences could be useful for research into biomarkers predicting surgery-induced early effects of intraoperative complications . For exaAnother example is the prediction of early postoperative seizures after supratentorial brain tumor surgery with intraoperative ASL and APTw imaging. These seizures have been associated with hemorrhage in the resection cavity . As the Reports focusing on the added value of intraoperative use of advanced MRI, particularly metabolic MRI, in the pediatric brain tumor population are scarce to date. The existing literature was often of a descriptive nature, and randomized controlled trials are lacking. Whilst we could learn from proof of concept reports that use advanced ioMRI in the adult population , a speciin vivo microscopy and high-speed histopathological diagnostics (To study the effect of surgery on a child’s brain, a multiparametric approach, including diffusion, perfusion, and metabolic ioMRI could be useful. Especially with the opportunity of tissue pathological validation on the spot. Recent developments regarding gnostics  may faciFurther development of accelerated imaging techniques could also be explored to reduce the acquisition time or to improve image quality. Particularly image acceleration techniques that could be used with the limited number of single-loop receiving coils that are currently available in the intraoperative setting. Alternatively, recent hardware development of thinner, more flexible, or multi-channel coils could improve image quality and reduce the transition time to and from the ioMRI suite.The application of advanced ioMRI could be potentially valuable in providing new relevant information of the brain in the peroperative setting. The implementation might be challenging and involves close collaboration between neurosurgeons, neuroradiologists, and physicists. Collaboration among professionals from different ioMRI centres will contribute and support progress in this field.The potential added value of advanced MRI in the intraoperative setting for pediatric brain tumors is to prevent damage to important structures, to provide additional physiological or metabolic information, or to indicate the onset of postoperative changes. Current developments within various advanced ioMRI sequences are promising with regard to providing in-depth tissue information.


To review current literature examining the presence of subclinical micro- and macrovascular alterations in normotensive individuals and their clinical significance in terms of hypertension prediction. Emphasis is placed on alterations that can be detected in peripheral vascular beds using non-invasive, easily applicable methodology, as these are in general easier to capture and evaluate in clinical practice compared to more complex invasive or functional tests.Arterial stiffness, increased carotid intima-media thickness, and altered retinal microvascular diameters predict the progression from the normotensive to the hypertensive state. By contrast, there is substantial lack of relevant prospective studies for skin microvascular alterations. Although conclusions regarding causality cannot be safely deduced from available studies, detection of morphological and functional vascular alterations in normotensive individuals emerges as a sensitive indicator of progression to hypertension and hence increased CVD risk.An increasing amount of evidence suggests that early detection of subclinical micro- and macrovascular alterations would be clinically useful for the early identification of individuals at high risk for future hypertension onset. Methodological issues and gaps in knowledge need to be addressed before detection of such changes could guide the development of strategies to prevent new-onset hypertension in normotensive individuals. Cardiovascular disease (CVD) remains the major cause of mortality globally among adults aged 35–70 years . It has Endothelial dysfunction is perceived as the earliest precursor of CVD, triggering the cascade of accelerated atherosclerosis, subclinical vascular injury, and subsequently clinically evident CVD manifestations . In thisHowever, the wide applicability of such markers of vasculopathy has revealed the presence of micro- and macrovascular alterations in a considerable portion of individuals with normal blood pressure levels as well. Nevertheless, the clinical importance of such changes in normotensive individuals has received much less attention compared to other high CVD risk populations, and their presence is frequently overlooked in clinical practice as many physicians remain unaware of their prognostic significance.Keeping in mind the pathophysiological and prognostic implementations of early vascular changes, the present review aims to summarize subclinical vascular alterations and their clinical significance in normotensive individuals. We will focus on changes that can be detected non-invasively in peripheral vascular beds as presented in Fig. Large artery stiffening corresponds to a degradation of the anatomical properties of the arterial wall resulting in a decrease of the high elastin to collagen ratio under the impact of various systemic diseases, particularly those primarily affecting the cardiovascular system . Evaluatβ stiffness index from ultrasound) was an independent predictor for the development of hypertension over a 6-year follow-up period. A decrease in arterial elasticity by one standard deviation was associated with a 15% increased risk of hypertension, and this association was independent of established risk factors for hypertension and the level of baseline blood pressure. In the Baltimore Longitudinal Study of Aging, increased PWV was an independent predictor of incident hypertension . Finally, a study in 1318 participants, without a history of diabetes mellitus, hypertension, coronary artery disease, and chronic kidney disease, showed an association between UACR and coronary atherosclerosis, as detected by coronary artery computerized tomography [Furthermore, MIA has been associated with incidence of CVD events and all-cause mortality in healthy individuals , 50. Anaollow-up  in 1568 mography .The retina provides an easily accessible window for the study of microcirculation . While cst quadrant), compared with those with the highest (4th quadrant), were 80% more likely to develop high blood pressure [Qualitative fundus evaluation using traditional classification systems may reveal arteriosclerotic changes in the retinal microvasculature. For instance, signs of retinopathy assessed with the Scheie classification in fundus photographs were associated with future onset of hypertension in a huge population of 34,649 normotensive individuals without any CVD, after a mean follow-up period of 3.18 years . Howeverpressure . This reConcordant results emerge from a 3-year follow-up of 5628 normotensives (49 to 73 years old) , as wellMore recent studies have tested the efficacy of novel computerized approaches to predict of hypertension and CVD outcomes. Complementary machine learning-based assessment of the retinal vasculature with phenome-wide and genome-wide analyses was leveraged across 97,895 retinal fundus images from 54,813 UK Biobank participants. Vascular density and fractal dimension as a measure of vascular branching complexity were calculated using convolutional neural networks to segment the retinal microvasculature. Both were significantly associated with a higher risk for incident mortality, hypertension, congestive heart failure, renal failure, type 2 diabetes, sleep apnea, anemia, and multiple ocular conditions epidemiologically, while lower microvascular density was further associated with genetically higher risk for hypertension and diabetes ••. ThougThe skin represents an easily accessible vascular bed, and as such, several techniques have been developed for the study of skin microcirculation. For instance, traditional laser Doppler techniques (laser Doppler imaging and laser Doppler flowmetry) that evaluate and quantify red cell flux in small areas of tissue, and more recently the laser speckle contrast imaging, have been applied to document altered microvascular reactivity in systemic diseases such as autoimmune rheumatic disorders and hypertension –64, 65•.Capillaroscopy is another non-invasive technique that can visualize by use of a stereo microscope the capillary network of the studied organ, usually the skin of the dorsum of the nail of the upper extremities. Nailfold capillaroscopy has been primarily used in rheumatology for the diagnosis and monitoring of autoimmune rheumatic diseases. It can provide valuable information for multiple microcirculatory parameters, including the morphology and density of the capillary network, under the influence of various systemic diseases . Using nn = 18). This correlation of insulin resistance, however, was not confirmed in a larger (n = 105) group of healthy individuals, using the HOMA index, an indirect and less accurate way of assessing insulin resistance compared to the euglycemic clamp and only fasting glucose correlated with capillary rarefaction [Nevertheless, the predictive role of capillary rarefaction for the development of hypertension in normotensive individuals has not been investigated to date with prospective studies. Relevant data come from cross-sectional studies in patients with hypertension  and in hefaction . Finallyefaction .Evaluation of arterial stiffness, carotid atherosclerosis, and retinal and skin microvascular changes is non-invasive, prompt, and feasible in routine clinical practice. Nevertheless, these measures are not routinely applied as part of the general check-up of healthy individuals. First, these methods necessitate expertise and relevant infrastructure, although once obtained consumables are not required. Second, with the exception of arterial stiffness measures and urine albumin excretion (UAE), methodological standardization remains the Achilles’ heel. This is particularly true for cIMT despite several decades of experience, which is therefore not recommended by current European Society of Cardiology guidelines on CVD prevention in clinical practice . While sMost importantly, the added value of the aforementioned subclinical vascular alterations in predicting future CVD events in low- and intermediate-risk individuals with normal blood pressure levels needs to be clarified in appropriately conducted prospective studies. There is evidence suggesting that accumulation of subclinical vascular alterations may be linked with increased CVD risk, but this needs to be verified in prospective cohort studies . FinallyA growing amount of prospectively obtained data supports that altered morphology and function of the micro- and microvasculature predict the development of new-onset hypertension in normotensive individuals. Specifically, arterial stiffness, increased cIMT and UAE, and altered retinal microvascular diameters predict the progression from the normotensive to the hypertensive state, whereas there is substantial lack of relevant prospective evidence for skin microvascular alterations. Although research reports suggest that such alterations precede and predispose to hypertension, through the remodeling of blood vessels and the increase in vascular resistance, conclusions regarding causality effects cannot be safely driven from available studies. Thus, identification of subtle vascular alterations in normotensive individuals could serve as a sensitive indicator of future hypertension onset and hence an increased CVD risk. It could be reasonably assumed that in these individuals, closer monitoring for the timely diagnosis of hypertension, intensification of lifestyle interventions, and perhaps early initiation of medical treatment would alleviate the global CVD burden. Appropriately designed longitudinal studies are eagerly warranted to delineate the potential role of subtle vascular alterations in primary CVD prevention.


Significant progress has been made in the elucidation of human antibody repertoires. Furthermore, non-canonical functions of antibodies have been identified that reach beyond classical functions linked to protection from pathogens. Polyclonal immunoglobulin preparations such as IVIG and SCIG represent the IgG repertoire of the donor population and will likely remain the cornerstone of antibody replacement therapy in immunodeficiencies. However, novel evidence suggests that pooled IgA might promote orthobiotic microbial colonization in gut dysbiosis linked to mucosal IgA immunodeficiency. Plasma-derived polyclonal IgG and IgA exhibit immunoregulatory effects by a diversity of different mechanisms, which have inspired the development of novel drugs. Here we highlight recent insights into IgG and IgA repertoires and discuss potential implications for polyclonal immunoglobulin therapy and inspired drugs. These pisorders , 3. Pathisorders . In the Activities of antibodies are executed by both major regions of the immunoglobulin, the fragment antigen binding (Fab), consisting of the light chain and part of the heavy chain, and the constant fragment crystallizable (Fc) domain. The variable Fab fragment is responsible for specific molecular recognition and binding of the antigen (epitope-paratope interaction). Beyond targeting specific antigens, the Fab fragment is responsible for the execution of important effector functions, such as direct neutralization of toxins or pathogens, agglutination, or blocking of ligand-receptor interactions , 5. Fab-While these classical functions of antibodies are commonly known, a large body of research revealed a range of alternative antibody activities, which have also been referred to as ‘noncanonical’ functions . Such novia tumor-specific antibodies that restore aberrant immune surveillance in immunodeficient patients. Profiling of individual patient antibody repertoires may lead to more personalized treatments  exhibits anti-inflammatory effects in the endogenous K/BxN mouse model of rheumatoid arthritis, which seems to involve the inhibition of neutrophil infiltration into the joints and the suppression of IL-1β production . A numbe+ NK cells are scarce  at mucosal surfaces of the  intestine, in particular. The extensive glycosylation of IgA is thought to confer protease resistance, binding to bacterial adhesins, or serve as carbon source for microbiota  can be derived . SurprisWhile the role played by IgA in the regulation of gut bacterial homeostasis appears now clearer, in our perspective there are currently no optimalcommercial IgA antibody preparations for oral use to test for their ability to regulate gut microbiome dysbiosis associated with primary antibody deficiencies. Despite IgA’s role in the growth and persistence of different species in the gut, only few studies have addressed the composition of the gut microbiome in the absence of IgA in PAD , 75–77. Current systemic Ig replacement therapy in CVID restores only serum IgG levels eventually resulting in reduced numbers of respiratory infections. Yet, it still fails to protect against autoimmunity, allergy, gastrointestinal disorders and fatal liver disease . In addiClinical studies using oral administration of a plasma-derived and IgA-enriched IgG preparation, IgAbulin, has revealed promising results in children with chronic diarrhea , but mixSerum monomeric IgA exhibits anti-inflammatory properties –88, and de novo binding to bacterial antigens and intact bacteria (in vivo models of sepsis and aseptic systemic inflammatory response syndromes (Escherichia coli and Staphylococcus aureus) and to viral antigens   . It remaHigher degree and non-site-specific antibody modification such as used for the generation of radioimmunoconjugates can be associated with reduced binding to the targeted antigen, eventually due to occupancy of the antigen-binding site . HoweverMild chemical modification of antibodies seems to provide a mechanism for further diversification of antibody specificities, beyond somatic recombination, junctional diversity and somatic hypermutation . HoweverIn the last decade, methodological advances have enabled greater insights into the human repertoire of antibodies, including sIgA. Research revealed that canonical and non-canonical effector functions of antibodies often act in concert to complete manifold tasks in immune defence, host-microbial symbiosis, or even immunoregulation. Insight into anti-inflammatory MoAs of IVIG has revealed the potential benefit of its pluripotency in targeting heterogenous disorders and served as a source for novel therapeutics. Nevertheless, research on IVIG mechanisms is often challenging due to the inherent complexity of polyclonal preparations . Limitations include the availability of suitable disease models , 105, anAll authors contributed to the article and approved the submitted version.


The process of inflammation is an intricate and essential response to biological, chemical, or physical stimuli. Ongoing research aims to unravel the cellular and molecular events that initiate and regulate the interactions among the various components involved in the inflammatory process. Numerous diseases, including arthritis, asthma, atherosclerosis, diabetes, cancer, and age-related conditions, have been associated with chronic inflammation.To address the challenges of high attrition rates, significant costs, and slow pace of new drug discovery and development, it is crucial for current research to focus on repurposing existing drugs. Given the multiple pathways involved in the development of inflammation, drugs already available on the market for other therapeutic purposes that can intervene in resolving inflammation may hold potential for mitigating the impact and consequences of other disorders with similar underlying mechanisms. However, repurposing drugs presents challenges due to the need to identify suitable candidates that can effectively compete with the numerous drugs commonly used in clinical practice for specific diseases. Overcoming this hurdle requires extensive research.The objective of this Research Topic was to gather research and review papers that demonstrate the significant interplay between inflammation and disease progression, as well as provide a deeper understanding of the molecular mechanisms involved in the assessment of repurposed drugs.Xu et al., entitled “Detecting potential mechanism of vitamin D in treating rheumatoid arthritis based on network pharmacology and molecular docking”, delves into unraveling the potential mechanism by which vitamin D treats rheumatoid arthritis (RA). Vitamin D plays a pivotal role in RA; however, the mechanism of vitamin D in RA is still unclear. Therefore, this study employs network pharmacology and molecular docking strategies to investigate its mechanism. By examining 1,139 RA targets and 201 vitamin D targets, the researchers identify 76 overlapping genes. Enrichment analysis reveiled that cell proliferation, immune response, and apoptotic process are critical biological processes influenced by vitamin D in treating RA. The study also identifies antifolate resistance, osteoclast differentiation, and the nuclear factor-kappa B (NF-κB) signaling pathway as fundamental mechanisms involved in the treatment of RA with vitamin D. Further molecular docking analysis suggests that ALB, TNF, CASP3 and TP53 may serve as crucial checkpoints or diagnostic markers for future RA treatment.This Research Topic begins with an article by Chaffey et al., titled “Drug repurposing in cardiovascular inflammation: Successes, failures, and future opportunities”. Drug repurposing is a pragmatic and appealing approach to drug discovery that has shown success in various medical fields over the years. Repurposing existing medications for new indications can significantly reduce development costs and timelines compared to de novo drug discovery process, making it a promising strategy in the field of cardiovascular disease, where new drug approvals lag behind other areas. Chronic inflammation has been established as a driving factor in cardiovascular disease pathology, leading to numerous attempts to therapeutically target cardiovascular inflammation. However, this approach has faced challenges, particularly off-target effects associated with broad-spectrum immunosuppression, which is especially problematic in long-term conditions like cardiovascular disease. Nevertheless, multiple anti-inflammatory drugs, repurposed from their original indications in autoimmune conditions such as rheumatoid arthritis, have been evaluated for efficacy in cardiovascular clinical trials. This review discusses the mixed outcomes of clinical trials investigating the use of anti-inflammatory drugs like anti-cytokine monoclonal antibodies, colchicine, and methotrexate in cardiovascular disease. Additionally, the authors highlight potential new directions for drug repurposing in cardiovascular inflammation, including the emerging concepts of drug re-engineering and chrono-pharmacology.Another noteworthy contribution to the Research Topic is a review article by Song et al. published a review titled “Natural drugs targeting inflammation pathways can be used to treat atherosclerosis” focusing on the use of natural fdrugs for treating atherosclerosis (AS). AS involves the gradual degradation of arteries accompanied by inflammation While current research has primarily focused on the interactions among inflammatory cells, inflammatory mediators and immune mechanisms, some studies have reported the efficacy of natural drugs against AS. However, the utilization of natural drugs is often limited by factors such as poor penetration across biological barriers, low bioavailability, and unclear mechanisms.Zhang et al., titled “The emerging roles of TLR and cGAS signaling in tumorigenesis and progression of ovarian cancer”. The authors highlight the significance of uncovering essential molecular biomarkers associated with the progression of ovarian cancer, considering its complex pathogenesis. Inflammation has been reported to play a crucial role in the initiation and progression of ovarian cancer, with both microenvironmental and tumor cell intrinsic inflammatory signals contributing to its malignancy. The review discusses the roles of receptors related to innate immune system, particularly Toll-like receptors (TLRs) and cyclic GMP-AMP synthase (cGAS), in the development and treatment of ovarian cancer. It sheds light on the intrinsic and exogenous inflammatory responses triggered by various stimuli and emphasizes the potential implications of these pathways in the management of ovarian cancer.In addition to the articles mentioned above, another valuable contribution to this Research Topic is the review article by El-Khateeb et al., entitled “Evaluating the safety and efficacy of the leukotriene receptor antagonist montelukast as adjuvant therapy in obese patients with type 2 diabetes mellitus: A double-blind, randomized, placebo-controlled trial” provides valuable insights into the treatment of obese patients with type 2 diabetes. The study aimed to evaluate the effectiveness, tolerability and safety of combining montelukast therapy with metformin in this patient population. The results demonstrated that the combination therapy of metformin and montelukast significantly improved diabetes control and induced weight loss. This improvement was attributed to the increased insulin sensitivity and anti-inflammatory properties of montelukast. The study concluded that the combination therapy was well-tolerated and safe throughout the duration of the trial. These findings suggest a potential role for montelukast as an adjuvant therapy in obese patients with type 2 diabetes.Finally, an important clinical trial article by In conclusion, the papers included in this Research Topic emphasize the strong relationship between inflammation and disease progression, providing a deeper understanding of the molecular mechanisms involved in the assessment of repurposed drugs. Additionally, they address concerns regarding potential adverse effects and pharmaceutical interactions associated with the use of repurposed drugs.


Aging-related phenotypes span many different tissues and cell types, and start to occur at different ages- a different typical age for every cell type. Among the earliest occurring aging events in the human body is the beginning of female reproductive aging and deterioration. The clinical cut-off for advanced maternal age (AMA), a condition associated with poor reproductive outcomes, is 35 years old. The early onset of reproductive aging poses a significant challenge to clinicians since a global consistent increase in maternal age at first birth has occurred in recent decades, effectively shortening the available time window for reproduction . As the Our recent publication  addresseIn a second stage of this project- we show that aged mouse oocytes lose their control over RNA transcription- showing elevated levels of dsRNA and Dicer- both markers of transcriptional control. We also investigated the levels of retrotransposon transcripts- which are elevated in aged oocytes, as well as Line1 retrotransposon protein L1ORF1p. Consequently, older oocytes show elevated levels of DNA damage markers such as Rad51 and gamma-H2AX.in-vitro compared to untreated oocytes.To causally link the two phenomena, we have disrupted heterochromatin structure in young (2 months old) oocytes using two known heterochromatin damaging agents- Chaetocin and TSA. As a result, the treated oocytes showed elevated retrotransposon transcription, elevated retrotransposon protein synthesis, elevated DNA damage, and reduced maturation ability in-vitro maturation ability in these old oocytes. Interestingly, the same result is achieved when retrotransposon activity is blocked by inhibiting reverse-transcriptase activity using AZT. This result shows that retrotransposon activation is a significant event during oocyte epigenetic aging.Finally, to demonstrate the central role of epigenetic changes in oocyte aging we show that the reversal of heterochromatin loss leads to a dramatic improvement in oocyte quality and function. Using heterochromatin enhancing small molecules or overexpression of heterochromatin genes , we elevated heterochromatin levels in old (9 months) mouse oocytes. This led to a decrease in retrotransposon expression levels and a decrease in DNA damage. It also led to a dramatic increase (2 fold) of Our work reveals unknown aspects in oocyte aging. It mainly offers two substantial and innovative advances: first, it investigates chromatin aging processes in the early stages of oocyte aging. The distinction of early aging allowed us to define and analyze previously unknown molecular characteristics of early-aging oocytes. In oocyte aging research, we can use these insights to better understand the damage and deterioration linked to later stages of aging. By following the escalation of the mild events described in this paper, we could determine how they lead to the total loss of function that defines the later stages of oocyte aging. The aspect of heterochromatin loss is a great example in this context- heterochromatin is a dynamic and important component of chromatin, that influences many aspects of the cellular environment through direct or indirect interactions, including cytoskeleton, spindle establishment, oocyte-to-zygote transition, RNA processing and transcription, and more. Heterochromatin loss during the early aging period could explain the deterioration of these systems later in the aging timeline. The second important insight provided in this paper is the concrete aging pathway it describes. Starting with a molecular event of heterochromatin loss, we identify downstream events that lead to the outcome of maturation failure. Tracking the direct chain of events allowed us to not only better understand the molecular features of oocyte aging, but also to therapeutically target them. If this approach can be applied in the IVF clinic, it will provide a concrete therapy option for age-related fertility loss, which is currently unaddressed in the clinic.In summary, our group demonstrates basic principles in the early aging of mammalian oocytes. This demonstration provides us with an improved understanding of how oocyte aging processes are initiated, and which events lead to deleterious outcomes, damaging oocyte maturation and quality.


Mitochondria are key structures providing most of the energy needed to maintain homeostasis. They are the main source of adenosine triphosphate (ATP), participate in glucose, lipid and amino acid metabolism, store calcium and are integral components in various intracellular signaling cascades. However, due to their crucial role in cellular integrity, mitochondrial damage and dysregulation in the context of critical illness can severely impair organ function, leading to energetic crisis and organ failure. Skeletal muscle tissue is rich in mitochondria and, therefore, particularly vulnerable to mitochondrial dysfunction. Intensive care unit-acquired weakness (ICUAW) and critical illness myopathy (CIM) are phenomena of generalized weakness and atrophying skeletal muscle wasting, including preferential myosin breakdown in critical illness, which has also been linked to mitochondrial failure. Hence, imbalanced mitochondrial dynamics, dysregulation of the respiratory chain complexes, alterations in gene expression, disturbed signal transduction as well as impaired nutrient utilization have been proposed as underlying mechanisms. This narrative review aims to highlight the current known molecular mechanisms immanent in mitochondrial dysfunction of patients suffering from ICUAW and CIM, as well as to discuss possible implications for muscle phenotype, function and therapeutic approaches. Skeletal muscle weakness and muscle wasting are frequently observed phenomena in the context of critical illness . The pheTherefore, this narrative review aims to highlight the current known molecular mechanisms immanent in mitochondrial dysfunction of patients suffering from critical illness-induced muscle wasting, as well as to discuss possible implications for muscle phenotype, function and therapeutic approaches.Mitochondria can be found in the majority of eukaryotic cells, as they are essential organelles to maintain cellular metabolism and energy supply. In skeletal muscle fibers, their subcellular localization can be either subsarcolemmal (SSM) or intermyofibrillar (IMF) . AccordiIrrespective of differences in the reductions in mitochondrial content and shifts between different fiber types in skeletal muscle, it is clear that the increased ATP hydrolysis during contractions needs to be balanced by ATP synthesis at some point: Whereas this task is reached at the local level in type I fibers, it is delegated to the liver by type II fibers. Thus, whenever considering the energy demands of the muscles, it is mandatory to take account of the mitochondrial function of hepatocytes and—for the effort of transporting oxygen and metabolites—cardiomyocytes. In both organs, severe alterations occur during sepsis ,20.Likely due to their ancient evolutionary origin from bacterial predecessors, mitochondria contain their own genome consisting of a circular double-stranded deoxyribonucleic acid (DNA) with 37 genes encoding for mitochondrial proteins, especially subunits of the respiratory chain complex and several transfer ribonucleic acids (RNA) . They arMitochondria are highly dynamic organelles, undergoing permanent changes to their structural organization by fusion, fission and degradation in response to various stimuli to maintain and adapt their physiological function  22]. Th. Th22]. As mitochondrial integrity is crucial for cellular bioenergetics and mitochondrial dysfunction has been shown to participate in a great spectrum of disease entities, various approaches have been taken in the detection and monitoring of mitochondrial function. Thus, it can be roughly divided into in vitro and in vivo diagnostic methods, targeting different functional units within the mitochondria . In an a13C-MRS can be used to determine utilization of carbohydrates and lipids via the TCA cycle and 31P-MRS for evaluation of mitochondrial ATP metabolism via the assessment of phosphocreatine turnover [Magnetic resonance spectroscopy (MRS) is a specialized technique of common MR-imaging, which quantifies electromagnetic signals emitted by atoms or molecules within a certain region of interest . With MRturnover  and animturnover , revealiturnover . FurtherNear-infrared spectroscopy (NIRS) is an in vivo optical method that utilizes the ability of heme groups to absorb infrared light by different amounts depending on their oxygenation status for the quantification of the overall oxygen saturation in the local tissue volume below . This prThe protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) is another optical approach to assess mitochondrial function in vivo. The aim of this non-invasive method is to estimate the mitochondrial oxygen tension, which is assumed to be a surrogate parameter for the balance between mitochondrial oxygen supply and demand . TherefoDifferent attempts have been made to evaluate changes in mitochondrial structure and function following pure immobilization and unloading of skeletal muscles. Frequently used methods include cast immobilization, hindlimb suspension, denervation and neural cord dissection ,45,46,472O2 concentrations and overshooting activation or depletion of antioxidant defense systems [At the functional level, the majority of investigations seem to demonstrate an overall decrease in mitochondrial ETC enzyme protein concentration, enzyme activity, respiratory capacity and ATP production ,58,59,60 systems ,49,56,58 systems .Mitophagy also seems to be impaired after denervation, possibly promoting accumulation of dysfunctional mitochondria and further increasing oxidative stress . HoweverThe use of animal experiments is common practice in critical care research to mimic and study distinct features of critical illness, as it provides several advantages such as high reproducibility, control of environmental parameters and the absence of interfering chronic disease or therapeutic interventions . In an aIn an attempt to replicate hallmark features of muscle weakness and wasting in critical illness, several different methods, including mechanical denervation, with or without steroids ,65,66,67The majority of experimental studies have used mice ,69, ratsComparing animal models and human studies of ICUAW and CIM also demands discussion of individual skeletal muscle properties. In clinical studies of CIM and mitochondrial dysfunction, the majority of biopsies have been taken from the vastus lateralis muscle ,88,89,90Both biomechanical and metabolic properties may vary between human and animal skeletal muscles. Early works demonstrated the different activity levels of ETC complex II between different animal species within the same muscle fiber types ,98. FurtBioenergetic failure related to critical illness seems to be accompanied by an overall reduction in mitochondrial content and function . In an aFurthermore, not only a diminished number of mitochondria, but also ultrastructural changes in the remaining organelles point to severe alterations in mitochondrial integrity during critical illness. Mitochondrial swelling, a fragmented or nearly absent cristae structure, matrix space enlargement and vacuolization are the most frequent morphological pathologies that have been reported in animal ,80,103 aDysregulation of mitochondrial dynamics has already been linked to critical illness-induced organ dysfunction. In sepsis, dysfunction of multiple regulating proteins contributes to impaired mitochondrial biogenesis and recovery. Inhibition of mitochondrial fusion by suppression of MFN2 and extensive mitochondrial fission by upregulation of DRP1 may promote multiple organ failure . In relaMitophagy is a specialized form of autophagy which allows the removal of dysfunctional or damaged mitochondria and related protein complexes to maintain cellular homeostasis. This process is facilitated by the parkin E3 ligase, an enzyme promoting degradation of dysfunctional mitochondria . ImpairmAlterations to the ETC enzyme complexes in skeletal muscle have frequently been observed in connection to critical illness and muscle wasting . StudiesAlthough it seems obvious that reduced ETC enzyme activity is related to a reduction in muscle strength and function, studies investigating the relationship between integrity of the OXPHOS system, muscle strength and function in critically ill patients with ICUAW are sparse and partly inconsistent. Some studies examined the function and integrity of the OXPHOS system in biopsies from limb skeletal muscle in critically ill patients. Early investigations by Brealey et al. demonstrated significantly reduced ATP concentrations, but no differences in skeletal muscle citrate synthase-normalized ETC enzyme activities between sepsis survivors and non-survivors .Duceau and coworkers used proteome and metabolome analysis to demonstrate a marked downregulation in OXPHOS gene expression in septic patients . HoweverBesides its function as the most important source of ATP, the mitochondrial respiratory chain also contributes to the production of reactive oxygen species (ROS), which are thought to participate physiologically in cell signaling . HoweverAt the level of the neuromuscular junction (NMJ) of motor nerve terminals, a ROS-mediated inhibition of transmitter release has been described . WhereasIn the skeletal muscles of patients suffering from critical illnesses, the ROS-associated damage may persist even after sepsis has been overcome . FurtherCalcium ions are indivisibly linked to proper skeletal muscle structure and function by fueling the contractile apparatus, acting as intracellular signaling molecules and impacting muscle fiber plasticity . In receVarious conditions of critical illness have been linked to mtDNA damage and profound alterations in gene expression encoding for key regulators in mitochondrial function ,159,160.Another mechanism proposed to disrupt mitochondrial function in critical illness might be mediated through microRNAs (miRNA). These non-coding RNA molecules are assumed to regulate protein expression by translational repression, a post-transcriptional mechanism in the modulation of gene expression . Their iMitochondria are essential components for generating energy to fuel cellular function by integrating the final steps in the metabolism of glucose and lipids. Acetyl-CoA, the main substrate for the TCA cycle, is generated by either pyruvate from glycolysis or β-oxidation of fatty acids. Disturbances in the metabolism of glucose or fatty acids have frequently been observed in critical illness, and are inextricably linked to altered mitochondrial function and skeletal muscle homeostasis .Adequate glucose utilization is vital for preserving mitochondrial function in skeletal muscles, and is mediated via the action of insulin-activating subsequent signaling pathways . RegularThe term “insulin resistance” describes the failure of insulin to induce subsequent metabolic pathways, resulting in impaired cellular glucose uptake, inadequate inhibition of gluconeogenesis and downregulation of anabolic processes. It is frequently associated with hyperinsulinemia, hyperglycemia and increased concentrations of FFAs. In reference to mitochondrial integrity, states of chronic hyperglycemia and insulin resistance have been directly linked to skeletal muscle mitochondrial dysfunction, including reduced mitochondrial content, reductions in OXPHOS activity and OXPHOS gene expression, dysregulation of mitochondrial biogenesis through suppression of PGC1α and ROS overproduction in diabetes mellitus ,178. In The metabolism of lipids provides another essential pathway for the generation of ATP that converges in mitochondria. Glucose and lipid metabolism are inextricably interlinked with each other, preserving mitochondrial energetic integrity and muscle health. Therefore, in conditions of critical illness, alterations in glucose metabolism are frequently accompanied by disturbances in lipid homeostasis, aggravating skeletal muscle wasting. In states of increased energy demands, lipids provide a quickly available fuel source for the organism. When glucose utilization is impaired and glycogen stores are depleted, skeletal muscle derives energy from the metabolism of FFAs and ketone bodies . LipolysAlterations in the skeletal muscles’ mitochondrial structure and function can be observed within the first few days after ICU admission. Mitochondrial swelling, decreased mitochondrial content, a significant decline in muscle ATP and phosphocreatine concentrations as well as pathologically reduced activities of the ETC enzymes I and IV have been described ,89,90. IAs insulin resistance and impaired glucose utilization have been linked to mitochondrial dysfunction promoting the development of neuromuscular failure in critically ill patients, glycemic control has become a promising treatment option. The use of an intensified insulin treatment has been shown to reduce the incidence of CINM and CIM ,198. TheBeyond glucose metabolism, a differentiated and personalized nutritional regime might help to restore mitochondrial function in patients with critical illness, preventing ICUAW and CIM. The amino acids leucine and glutamine have been shown to promote activation of PGC1α and ETC complex I activity in skeletal muscles in a recent animal model . The infExercise might be an important non-pharmacological approach to improve mitochondrial function in critically ill patients. Early mobilization is safe, maintains muscle mass and prevents against ICUAW . On the In order to reduce oxidative stress and counteract free oxygen radical damage, various antioxidative pharmacological agents have been evaluated to improve mitochondrial function and counteract muscle wasting and dysfunction. Thus, extensive research has been conducted on sepsis-induced diaphragm dysfunction using rodent animal models ,209,210.Another approach to restore mitochondrial function in critical illness-induced muscle weakness is to promote mitochondrial biogenesis and to preserve the fusion–fission balance in mitochondrial dynamics. Pharmacologically-induced activation of NRF2 has been evaluated in an attempt to promote mitochondrial biogenesis and, therefore, to restore mitochondrial count, with positive results ,214. ThiIn this narrative review, we illustrated the molecular patterns of mitochondrial function in skeletal muscle homeostasis, in both health and critical illness. Mitochondrial dysfunction seems to be involved in different aspects of neuromuscular failure in critical illness, contributing to critical illness-induced muscle weakness and, at least in part, to muscle wasting. Thus, impairment is present at different levels of mitochondrial structure and function. As mitochondria are involved in various cellular pathways, their failure leads to bio-energetic crises, which impair cell integrity. However, the majority of data on mitochondrial dysfunction related to neuromuscular failure in critical illness were derived from animal studies, whereas evidence from critically ill patients was sparse. Recent advances in diagnostics and monitoring of mitochondrial function deepened our understanding of the pathological mechanisms related to mitochondrial dysfunction in neuromuscular failure, although their value for daily clinical practice remains to be approved. Interesting therapeutic approaches have been made to preserve or restore mitochondrial metabolism and, therefore, muscle function, representing one important factor in the multiprofessional care of the critically ill. Future efforts should not only focus on the molecular patterns of skeletal muscle mitochondrial dysfunction in the acute phase, but also need to evaluate the adaptive processes in chronic states of critical illness.


HIV incidence has recently increased among people who inject drugs (PWID) across the United States, with outbreaks occurring in states with long-standing syringe service programs (SSPs) including Massachusetts (MA). Antiretroviral pre-exposure prophylaxis (PrEP) is an evidence-based HIV prevention strategy recommended for PWID, but uptake in this marginalized population is extraordinarily low.We describe the design and procedures for a National Institute on Drug Abuse (NIDA)-funded (R01) randomized controlled trial (RCT) testing the efficacy of “PrEP for Health,” a multicomponent behavioral intervention to increase PrEP uptake, adherence, and persistence among HIV-negative PWID attending SSPs in two areas of the U.S. Northeast that are heavily affected by injection-related HIV transmission. Participants are equally randomized to receive the “PrEP for Health” intervention  or an enhanced standard of care (eSOC) control condition involving a brief educational video on the utility of PrEP for HIV prevention. Co-primary outcomes are PrEP uptake  and adherence (using tenofovir quantification in hair samples); a secondary outcome is PrEP persistence  over 12 months. Major assessments occur at baseline, 1-, 3-, 6-, and 12-month follow-up visits. Planned analyses will examine intervention efficacy, specific hypothesized conceptual mediators of the intervention effect  and epidemiologically linked moderators .Findings from our extensive preliminary research with the study population revealed that a multicomponent, theory-based intervention targeting PrEP knowledge, motivation, self-efficacy, behavioral skills, and structural barriers to PrEP access is urgently needed for PWID who are at risk of HIV acquisition. We also learned that SSPs represent a highly acceptable service setting for delivering such interventions. In this study, we are evaluating the efficacy of the “PrEP for Health” intervention. If efficacious, findings from our implementation evaluation could help guide its dissemination to diverse SSPs and possibly other community-based settings accessed by this population.ClinicalTrials.gov number NCT04430257, registered June 12, 2020. In the context of the ongoing opioid and polysubstance use crises in the United States, Human Immunodeficiency Virus (HIV) transmission among people who inject drugs (PWID) is a critical public health challenge , 2. IncrAntiretroviral pre-exposure prophylaxis (PrEP) is an evidence-based HIV prevention strategy that is recommended for PWID at risk of HIV acquisition,  yet PrEPAs PWID may have limited or ineffective interactions with traditional healthcare systems, , 37, 38 Social Cognitive Theory (SCT) provides a valuable theoretical basis for behavioral interventions targeting the multilevel barriers to PrEP uptake, adherence, and persistence for PWID. SCT posits that people, their behaviors, and surrounding environments interact to influence how and when behaviors are performed,  with a fPatient navigation is an evidence-based, patient-centered healthcare delivery strategy that provides individually tailored supports to help people overcome barriers to accessing and staying engaged in healthcare services . PatientPrEP for Health is a two-arm RCT aiming to enroll 200 HIV-negative PWID attending two SSPs in MA (n = 100 per site). Based on the locations of recent HIV outbreaks and clusters among PWID, we partnered with two SSPs  in Lawrence and Boston/Cambridge, MA, jurisdictions heavily affected by opioid and stimulant use and injection-related HIV transmission. The SPIRIT figure of this trial, including the schedule of screening, enrollment and follow up visits, is illustrated in Fig. Participant recruitment involves active and passive methods. At each SSP, research team members  and SSP personnel  approach potential participants seeking SSP services to briefly describe the study and assess interest in participating. At both study sites, screening is integrated into standard SSP intake processes to facilitate efficiency and reduce participant burden. Active recruitment also occurs when research staff accompany SSP personnel during wider community outreach . Passive recruitment involves posting flyers and sharing business cards at the SSPs, nearby health centers, substance use treatment facilities, churches, and other places where PWID congregate. Recruitment is currently ongoing.With support from SSP personnel, who often know potentially eligible members of the target population and can refer them to the study, research staff conduct brief eligibility screenings. Detailed eligibility criteria are described in Table After confirming eligibility, trained study staff either conduct informed consent immediately or schedule an appointment for a later date, depending on participant preference. Study staff conduct informed consent in a private room using a consent document written according to Federal standards at or below an eighth-grade reading level that describes the study rationale, procedures, risks, benefits, measures to protect confidentiality, and participants’ rights and responsibilities. After explaining all study procedures, study staff then answer questions and gauge comprehension using a brief, structured assessment. Individuals who consent to participate sign the informed consent form, which is reviewed by the Site PI or their designees.Following baseline assessments  and 1-, 3-, 6-, and 12-month follow-up visits (post-randomization). Assessments are not blinded, however only research staff uninvolved in intervention delivery administer follow-up assessments to reduce social desirability bias. Moreover, study staff use standardized survey instruments and are instructed in how to mitigate bias in survey administration. Between 6- and 12-month assessments, check-ins (by phone or in-person) are incentivized ($10/check-in) to update locator information, support retention, and facilitate ongoing communication between major assessments.PrEP for Health is a two-session, manualized, multicomponent intervention grounded in SCT and informed by prior interventions with similar populations. Given that a key strategy is MI, draft manuals were reviewed and refined by a certified Motivational Interviewing Network of Trainers (MINT) trainer. The intervention is delivered by trained PrEP Navigators through up to two in-person intervention sessions at SSP study sites, followed by ongoing navigation support for a period of three months. The two in-person intervention sessions cover PrEP uptake , and PrEP Adherence and Persistence . Navigators are individuals without healthcare training who have personal or professional experience working with PWID .Interventionist training and supervision: PrEP Navigators receive extensive training in the intervention manual content (including detailed background information on HIV transmission and PrEP); MI ; principles of harm reduction (including overdose prevention), cultural humility, and trauma-informed services; problem-solving and de-escalation techniques; and research ethics. An MI-experienced licensed clinical social worker conducts ongoing supervision and training to enhance intervention delivery and ensure fidelity to the intervention manual and principles of MI.Module 1: PrEP uptake: In the first in-person intervention session, PrEP Navigators use specific strategies to deliver individually tailored HIV and PrEP education and MI to address HIV risk perceptions and increase PrEP interest and motivation. PrEP Navigators also use this session and ongoing follow-up interactions (described below) to engage participants in problem-solving and planning to build their self-efficacy and behavioral skills to facilitate the process of obtaining initial PrEP screening and prescriptions. PrEP Navigators use patient navigation techniques to assess barriers to PrEP uptake, navigate the PrEP medical care system , and engage participants in problem-solving and planning around anticipated challenges in obtaining PrEP prescriptions . If participants do not initiate PrEP within one month of this initial intervention session, this PrEP uptake-focused content  can be repeated in participants’ second intervention session to help trouble-shoot around initial uptake.Module 2: PrEP adherence and persistence: For participants who initiate PrEP, in the second in-person intervention session, PrEP Navigators use similar strategies described above  to motivate and support participants in achieving optimal PrEP adherence and persistence. This includes problem-solving around adherence challenges and anticipated adherence facilitators informed by evidence-based interventions and formative research . Finally, PrEP Navigators support participants in developing an individually tailored PrEP routine, including planning for future PrEP prescription refills and care appointments, and devising plans for handling missed doses and lapses in PrEP care.Ongoing PrEP navigation support: For three months after the initial intervention session, PrEP Navigators provide additional, ongoing MI and PrEP navigation, as needed, through in-person, phone, and text-based interactions to support participants in coping with ongoing or new challenges with PrEP uptake, adherence, and persistence. Near the end of this three-month period, PrEP Navigators’ help participants plan for ongoing PrEP adherence and persistence.Following randomization, participants in both the PrEP for Health intervention and eSOC control conditions watch a brief educational video on PrEP that was adapted by our research team from existing resources . The video is available in English and Spanish and uses non-technical terminology to describe what PrEP is and how it works to prevent HIV via sexual and injection exposures. Additionally, all participants, regardless of study condition, are eligible to receive any services offered at the local SSP study sites, which includes referrals to local PrEP clinicians and other healthcare providers. Moreover, participants who attend local PrEP clinics and are deemed clinically eligible for PrEP may be prescribed PrEP according to standard clinical practices.At all major assessment visits, participants complete interviewer-administered assessments of HIV and PrEP knowledge, perceived HIV risk, PrEP interest and self-efficacy, and patterns of substance use, sexual behaviors, physical and psychosocial health conditions, and health service utilization. Socio-demographics  are assessed at baseline only. Medical record review is conducted throughout the course of the study. Biological measures of adherence to oral PrEP  areCo-primary outcome measures: Our first co-primary outcome, PrEP uptake, is primarily assessed via visual confirmation that a PrEP prescription was filled and received. At each follow-up assessment, we also request that participants bring in their PrEP prescription packaging to confirm that the prescription was picked up. For injectable PrEP, uptake will be defined as receipt of their first injection,Our second co-primary outcome, PrEP adherence, is assessed only among those who report initiating PrEP (i.e. taking at least one pill or receiving at least one injection). We assess adherence at 3-, 6-, and 12-month follow-up visits by collecting hair samples to test for the presence of detectable levels of tenofovir, which reflect adherence to either tenofovir disoproxil (TDF) or tenofovir alafenamide (TAF) over approximately the prior month. Samples are ~ 50–100, 1-2 mm-long strands of hair cut with scissors. Samples will be analyzed at the University of California, San Francisco, Hair Analysis Lab for quantification of tenofovir levels . We suppSecondary outcome: We assess our secondary outcome, PrEP persistence, using pharmacy and medical records to confirm prescription refill maintenance and attendance at PrEP care follow-up visits. PrEP persistence will be defined as having ≥ 16 days of oral PrEP medication filled per 30-day period, or receiving PrEP injections within one month of their target dates, for at least three-quarters of the months since initiation to study completion (approximately 9/12 months) [ months) .Conceptual mediators: Informed by our preliminary studies, conceptual mediators of intervention effects are measured by self-report at each major assessment visit and include HIV knowledge , peionnaire ), and Prionnaire ). PrEP uionnaire  and antiionnaire . Structuionnaire ) and stiionnaire ).Epidemiologically linked moderators: Also based on our formative research, we hypothesize observing heterogeneity in intervention effects according to participant age, gender, and baseline sexual risk  and polysubstance use (including stimulants), assessed by self-report at baseline and follow-ups assessment visits.Planned preliminary analyses include assessing distributions and autocorrelations across time for all variables. As we are utilizing interviewer-administered survey assessments, the primary anticipated reason for missing data is participant attrition. Based on our preliminary studies, we are anticipating and accounting for 20% attrition. Attrition effects will be evaluated by testing whether systematic differences exist between participants who complete the research study and those who discontinue participation. We will explore strategies  for imputing missing data based on patterns of missingness, using sensitivity analyses to determine the optimal method of handling missingness. Dependent variables will be examined to determine which distributional models are most appropriate for subsequent statistical procedures. We will examine the equivalence of the random assignment with regards to key baseline characteristics, including socio-demographics and risk behaviors.Aim 1: Our primary analysis will compare PrEP uptake and adherence (co-primary outcomes) between the PrEP for Health intervention and eSOC conditions. For PrEP uptake, we will perform chi-square tests to examine group differences in proportions over the course of the study, and Cox proportional hazard models to assess differences in time to PrEP initiation. For PrEP adherence, we will initially assess differences in adherence at 3-month follow-up visits. We will also conduct longitudinal analyses to assess differences in adherence over the course of the study (12 months). We will use generalized linear models (GLMs) with properly chosen link functions. GLMs will be estimated using generalized estimating equations with robust standard error estimates (GEE), which provide an extension of regression analysis to the case of correlated or repeated observations and allows for inclusion of both categorical  and count-dependent variables  and appropriate modeling of covariance structures when observations are correlated across time. We will follow an intent-to-treat model, analyzing participants according to the study arm to which they are assigned, regardless of fidelity to assigned group. Sensitivity analyses will compare those who receive the intervention session(s) to those who do not. All analyses will use two-tailed tests of significance, with significance at alpha level 0.05.Aim 2: For mediation analyses, we will explore the role of hypothesized mediators using structural equation modeling (SEM) to determine whether the intervention impacts PrEP uptake and/or adherence through our hypothesized mediators. SEM will allow for the simultaneous estimation of total, direct, mediated, and indirect effects of the intervention on the outcome(s) via a set of multiple mediators, simultaneously. The fit of the overall model will be assessed, and individual paths will be assessed using path coefficients, standard errors for each path coefficient, and tests of significance of each coefficient [fficient . InferenAim 3: To assess heterogeneity of intervention effects, we will examine interactions between the intervention and hypothesized moderators on both the multiplicative and additive scales. Multiplicative interaction will be estimated by including a treatment x moderator term in primary analyses described in Aim 1 above. Additive interaction will be estimated by calculating the Attributable Proportion due to Interaction (API), the proportion of the excess risk that is due to the presence of both the intervention and the moderator simultaneously. Significant or large interaction terms suggest that intervention effects differ by subgroups of the moderators. We will then perform stratified subgroup analyses to further investigate and describe heterogeneity between groups of individuals.The primary power analysis was based on differences between the PrEP for Health intervention and eSOC conditions in PrEP uptake (co-primary outcome). Effect sizes are estimated from our pilot studies, as well as results of a large mathematical modeling study examining PrEP use among U.S. PWID . AssuminAll survey data is inputted directly into REDCap, a HIPAA-compliant, comprehensive data management system. Hard and soft-copy participant data is identified by an ID number only, and a link between names and ID numbers is kept separately in a password-protected file. Name-based files are stored separately from survey data. Soft copy data is stored on study-specific secure and password protected network drive folders and is only accessible to study staff. Hard copy data is stored in locked cabinets within restricted and secure areas at study sites.All study staff are trained in confidentiality and have signed confidentiality agreements. Study staff have been trained in ethical human subject research practices to minimize participant risk. The investigators report unanticipated problems, safety monitors’ reports, and adverse events to the Fenway Health IRB in accordance with IRB policies.Given that this is a behavioral intervention with minimal risk, the study has no stopping rules and interim analyses will not be conducted. An independent Data Safety Monitoring Board has been assembled and reviews study progress, including safety concerns and adverse events, twice per year. All reports are shared with the IRB and the funder at least annually.In addition to reporting on clinicaltrials.gov, findings from this study will be disseminated via peer-reviewed publications and conference abstracts/presentations. Moreover, presentations at community organizations, including our partner SSP sites, and government entities  will be conducted. We will also create easy-to-read infographics to share via social media and our research group website(s).In the context of rising HIV transmission among PWID nationally, –4 improvFirst, in addition to selecting intervention strategies and a delivery format supported by behavioral theory and evidence from our formative research, we also received critical input from our community research partner organizations (SSPs). Importantly, we learned that most—if not all—of the theory- and evidence-based strategies we selected were feasible and culturally appropriate within SSP contexts. For example, many SSPs already provide PrEP information and screening services  that can support the initial stages of the PrEP care continuum. Similarly, we learned that MI is well-aligned with SSPs’ harm reduction-orientation and models of client-centered service delivery. In terms of intervention delivery, we also received community input on the need for primarily in-person research activities. Despite the restrictions imposed by the COVID-19 pandemic on research activities and SSP services, we were aware that a large proportion of our study population lacks consistent phone and Internet access. Thus, to reach a wide swatch of local PWID and ensure that some of the most marginalized individuals  were not excluded, we temporarily delayed study initiation during COVID-19-related closures of our SSP sites to eventually launch an in-person study with appropriate safety measures in place.Similarly, while we initially considered delivering all intervention content and strategies within a single session, our SSP research partners advised us to split the content across two sessions of more limited length  to reduce participant burden. Upon implementing the study, we also learned that referrals from SSPs to nearby PrEP prescribers, along with participants’ decisions to initiate PrEP, can often require days to weeks, so our separation of the intervention content into two sessions, spaced approximately one month apart, enabled the first session to focus on PrEP uptake and the second session to either revisit that content or proceed on to PrEP adherence and persistence, depending on participants’ needs. These two sessions, in addition to being more manageable for interventionists and participants, thus enabled more specific attention to key barriers and plans relating distinct steps in the PrEP care continuum. Anecdotally, we have also observed how the scheduling and planning required for the second session promotes continued interaction and rapport between interventionists and participants.Second, to enhance scalability if PrEP for Health is found to be efficacious, we designed a rigorous yet “real-world” intervention to help maximize widespread implementation. Our study takes place in two distinct sites in terms of inner  and outer contextual  factors. While our intervention and assessment procedures are identical across sites, we worked with our SSP research partners to develop recruitment and retention protocols to maximally engage participants in the study; while we use most of our recruitment and retention activities in both sites, differences in the communities  have required regular reconsideration of which strategies to prioritize in each site. By being in constant community with our SSP research partners through regular all-team meetings and daily interactions with study staff, we have also been able to improve our referral processes, for example, by ensuring that staff are present on days when PrEP prescribers are onsite or in nearby mobile clinics. Maintaining flexibility in some aspects of our approach based on our understanding of real-world organizational and community factors  has supported implementation of the trial so far.Third, we have remained aware of recent advances in biomedical HIV prevention technologies and were pleased that cabotegravir received U.S. FDA-approval for use as long-acting injectable as PrEP (LAIP). Research by our team and others has found LAIP to be preferred over daily oral PrEP in several PWID samples because it would eliminating the need for adherence to a daily medication, which is particularly challenging for PWID who frequently experience homelessness, forced residential relocation, incarceration, and loss or theft of personal belongings including medications , 88, 89.There are potential limitations of the proposed project. First, the RCT may not be powered to determine intervention efficacy for our secondary outcome of PrEP persistence; however, findings may still help inform subsequent research. Second, by situating this study within two urban SSPs in Massachusetts, findings may not generalize to other geographic regions or PWID who do not access SSPs. However, our selected SSP sites cover distinct metropolitan areas and are community-based venues frequented by large numbers of PWID who are diverse in terms of race, ethnicity, age, gender, and sexual orientation, possibly enhancing the generalizability of findings. Moreover, our mediation and moderation analyses will provide more insight into both mechanisms of action and subgroup differences that can inform future broadening in scope. Finally, although this study will not directly address issues of cost-effectiveness of PrEP for PWID, implementation-related findings could inform research on other health interventions for this medically underserved population.In summary, based on formative work and community collaboration, we developed the theory-based, multicomponent “PrEP for Health” intervention targeting PrEP knowledge, motivation, self-efficacy, behavioral skills, and structural barriers to access among PWID at risk of HIV acquisition. Importantly, this RCT is based in two SSPs in a region experiencing ongoing HIV transmission associated with injection drug use, making our study sites ideal settings for the evaluation of PrEP interventions for PWID. If our PrEP navigator-delivered behavioral intervention is found to be efficacious in improving PrEP uptake, adherence, and persistence, findings could inform the dissemination of this model to diverse SSPs and possibly other community-based settings. Future research could also investigate whether components of PrEP for Health, if successful, could be adapted to support utilization of HCV treatment, medications for substance use disorders, or other bio-behavioral prevention services for this marginalized population.