Delivery of clinical dental education, as opposed to clinical medicine, is particularly challenging due to the obligatory aerosol-generating procedures (AGPs) used in dentistry, which are known to facilitate the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, using AGPs and working in close proximity to patients for extended periods in dental hospital/university settings with multiple teaching clinics have been a formidable prospect for all stake holders. Therefore, several professional and governmental organizations have promulgated variations of infection control guidelines for general practice dentistry in the pandemic era to mitigate SARS-CoV-2 transmission.In the absence of unified guidelines for modified infection control/clinical procedures for dental education. We implemented a clinical protocol template and modified operating procedures (MOP) for teaching clinical dentistry to fit the infection control requirements during the pandemic/post-pandemic period at the Sharjah University, College of Dentistry, UAE. MOPs ranged from various engineering control measures  to administrative control measures featuring post-procedure fallow periods of treatment-abeyance between patient sessions.The new MOPs for clinical dentistry in the COVID-19 pandemic era, trialled in a UAE dental teaching hospital, have successfully eliminated infection transmission amongst the students, clinicians, ancillary staff, or attending patients, thus far.The proposed MOPs that complement the standard operating protocols in clinical dentistry were an attempt to mitigate nosocomial infection transmission and protect four different groups of stakeholders, i) the patients, ii) the dental students, iii) the clinical academics, and iv) the para-dental personnel/assistants. Due to the endemicity of the COVID-19 in many regions of the World, the suggested MOPs need periodic review and revision, to fit the emerging data on the disease. Finally, as there are no studies to date comparing the relative efficacy of the MOPs in various dental academic institutions, there is an urgent need for future workers to address this issue. World Health Organization (WHO), in January 2020, announced a new human pneumonia-like outbreak in Wuhan, China, termed the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a global pandemic of unprecedented proportions. The ravaging impact of SARS-CoV-2 was profound, with the global population under ‘lockdown’ conditions in several countries  into unsThe major impediment for reining in the COVID-19 pandemic was the high prevalence of up to 40% asymptomatic disease carriers in the community. Radical containment measures to limit disease transmission have included social distancing, thorough hand washing, quarantine, and the use of personal protective equipment such as masks . In addiCompared to the general population at large, healthcare workers (HCWs), including dental professionals, run a greater risk of contracting respiratory pathogens by the nature of their profession. This was evident in the SARS epidemic of 2013, which led to numerous deaths of HCWs  and in tIt is now clear that COVID-19 will be entrenched in the global community for the foreseeable future, as an endemic disease. Given this scenario, dental educators need to rethink and reinvent effective and safe delivery modes of clinical dental education. As opposed to clinical medicine, the latter is particularly challenging in this context due to the inevitable and obligatory aerosol-generating procedures (AGPs) used for dental patient management while working for prolonged periods in close proximity to patients who may be unknowingly harbouring the SARS-CoV-2. For instance, it is well known that in dentistry, the routine use of high-speed handpieces with water jets, air/water syringes, ultrasonic scaling, and air polishing causes such aerosolization of microbes into the ambient clinic air .After the pronouncement by the World Health Organization that SARS-CoV-2 infection is a pandemic, on 11th of March, 2020, the Ministry of Health and Prevention, in liaison with the Ministry of Education of the United Arab Emirates (UAE), advised the cessation of clinical dentistry, apart from urgent procedures, in all primary care dental practices in the country. The university dental colleges followed these instructions in UAE, and all non-clinical teaching was moved to virtual platforms. Furthermore, university examinations were rescheduled and remodelled, and criteria for the successful graduation of students were reviewed and modified considering the prevailing conditions.This situation persisted until mid-July 2020, after which UAE authorities decided to resumption dental care delivery in accordance with stringent guidelines provided by international and national health regulatory bodies . The NewIn the absence of standard universally promulgated guidelines on obviating COVID-19 nosocomial transmission in the dental educational environment, we provide salient features below of a new operational model for a dental hospital teaching facility in UAE. These guidelines could be used to offer protection to all stakeholders while simultaneously delivering safe and efficacious clinical care for the patient and uninterrupted education for aspiring dentists.University Dental Hospital Sharjah-UAE (UDHS-UAE) is a primary dental healthcare facility equipped with 124 dental units. It is home to more than 125 academic and non-academic staff and approximately 186 undergraduate clinical students. During the pre-COVID era, the hospital provided comprehensive dental care to some 150 patients daily, during two clinical sessions of 3 hours each. Using a curriculum based on the comprehensive dental care delivery teaching model, the undergraduate trainees and residents were mentored to care for all walk-in patients irrespective of their socio-economic status.In considering infection control in an institutionalized setting or otherwise, the analytical and subsequent decision-making processes should generally be based on the following three main principles.Administrative controls offer work policies and procedures to reduce the exposure of all stakeholders to the identified hazard and change the way people work.3.1. Engineering controls entail changes in the physical features of the workplace, such as hospital design and configuration, to mitigate pathogen exposure at the source and improve compliance.3.2. Personal protective equipment (PPE) utilization was considered with engineering solutions and administrative controls (Note: PPE is not described here as it is beyond the remit of this article).3.3. The following describes in detail how the MOPSs entailing engineering and administrative control measures were planned and implemented at the University Dental Hospital Sharjah, UAE (UDHS-UAE) Fig. .Fig. 1NoThe administrative controls were sub-categorized as a) general measures and b) those appertaining to work practices. These are outlined below.The UDHS-UAE dental clinic’s architectural profile is the standard model, featuring multiple, partially isolated cubicles set in clusters and rows, with separate clinics devoted to specialized care. The new guidelines for infection mitigation entailed alternate colour-coding of each cubicle, either as blue or yellow, with 18-blue-coded functioning units alternating with an equivalent number of yellow-coded units Fig. . To mainAs a consequence of this administrative control, the clinic’s functional capacity was reduced by 50%, leading to knock-on issues that entailed longer teaching hours extending well into the late evening with modified, sub-divided, multiple clinical sessions for each student group. In practical terms, the blue/yellow coded cubicle arrangement permitted a more than 4-m space between two functioning units, thus maintaining a safe, social distancing system to obviate infection transmission. This arrangement also means that the alternate cubicles can be deep cleansed with the increased fallow period in between patient sessions (see below).During the pre-COVID era, the students of BDS years four and five were paired (the buddy system) to deliver clinical care guided by four-handed dentistry philosophy from 9 am to 4 pm. However, these new teaching arrangements of split teaching sessions and stretched teaching periods and clinical sessions well into the evening hours (8 am to 8 pm) necessitated an increased workforce, such as general practitioners and additional specialists from different dental specialties Fig. .Fig. 3ClIn the UAE, the government has mandated a contact tracing application, ALHOSN, which utilizes BlueTooth technology to swiftly monitor and alert individuals of their COVID-19 health status . This waA unique QR code may comprise one of four colours . An untested individual displays a ‘grey’ QR code. In contrast, a negatively tested or healthy individual with a ‘green’ code grants the individual access to all public spaces. On the contrary, a ‘red’ QR code signifies a ‘COVID-positive’ individual. An ‘amber’ QR code implies a test or re-test for possible COVID-19 exposure. Accordingly, only the ‘green’ QR-coded faculty members, administrative and clinical staff, students, and resident dentists are eligible to enter the hospital premises after they are temperature scanned at the hospital’s designated, dedicated entry point.Besides temperature checks, hand sanitization at the designated entrance for DHCWs, teachers, and students were required. In addition, all DHCWs were mandated to wear full enhanced personal protective equipment (PPE), including an N95 respirator, protective eyewear, face shield, disposable gowns, and protective layering on footwear in designated PPE donning areas (see below).Patient entrance to the hospital was organized via a designated entry point after passing through an installed ‘sanitizing tunnel,’ leading to a ‘fever check’ booth. Following hand sanitization and fever check, the designated resident dentist in full enhanced PPE recorded COVID-19-related symptoms (if any) from the patients using a ‘patient triage questionnaire.’ Only patients that showed apparent COVID-negative symptoms were allowed in institution.In the event of a COVID-19 suspect or positive patient being identified, emergency care was provided. The designated nurse escorted these patients to the negative pressure ‘isolation clinic’ dedicated for emergency dental procedures. Furthermore, a suspected COVID-19 case with non-emergency dental needs was referred to the adjoining, designated Emergency Care Department of the Medical Facility of the Sharjah University Hospital, which has special facilities for suspected COVID-19 patients.The communal seating in the waiting area was arranged to permit a social distance of 6-ft between two seated patients . AdditioWhere possible, students were encouraged to avoid AGPs, using hand excavators for carious dentine excavation and hand instruments for scaling and root planing. The use of a rubber dam was essential. If dental procedures such as ultrasonic scaling, subgingival restorations, or surgical procedures that generate copious bioaerosols cannot be avoided and rubber dam utilization was not possible –17, thenWearing (donning) personal protective equipment (PPE) before a clinical session and removing (doffing) them afterwards need to be done with extreme care, as the newly donned PPE should not be contaminated. In contrast, in the post-clinic session, contaminated PPE should be discarded not to contaminate any other source with which they come into contact. Hence separate donning areas (clean areas) and doffing areas (dirty areas) should be designated in any dental clinic. This situation was further complicated when numerous students and teaching and para-dental support staff work in a multi-unit teaching clinic. Consequently, distinct designated areas for the ‘donning’ and ‘doffing’ of personal protective equipment (PPE) were created in each teaching clinic. It was critical to have either adequate natural or negative pressure ventilation in such areas that should be large enough to accommodate social distancing.A period of treatment abeyance between two different patient sessions in a clinical unit is termed the fallow period. The fallow period needs to be determined and guided by the clinical procedure undertaken, as well as the use of AGP and ambient ventilation. UK dental authorities had provided extensive guidance on determining and calculating the fallow period . For insFollowing the fallow period, the assigned nursing team dons appropriate PPE and disinfects operatories with particular attention to high-contact surfaces, utilizing appropriate hospital-grade disinfectants  prior to the arrival of the next patient.All unnecessary shelving, furniture, brochures, pamphlets, and magazines that may surreptitiously act as fomites for vector transmission of infection were removed from the clinical areas and the hospital lobby. These inanimate objects or fomites frequently handled by clinic attendees and staff cannot be easily sanitized and are known to be potential vectors of viral transmission .COVID-era recommendations from local and international regulatory bodies are to defer or avoid intraoral radiographs as much as possible during the pandemic. Such procedures frequently evoke coughing or gagging reflex in the patient, thereby generating microbe-laden aerosols , 21. HenMany interventional procedures are known to aerosolize respiratory secretions in healthcare settings , 22. In It is possible to mitigate the burden of bio-aerosolization in the operatories by installing High-volume evacuators HVE for the operatories during AGP, along with retrofitted filters, which are integral components of the hospital’s air conditioning system .High-efficiency particulate air (HEPA) filters incorporated into existing centralized ventilation systems (HVAC) can decontaminate the clinics’ ambient exhaust air . HEPA fiCreating negative air pressure in clinics helps keep airborne viruses and other microbes from entering the surrounding rooms and hallways, thus decreasing the potential risk of COVID-19 spread from clinics .Ultraviolet (UV) germicidal irradiation utilizes short wavelength UV light to inactivate microbes, including viruses, essentially disrupting their nuclear DNA/RNA. They are generally harmless for humans unless on exposure for prolonged periods .Further engineering measures to restrict close contact between administrative staff and patients in the reception areas included installing a transparent plastic/glass barrier between the receptionist and the patient with a communication portal.Another ancillary engineering control measure was to install an indicator lighting system in each cubicle where a green light implied non-AGPs, and a red light indicated AGPs being in current use.Last but not least, behavioural changes of the dental health care workers (DHCWs), including students, were a prerequisite for the proper execution of infection control measures . AwareneWhile many dentistry colleges across the globe chose to close the clinics and delay student graduations due to the pandemic, ours did not. Alternatively, efforts were invested in continuing the clinical and pre-clinical training sessions for our students in accordance with the constraints. Abiding by social distancing practices, students were distributed into subgroups, with labs and clinical sessions spread out, which stretched the teaching hours considerably. Supervision was further individualized due to student subgrouping. Therefore, hiring additional part-time staff was prioritized, so that the overall competency requirements were not compromised.In consideration of the reduced patient flow consequent to the pandemic, clinical requirements were adjusted with the inclusion of case-based scenarios performed using ‘dummies’ in clinics. As a result, additional lab and clinical manuals were prepared by faculty, seen illustrated in Fig. Furthermore, as illustrated in the timeline in Fig. Students who contracted COVID were not marked as absent and were provided extra resources to complete their requirements missed during the quarantine period. In addition, the off-site examinations were arranged for them with additional invigilation staff devoted for the task.Such extension of clinical periods positively impacted the continuity of patient care and the management of dental emergencies. It is noteworthy that our logistic policies and procedures were guided by the regulations promulgated by a number of other global health regulatory bodies and institutions , 30–32. COVID-19 pandemic, has now taken root as an endemic infection with much less severity in many regions of the world due to the efficacious vaccination programs and a plethora of other social restrictions. This ‘new normal’ implies that the community as a whole need to live and conduct their daily business in the presence of a relentlessly mutating virus with variants emerging periodically. Therefore, a correct balance needs to be struck to offer novice students the necessary training while not compromising on the infection control measures needed for their safety as well as the teaching staff, and the patients. Hence the structure of the modified operative procedures (MOP) implemented in the University Dental Teaching Hospital Sharjah, described in detail here, should serve all stakeholders in the ‘dental’ community as a prudent template to mitigate infection spread now and into the future. The foregoing MOP describes in detail, how the various challenges were addressed, particularly the administrative and engineering controls required to prevent nosocomial spread of SARS-CoV-2 in a medium-sized dental teaching hospital. Although, the financial resources needed for engineering controls are substantial and burdensome, the administrative controls did not require similar outlays.


Myocardial fibrosis is a remodeling process of the extracellular matrix (ECM) following cardiac stress. “Replacement fibrosis” is a term used to describe wound healing in the acute phase of an injury, such as myocardial infarction. In striking contrast, ECM remodeling following chronic pressure overload insidiously develops over time as “reactive fibrosis” leading to diffuse interstitial and perivascular collagen deposition that continuously perturbs the function of the left (L) or the right ventricle (RV). Examples for pressure-overload conditions resulting in reactive fibrosis in the LV are systemic hypertension or aortic stenosis, whereas pulmonary arterial hypertension (PAH) or congenital heart disease with right sided obstructive lesions such as pulmonary stenosis result in RV reactive fibrosis. In-depth phenotyping of cardiac fibrosis has made it increasingly clear that both forms, replacement and reactive fibrosis co-exist in various etiologies of heart failure. While the role of fibrosis in the pathogenesis of RV heart failure needs further assessment, reactive fibrosis in the LV is a pathological hallmark of adverse cardiac remodeling that is correlated with or potentially might even drive both development and progression of heart failure (HF). Further, LV reactive fibrosis predicts adverse outcome in various myocardial diseases and contributes to arrhythmias. The ability to effectively block pathological ECM remodeling of the LV is therefore an important medical need. At a cellular level, the cardiac fibroblast takes center stage in reactive fibrotic remodeling of the heart. Activation and proliferation of endogenous fibroblast populations are the major source of synthesis, secretion, and deposition of collagens in response to a variety of stimuli. Enzymes residing in the ECM are responsible for collagen maturation and cross-linking. Highly cross-linked type I collagen stiffens the ventricles and predominates over more elastic type III collagen in pressure-overloaded conditions. Research has attempted to identify pro-fibrotic drivers causing fibrotic remodeling. Single key factors such as Transforming Growth Factor β (TGFβ) have been described and subsequently targeted to test their usefulness in inhibiting fibrosis in cultured fibroblasts of the ventricles, and in animal models of cardiac fibrosis. More recently, modulation of phenotypic behaviors like inhibition of proliferating fibroblasts has emerged as a strategy to reduce pathogenic cardiac fibroblast numbers in the heart. Some studies targeting LV reactive fibrosis as outlined above have successfully led to improvements of cardiac structure and function in relevant animal models. For the RV, fibrosis research is needed to better understand the evolution and roles of fibrosis in RV failure. RV fibrosis is seen as an integral part of RV remodeling and presents at varying degrees in patients with PAH and animal models replicating the disease of RV afterload. The extent to which ECM remodeling impacts RV function and thus patient survival is less clear. In this review, we describe differences as well as common characteristics and key players in ECM remodeling of the LV vs. the RV in response to pressure overload. We review pre-clinical studies assessing the effect of anti-fibrotic drug candidates on LV and RV function and their premise for clinical testing. Finally, we discuss the mode of action, safety and efficacy of anti-fibrotic drugs currently tested for the treatment of left HF in clinical trials, which might guide development of new approaches to target right heart failure. We touch upon important considerations and knowledge gaps to be addressed for future clinical testing of anti-fibrotic cardiac therapies. Reactive fibrosis is a process that accompanies cardiac remodeling in a wide range of heart diseases. Fibrotic remodeling of the heart affects the extracellular matrix (ECM), which is a three-dimensional elastic network of collagen fibers and non-structural proteins that embeds cardiomyocytes, vessels, cardiac fibroblasts, and immune cells, and represents the scaffold of the healthy heart. Depending on the etiology and pathological context, ECM remodeling can primarily affect the left or the right ventricle, respective atria, or all chambers of the heart at once.Historically, cardiac fibrosis has been broadly categorized into two types: reparative, also called replacement fibrosis, and reactive fibrosis, also called diffuse myocardial fibrosis, given the fact that it manifests as excessive and diffuse deposition of ECM components in interstitial and perivascular areas . ReplaceThe notion that changes in the ECM of the human heart occur in cardiac disease is based on a very old idea and was first interrogated in 1957 , 6–11. OIn 1988, the cardiac fibroblast was identified to be the major source of collagen fibers, and the predominant type I collagen producing cell in the heart , 28. A lIn this review, we focus on reactive fibrosis affecting the LV and the RV in chronic cardiac conditions and discuss the phenotypic and clinical consequences of adverse ECM remodeling in the pressure- overloaded heart. We reflect on therapeutic approaches aimed at improving detrimental effects of the fibrotic program of reactive fibrosis, and the potential relevance of preserving required functions of fibrosis. To facilitate anti-fibrotic drug development in the future, we identify open questions to be addressed by research, and potential considerations for clinical trial design.Despite extensive research, the exact role of cardiac fibrosis in heart diseases remains largely elusive. The extent to which fibrosis is a causal, contributing or an adaptive mechanism in the pathogenesis of cardiac conditions is likely context dependent, or co-exists, considering its heterogeneous nature as well as functional and phenotypic diversity . ReparatHF is characterized by a progressive deterioration of LV function. Reactive fibrosis in the LV develops in chronic pressure overloaded conditions, such as hypertensive heart disease or aortic stenosis . ConsideIn addition to its impact on LV function, reactive fibrosis increases the risk of ventricular arrhythmias  Figure . As suchPerivascular fibrosis, specifically, in addition to diffuse interstitial fibrosis, has implications for ischemia in the heart . For exaThe hypothesis that reactive fibrosis might lead to hypoxic and dysfunctional collagen-encircled cardiomyocytes, was first tested 1995 in dogs with chronic HF induced by sequential intracoronary microembolizations . Even thAs described above, reactive fibrosis is associated with distorted myocardial architecture, physiology, and cardiac function, and as such might play a critical role in the development of pressure-overload induced HF and adverse clinical outcomes in patients . Reactivi) Reactive fibrosis plays a prognostic role in HF. The severity of fibrosis assessed with cardiac magnetic resonance imaging (CMRI) correlates with a risk for sudden cardiac death, as well as cardiac and all-cause mortality in patients with non-high-risk hypertrophic cardiomyopathy (HCM) ((hy (HCM) . Moreovehy (HCM) . It shouhy (HCM) . Howeverhy (HCM) . In hypehy (HCM) , 53. As hy (HCM) , 54, 55.ii) Reactive fibrosis defines the response to treatment modalities in HF. Weidemann et al. showed a correlation of severity of reactive fibrosis at the time of surgery and parameters of longitudinal systolic function with clinical outcome post-surgery by assessing standard and tissue Doppler echocardiography and late-enhancement cardiac magnetic resonance imaging (CMRI) at baseline and 9 months after aortic valve replacement in patients with severe aortic stenosis, as well as endomyocardial biopsies obtained during valve replacement surgery   but alsoSeveral histology studies have reported mild but significant increase in RV fibrosis in patients with PAH , 66. CMR2+ responsiveness was lower compared to sham animals in the RV papillary muscles  kinases and the extracellular signal-regulated kinase (ERK) 1/2 that is activated by angiotensin II  is a vasoactive factor predominantly secreted by endothelial cells, but also by fibroblasts, cardiomyocytes, and macrophages. ET-1 can bind to ET receptors that are present on cardiac fibroblasts , therebyFinally, it should be noted that both age and sex are factors that seem to mediate diffuse myocardial fibrosis , 106–111In addition it should be noted that non-fibroblast cardiac cells such as cardiomyocytes, inflammatory, smooth muscle and endothelial cells present in both ventricles can also modulate cardiac fibroblasts by secreting factors directly impacting on fibroblast behaviors , 114–118The cardiac ECM provides a three-dimensional scaffold that is paramount for the healthy architecture, structure, mechanics, myocyte orientation, and transmission of contractile force of the heart –121. TheExcessive deposition of thick, highly cross-linked collagen type I fibers as seen for example in pressure-overloaded conditions , 9, 21 ivia enzyme-linked immunosorbent assay (ELISA) in serum was inversely correlated with the ejection fraction (EF) and directly correlated with LV end-diastolic diameter (LVEDD). Another interesting finding was that patients with aortic stenosis and HFrEF had higher levels of MMP1 and other MMPs and a lower level of TIMPs in the myocardium as compared to patients with HFpEF with the same etiology , and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs)  have beeetiology . The degetiology . It was etiology .With in-depth reviews of the role of MMPs and TIMPs in cardiac extracellular matrix remodeling already existent in the literature , it shouAlthough the role and composition of the ECM in the RV are considered to be similar to that of LV, few studies have investigated the difference between the LV and RV. A study using autopsy samples from patients who died of a non-cardiac cause showed that normal RV has higher collagen content than the LV: 7.4% in RV vs. 5.5% in LV . These dAs research uncovers the mechanisms involved in reactive fibrosis, new targets and strategies are identified. We here briefly highlight preclinical studies modulating some of the most relevant targets involving extracellular collagen turnover, and cardiac fibroblast responses in the LV and the RV.Although numerous preclinical studies using animal models of PH  have shown a reduction of RV fibrosis in response to therapeutic interventions, most of these interventions also attenuated the pulmonary vasculopathy and decreased the pulmonary arterial pressure. Thus, it is not clear whether these interventions directly or indirectly reduced fibrosis by decreasing the RV afterload in these animal models. In this section, we will only discuss preclinical studies done in the pure RV pressure overload model (PAB) and the interventions aimed to directly target the RV.in vivo preclinical model of LV reactive fibrosis due to pressure-overload mimicking systemic hypertension in patients  antagonists led to attenuated angiotensin II-induced fibrosis without affecting the blood pressure level in the mouse, halted progression of cardiac fibrosis and ameliorated myocardial global longitudinal strain in the hypertensive rat , 148.As activation of the neurohormonal system has been also proposed as a key player in maladaptive RV failure, inhibition of the renin-angiotensin-aldosterone system by angiotensin receptor blockers (ARB) losartan  and NeprConsidering the strong correlation between the extent of collagen cross-linking with impairment of cardiac function and with a poor prognosis in HF patients, the enzymes catalyzing covalent cross-links between polypeptide chains of adjacent fibrils, lysyl oxidase (LOX), and lysyl oxidase homolog 2 (LOXL2), could be of major interest as therapeutic target. However, it was suggested that on the one hand a healthy degree of collagen cross-linking is needed for the adequate formation of collagen fibers and on the other hand that a stable ECM might be beneficial in overloaded conditions. Reducing excessive LOX expression or restoring the balance in LOX activity might therefore be superior to the irreversible blockage of LOX enzymatic activity .Direct inhibition of LOXL2 by specific neutralizing monoclonal antibodies in mice with cardiac hypertrophy 2 weeks after TAC-surgery led to the elimination of cardiac fibrosis progression and major improvements of LV function without impacting hypertrophy . Direct Presumably indirect effects on LOX modulation were observed upon chronic blockade of the TGF-β type III receptor beta glycan using the peptide P144 in spontaneously hypertensive rats which led to a reduced expression of LOX and a decrease in collagen cross-linking .In vivo, chronic treatment with EXP3179 prevented LOX upregulation and collagen cross-linking without impacting the systemic blood pressure in a rat hypertensive model. Taken together, collagen cross-linking enzymes might be used as therapeutic targets of anti-fibrotic drugs.In human TGF-β1-stimulated fibroblasts, EXP3179, a non-antihypertensive metabolite of angiotensin II antagonist losartan, reduced over-expression and enhanced activity of LOX . In vivoThe role of the pro-fibrotic cytokine TGF-β1 was determined in animal studies using genetic and pharmacological interventions. TGF-β1 belongs to the TGF-β family of proteins, which have been described as the “master regulators” of fibrosis . Mice wiThe TGF-β1 inhibitor pirfenidone, which is an approved drug for the treatment of idiopathic pulmonary fibrosis, showed anti-fibrotic effects in pre-clinical animal models of fibrosis across organs, namely lung, liver, LV, RV, and kidney . PirfeniReduction of RV fibrosis by pirfenidone has also been reported in the PAB animal model of RV pressure overload. Crnkovic et al. demonstrated a significant reduction of RV fibrosis by treating the animals with pirfenidone after RV fibrosis was established ; however, they did not observe an improvement in RV function . The autET-1 has been shown to be compartmentalized in the myocardial interstitium, and ET-1 uptake was enhanced in a swine model of chronic HF . AngioteThe Endothelin receptor antagonist macitentan was shown to have anti-fibrotic effect in PAB rabbit model .Newest approaches aimed at inhibiting pro-fibrotic responses of activated cardiac fibroblasts in a non-cellular-target-specific fashion. Discovery screens were based on the impact of drug candidates on proliferation rates , 167 andWith the presumption that growth factor plays an important role in fibroblast proliferation, receptor tyrosine kinase inhibitors sunitinib and sorafenib have been examined in PAB rat model and have shown to reduce RV fibrosis .Several other factors that been suggested to influence the RV adaptation. Activation of the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cyclic guanosine 3’5’-monophosphate (cGMP) pathway by the phosphodiesterase type 5 (PDE-5) inhibitor sildenafil  and the As discussed above, research efforts aimed at understanding the initiation and progression of adverse ECM remodeling have generated awareness of a panel of responsible key-players, some of which have subsequently been tested as therapeutic targets in humans. It should be noted that most anti-fibrotic therapies for the heart that seemed promising in experimental models have not been evaluated in patients, while the ones that underwent clinical testing mostly amounted to data that are limited or conflicting. We here discuss clinical testing of drugs that target extracellular collagen processing and/or cellular responses of the cardiac fibroblast . No antiClinical evidence indicates that the mainstay therapy for LV systolic HF, inhibiting the RAAS axis, targets diffuse myocardial fibrosis. As outlined in section “Extracellular Matrix Remodeling in the Left vs. Right Ventricle,” cardiac fibroblasts express an AT1 receptor and respond to angiotensin II binding by ECM production and cellular proliferation , 93, 124via upregulation of MAPK pathways  antagonists are another class of established drugs for the treatment of LV systolic HF. Aldosterone can augment angiotensin II signaling, and also induce cardiac fibroblast proliferation independently of angiotensin II signaling, when binding to the MR pathways , 181. Thpathways . Howeverpathways . While tpathways , 185. Repathways . Above tDiuretics are in clinical use to unload the heart. In small studies with patients with hypertensive HF, addition of the loop diuretic torsemide to conventional HF therapy led to beneficial modulation of extracellular collagen processing that associated with improvements in ECM quality and LV function , 41, 131per se might represent a promising avenue for fibrosis treatment in the future and should be tested in large randomized controlled clinical trials. Of note, inhibition of LOXL-2 using the humanized monoclonal antibody simtuzumab has already been tested in phase II clinical trials in patients with hepatic or pulmonary fibrosis, demonstrating the feasibility direct targeting of collagen cross-linking  assessing RV fibrosis as one of the outcomes. This trial is a phase 4 clinical trial investigating the effect of the mineralocorticoid receptor antagonist spironolactone in patients with chronic RV failure caused by either PH or cardiomyopathy. RV fibrosis will be assessed i) Should therapeutic strategies block chronic maladaptive forms of fibrosis, while preserving its adaptive function to confer chamber stability, or locally replace dying cardiomyocytes upon overloaded conditions? (ii) Is established reactive myocardial fibrosis reversible? To aid in the development of effective and safe therapeutic strategies, we are discussing these important considerations in the following section.Despite pre-clinical successes, clinical testing of anti-fibrotic compounds has been unsuccessful. Endeavors to develop therapeutic approaches for reactive myocardial fibrosis go hand in hand with fundamental questions, such as , and indirect assessment of diffuse interstitial fibrosis by T1 mapping of the myocardial wall, including native T1 differentiating normal from infarcted myocardium, post-contrast T1, and extracellular volume fraction (ECV)  . Howeveron (ECV) , 211. Inon (ECV) , both foon (ECV) . The molon (ECV) –216. Foron (ECV) –220 and on (ECV)  by tracipecimens . Importapecimens , 213. FuIn vitro studies using rat cardiac fibroblasts demonstrated that cardiac fibroblasts expressed an angiotensin II receptor that controls proliferative activities and is sensitive to the antagonist losartan  as a means to bridge time until a heart transplant becomes available, or as a final treatment option for end-stage HF, sometimes is followed by cardiac recovery so that the LVAD can be weaned off in some cases. Interestingly, pre-clinical studies demonstrated that replicating mechanical unloading in a rat model of TAC by heterotopic heart transplantation led to complete regression of cardiomyocyte hypertrophy, while fibrotic remodeling was significantly exacerbated . The autMechanistically, reversal of reactive cardiac fibrosis likely includes degradation or modification of the fibrotic extracellular matrix, and changes in cellular behaviors of activated fibroblasts and myofibroblasts such as proliferation and collagen I synthesis, apoptosis, senescence, dedifferentiation, and reprogramming. Further research would be needed to understand mechanisms of fibrosis resolution, which might benefit from newest technical developments, such as single cell RNA sequencing (scRNAseq), or molecular imaging techniques.Even though regression of reactive myocardial fibrosis is possible in some scenarios as mentioned above, complete fibrosis resolution and regaining of healthy state levels have never been achieved in humans. This observation supports the notion that further regression would require the development of additional fibrosis-resolving treatments. However, as reactive fibrosis develops in response to a mechanical stimulus such as pressure-overload it may play additional important adaptive roles by providing structural stability. Unless there is evidence of ongoing, unrestrained, and non-beneficial fibrosis at some point in the cardiac remodeling process, there might be therefore also potential negative consequences of inhibiting fibrosis. In any case, treating the underlying condition in parallel by for example reducing RV pressure-overload or aortic stenosis would be paramount.via T1 mapping and ECV assessment and captures existing ECM deposits in the heart. Molecular imaging would additionally enable visualization of active fibrogenesis of reactive fibrosis occurring in real-time. In addition, T1 mapping would allow reactive fibrosis to be graded, and the question if there is a point of no return for reversal of reactive fibrosis could be answered.Reactive fibrosis in the pressure overloaded LV and RV accompanies cardiac remodeling that leads to heart failure. Most studies aimed at understanding the impact of reactive fibrosis on cardiac function, physiology and patient outcomes have been conducted in the LV. A limitation of probably all human correlational studies is the small patient number and occasional inconsistencies in readouts that range from histology, imaging modalities to fibrosis-biomarkers. In addition, up to one-third of HFpEF patients have normal measures of myocardial fibrosis, which might call for stratification for individuals that might benefit from anti-fibrotic therapies. Lastly, timing of anti-fibrotic drug administration might be an important factor to be considered for clinical trial designs to optimize efficacy and safety. Non-invasive imaging could be used to guide optimal timing and monitor treatment response. CMRI can be used to determine reactive fibrosis in vitro and in vivo experiments. The mayor limitation of in vitro studies is the lack of healthy fibroblasts in a culture dish due to immediate differentiation into myofibroblasts that is induced by the strain of the plastic surface. This imposes difficulties not only in understanding differences in the healthy vs. activated fibroblast, but also dissecting the role of myofibroblasts vs. proliferating resident fibroblasts in myocardial remodeling. Cardiac fibroblast markers used in vivo mostly lack specificity and for that reason, cardiac fibroblast-specific ablation of factors of interest remains challenging. However, it should be noted that emerging newer technologies, such as scRNA seq, might help in these quests. Most recently, a vast heterogeneity in cardiac fibroblast populations has been uncovered using scRNAseq (Fibrosis research has been hampered by technical limitations affecting both scRNAseq , and difMost of our perception on reactive fibrosis stems from studies focused on the LV. Further research is needed to understand the contribution of reactive fibrosis in the RV with regards to RV dysfunction, arrhythmias, and creation of hypoxic conditions. In addition, RV fibrosis as predictor of survival of patients is currently unknown. RV biopsies of the RV free wall would put patients at risk for perforation, therefore the use of non-invasive imaging and biomarkers to assess reactive fibrosis in addition to the meticulous study of human heart tissue at the time of transplant or surgery, would be the first-line modalities to address these questions.Despite all the above-mentioned challenges in fibrosis research, the observation that fibrosis regression is possible, is promising and a fascinating area to study. Understudied research areas include the process of intracellular collagen degradation, namely phagocytosis of macrophages and cardiac fibroblasts  as well KS and ES conceptualized the review. KS and KI wrote the manuscript. KS created the figures and table. ES, SR, and FH critically reviewed and provided feedback and guidance. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lactate is the basic blood parameter in the arsenal of an intensivist when managing a critically ill patient. A 62-year-old male presented with nausea and vomiting. He had been using an Ayurvedic medication, Insulin Management Expert (IME-9), for his type 2 diabetes mellitus and was found to have severe lactic acidosis that was resistant to initial fluid resuscitation and Ayurvedic medicine-induced liver injury. He required admission to critical care for organ support and ultimately recovered. Because current literature on the adverse effects of this Ayurvedic medication, particularly hepatotoxicity, is limited, causality was determined using the adverse drug association tool Roussel Uclaf Causality Assessment Method (RUCAM), which determined this as a probable cause with a strong score of seven. As a result, our case adds a vital gear to the wheel of current research literature. According to current literature, elevated lactate and poor lactate clearance are associated with poor prognosis in critical care medicine . The conA 62-year-old man with a history of type 2 diabetes mellitus was admitted to the intensive care unit after experiencing nausea and vomiting for two days but no trauma, convulsions, or infectious symptoms. He was a non-smoker and drank no alcohol. A thorough medication history revealed that he had not taken his metformin for approximately two weeks and believed that these medications did not work; instead, he had been taking a herbal medication known as Insulin Management Expert (IME-9) for approximately 25 days which he used to take two tablets three times a day, 30 minutes before meals. His clinical exam revealed mild tachycardia (117 beats per minute), normal blood pressure, diffuse abdominal tenderness with no guarding, and no icterus. The rest of his exam was uneventful, with no signs of chronic liver or kidney disease.InvestigationsHis bloodwork initially revealed severe metabolic acidosis, with lactate levels that were too high to measure using arterial blood gas (ABG) analysis. Formal blood tests provided to the biochemistry department revealed a lactate level of 25.02 mmol/L as well as abnormally high transaminitis but normal albumin levels and a normal prothrombin time Table . The toxDifferential diagnosisWe ruled out common causes of hyperlactatemia, such as sepsis, tissue hypoperfusion, regional ischemia, and cardiogenic shock. Imaging was performed in tandem to rule out obstructive shock, mesenteric ischemia, and any significant cancer. During interrogation, there was no strong indication of seizures, strenuous exertion, or shivering. His ketones were normal. There were no signs of carbon monoxide, cyanide, iron, or any other chemical overdose or poisoning. Other potential causes of liver disease were thoroughly investigated, including various hepatitis viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), and varicella-zoster virus (VZV); autoimmune hepatic conditions; and hepatic vascular diseases such as Budd-Chiari syndrome and sinusoidal obstruction. He had no previous hospitalizations in the previous six months.TreatmentFluid resuscitation was performed using four liters of intravenous 0.9% saline guided by focused ultrasound and other usual measures for predicting fluid responsiveness; however, a repeat ABG after six hours revealed unrecordable lactate Table . ContinuOutcome and follow-upThe patient improved after three days of critical care and was stepped down to the medical gastroenterology ward. His liver function tests (LFTs) improved while he was under their care. Following adequate electrolyte replenishment and blood sugar control, the patient was discharged after a total of 10 days in the hospital with pre-booked follow-up with his general practitioner to monitor his LFTs.Lactate is a by-product of glycolysis that is produced in the central nervous system, erythrocytes, skin, and skeletal muscles. In the presence of adequate oxygen, glucose enters the tricarboxylic acid (TCA) cycle and the electron transfer chain, resulting in the formation of 38 molecules of adenosine 5′-triphosphate (ATP). In the absence of adequate oxygen, pyruvate is converted to lactate rather than initiating the TCA cycle, resulting in decreased ATP production. The liver removes half of this lactate, while the kidneys and muscles handle the other half. Lactic acidosis  and sepsis have been connected for more than 200 years, as has tissue hypoxia for more than 100 years [Cohen and Woods have identified two types of hyperlactatemia: type A, which is linked to inadequate tissue oxygenation in illnesses like sepsis, shock, hypovolemia, and extreme hypoxia; and type B, which is seen with sufficient oxygenation and regular hemodynamics and can be associated with certain medical conditions such as liver failure or cancer, medications or toxins, or genetic metabolic disorders . In our According to a research report published in the United States of America, the majority of herbal remedies used are based on extensive clinical and traditional experiences and have not been subjected to safety analyses through the drug-development process .The Roussel Uclaf Causality Assessment Method (RUCAM) is a tool for quantifying causality in cases of suspected drug-induced liver damage (DILI) and herb-induced liver injury (HILI) . The revThe RUCAM-based assessment has a high sensitivity of 86% and specificity of 89% [Mangifera indica), Karela (Momordica charantia), Gudmar (Gymnema Sylvestre), Jamun (Syzygium cumini), and Shudh Shilajit , as described in a review article by Gajarmal, which also mentions a double-blind clinical human trial study of >800 patients, the details of which could not be sought on a thorough literature search with all possible keywords [Many people around the world seek traditional alternative medicine to treat their diabetes. Nearly half of the diabetic patients take both oral hypoglycemic medicines and herbal drugs; however, only around 5% of these people consult with an Ayurvedic practitioner before utilizing these herbs . This cokeywords . Furtherkeywords . The scakeywords .Lactate measurement and clearance are critical components in the prognosis and therapy of septic shock patients. Lactic acidosis includes many more differential diagnoses than septic shock and hypoxia, about which we should be very careful before prognosticating such patients, combined with an awareness of lactate metabolism physiology. Although the mechanism is uncertain, according to the RUCAM criteria for HILI and DILI etiology, IME-9 ingestion is associated with hepatotoxicity and hence lactic acidosis, but more research is needed to determine the specific mechanism of this.


Ovarian hormones, particularly 17β-estradiol, are involved in numerous neurophysiological and neurochemical processes, including those subserving cognitive function. Estradiol plays a key role in the neurobiology of aging, in part due to extensive interconnectivity of the neural and endocrine system. This aspect of aging is fundamental for women’s brains as all women experience a drop in circulating estradiol levels in midlife, after menopause. Given the importance of estradiol for brain function, it is not surprising that up to 80% of peri-menopausal and post-menopausal women report neurological symptoms including changes in thermoregulation (vasomotor symptoms), mood, sleep, and cognitive performance. Preclinical evidence for neuroprotective effects of 17β-estradiol also indicate associations between menopause, cognitive aging, and Alzheimer’s disease (AD), the most common cause of dementia affecting nearly twice more women than men. Brain imaging studies demonstrated that middle-aged women exhibit increased indicators of AD endophenotype as compared to men of the same age, with onset in perimenopause. Herein, we take a translational approach to illustrate the contribution of ovarian hormones in maintaining cognition in women, with evidence implicating menopause-related declines in 17β-estradiol in cognitive aging and AD risk. We will review research focused on the role of endogenous and exogenous estrogen exposure as a key underlying mechanism to neuropathological aging in women, with a focus on whether brain structure, function and neurochemistry respond to hormone treatment. While still in development, this research area offers a new sex-based perspective on brain aging and risk of AD, while also highlighting an urgent need for better integration between neurology, psychiatry, and women’s health practices. Sex differences in disease prevalence, manifestation, and response to treatment are rooted in genetic and hormonal differences between men and women. The effects of sex on neural aging phenotypes are often as large as, if not larger than the effects of other important variables . In factFor decades, the general mindset was that sex differences in brain structure and function were controlled by a unitary program: genetic sex as the determinant of gonadal sex, and gonadal hormones as the determinants of brain sexual differentiation and subsequent neurophysiological and behavioral outcomes . EvidencThe value of understanding sex differences in brain aging and neurodegenerative disease is as self-evident as it is underappreciated. Historically, for multiple reasons, including the purported safety of women and their offspring, women of childbearing age were excluded from clinical trials . As a reThe field of cognitive aging and AD is no exception as sex and gender are more likely to be used as confounders than predictors. As of 2022, of all clinical trials of AD, none has set out to examine sex differences in efficacy or outcomes . Recent This review explores the role of ovarian steroid hormones, especially 17β-estradiol, as contributors of brain aging and risk of AD or dementia. Endogenous exposures to ovarian hormones include chiefly pubertal timing, the menstrual cycle, pregnancy, and menopause. Exogenous hormonal exposures include chiefly use of hormonal therapy such as oral contraceptives and menopause hormone therapy (MHT). Throughout the review, the emphasis is on studies that used brain biomarkers of AD, primarily brain imaging, conducted in cisgender women with sound methodology.We conducted a systematic review of neuroimaging studies of the menstrual cycle, oral contraceptives, menopausal status, and randomized clinical trials of MHT, as well as of imaging studies of pubertal timing and pregnancy as related to cognitive aging and AD risk. We also provide a narrative review of psychometric studies of all exposures. We searched PubMed and the Web of Science for papers published in English between 1998 and 2022, using “estrogen,” “sex steroids,” “ovarian hormones,” or “sex hormones,” all exposures and outcomes as search terms. Although we tried to cite seminal studies as necessary, because of space limitation, representative reviews were also selected. We also provide general information on the actions of ovarian steroid hormones in brain to provide context for the research findings linking these hormones to brain aging and AD risk.The primary hormones secreted by the ovaries are 17β-estradiol , the most prevalent form of estrogen produced before menopause, and progesterone, a type of progestagen. Both hormones pass through the blood–brain barrier and have receptors throughout the brain . As reviEstradiol is a steroid hormone synthesized in a series of enzymatic steps beginning with the conversion of cholesterol into pregnenolone in the mitochondria . The finEstradiol is also locally synthesized in different tissues, including brain. Recent research demonstrates that the brain is a steroidogenic organ  expressiMoreover, there is some evidence that the brain upregulates synthesis of neurosteroids in response to the drop in estrogen following oophorectomy as a compensatory adaptive reaction . This suEstradiol has been called the “master regulator of the female brain”  due to iEstrogen therapy reduces such damage , normaliAdditionally, genetic studies have identified variants of the gene encoding for the aromatase enzyme that are associated with an increased risk for AD  These geEstrogen receptors (ERs) also coordinate multiple neuroprotective signaling cascades, either via direct activation or by the interaction of ERs with the receptors for other neuroprotective factors. Estradiol action in brain can be both delayed in onset and prolonged in duration (“genomic”) or rapid in onset and short in duration (“non-genomic”) . Both ERERα and ERβ are also implicated in modulating the immune system. Both receptors are expressed on microglia and astrocytes, both involved in neuroinflammation and implicated in Alzheimer’s disease . ActivatOvarian hormones affect the nervous system in ways that extend beyond their essential actions of regulating gonadotropin secretion and modulating sexual behavior. As reviewed below, at a neurological level, estrogens are involved in regulation of thermoregulation, mood, sleep, and cognitive abilities, among other factors . From a In what follows, we review research elucidating the role of ovarian steroid hormones in cognitive aging and AD risk across the female lifespan, including studies of puberty, menstrual cycle, hormonal contraceptive use, the menopause transition, and hormone therapy for menopausal symptoms.Puberty is characterized by surges in the production of sex hormones, which in turn prompt dramatic organizational changes in the brain, followed by transformative changes in cognition and behavior . For girThere is ample evidence that ovarian sex hormones influence brain development and cognition during adolescence. While reviewing these findings is beyond the scope of this review, we recommend prior reviews on the topic . Herein,A recent hypothesis posits that the brain has declining sensitivity to sex hormones throughout adolescence, such that females who mature early have greater effective ovarian hormone exposure than those who mature late . The age42/40 ratio and higher hyperphosphorylated tau levels among older post-menopausal women free of dementia  is reduced during the late follicular phase, opposite the pattern observed for the hippocampus . In the Functional MRI studies also provide evidence of differential activation patterns during the menstrual cycle. A recent systematic review of neuroimaging studies indicates increased prefrontal cortical activity during cognitive tasks during the mid-luteal phase . There iAdditionally, resting state fMRI studies indicate that some regions within the Default Mode Network (DMN) are more connected in the early follicular phase, when estradiol and progesterone levels are low . Instead18F]fluorodeoxyglucose (FDG) PET, cerebral glucose metabolism (CMRglc) was higher in thalamic, prefrontal, temporoparietal, and inferior temporal regions in the mid-follicular as compared to the luteal phase, whereas CMRglc in superior temporal, occipital, cerebellar, cingulate and anterior insular regions was higher in the luteal as compared to the follicular phase  binding during the follicular phase in naturally cycling women   . Howeverex (PFC) , thus adAltogether, neuroimaging results indicate that hormonal changes during the menstrual cycle may impact widespread networks involved in memory, learning, attention, and emotion. It is possible that, as effects of ovarian hormones on synaptic activity are generally subtle, neuroimaging might be more sensitive to detecting these changes than cognitive tests. Since most fMRI studies show no links to cognitive performance despite detecting activational changes during the menstrual cycle, it’s been hypothesized that not all effects of ovarian hormones might immediately translate to changes in cognition . It is aHormonal contraceptives consist either of a synthetic progesterone , or a progestin and a synthetic estrogen . These exogenous hormones control ovulation by inhibition of follicular development, and suppression of the production of endogenous estradiol and progesterone . HormonaGiven the effects of ovarian hormones on brain structure and function, examination of the effects of OCs on cognitive aging and AD risk provides important information for preventative efforts. Nonetheless, few studies have investigated whether OC use influences cognition. Most of these studies were conducted on young adult women, while a handful examined associations between OC use in young adulthood and midlife, and future risk of cognitive decline in older age. While some studies report no differences in cognitive performance between young adult women with natural cycles and OC users , others Neuroimaging studies of OC use are summarized in Most fMRI studies report an overall lack of performance differences between OC users and naturally cycling women during processing tasks , althougOverall, research concerning OC effects on cognitive aging is just emerging. Although samples are small and differences between OC formulations were not reported in most studies, there is some indication that exogenous hormones influence brain volumes among young adult OC users, and may play a role in verbal functions, consistent with research on the menstrual cycle. Future systematic work is needed to better elucidate androgenic vs. anti-androgenic OC effects on cognitive health, and to probe between OC use pre-menopause and cognition post-menopause. Given the widespread use of OC, this work carries significant implications.Pregnancy induces significant changes in endogenous estrogen levels, with reported effects on brain structure and function . High leNonetheless, the vast majority of studies have focused on the short-term effects of pregnancy and postpartum on brain structure, function, and cognition, with the longest follow-ups conducted at 2–6 years postpartum . StudiesThere is some evidence for a positive effect of pregnancy on cognitive aging. Several studies have reported that midlife women who had experienced pregnancy exhibited better cognitive performance in verbal and visual memory performance , and anoHowever, other studies report detrimental effects of pregnancy on cognitive aging. Compared to nulliparous women, parous women had greater cognitive decline on Mini-Mental State Examination (MMSE) scores , increasOther studies have reported null associations between parity and cognitive performance or dementia risk . In the Discrepancies may be in part due to how studies define parity. Studies defining parity as the number of childbirths or time spent pregnant more commonly report associations with cognition as compared to studies defining parity as parous vs. nulliparous. The number of children may play an important role, studies report having 1–4 children provides neuroprotection in women , having While neuroimaging results are also mixed, MRI studies generally report positive effects of pregnancy and parity on structural brain aging . Two larOverall, studies investigating the associations between pregnancy and later life cognition are limited by small samples, heterogeneity of cognitive assessments and diagnostic criteria, possible inclusion of non-biological children, and different exposure variables. Pregnancy-related factors, including age at first birth, breastfeeding, or complications such as gestational diabetes or pre-eclampsia, have rarely been considered yet may have significant contributions. Later life cognitive testing or dementia diagnosis may also contribute to contrasting results, as the effects of pregnancy are likely more apparent closer to the time of childbirth than many years later after cumulative experiences have affected the brain.Menopause represents the permanent cessation of ovulation and menstrual cycles, which is defined retrospectively, after 12 months of amenorrhea without obvious pathologic cause . HormonaThe average age at spontaneous menopause in industrialized countries is 49–51 years . TherefoSpontaneous menopause is a normal physiological event without long-term adverse effects for the majority of women . HoweverNonetheless, whether menopause-related cognitive complaints can be confirmed objectively is a topic of debate . The firOverall, studies including surgical and spontaneous menopause cases indicate measurable, yet modest declines in verbal episodic memory on delayed recall tests, or lack of improvement in verbal memory and processing speed with repeated testing . In someSome studies indicate that cognitive changes are possibly transient, as evidenced by longitudinal reports suggesting that they are mostly present at the peri-menopausal and early post-menopausal stages, with a rebound to almost pre-menopausal levels after menopause . In the Importantly, memory declines during perimenopause and early postmenopause ranged from subtle to moderate, and remained within normal limits for age and education in most studies . MoreoveAlthough neuroimaging research of menopause is scant, and the majority of studies has been carried out in women who had already transitioned through the menopause, recent translational neuroimaging studies corroborate animal findings by showing associations between menopause and biomarker indicators of AD risk in midlife women . NeuroimRecent multi-modality neuroimaging investigations targeting women at different menopausal stages , all carrying risk factors for AD, such as ApoE4 genotype and a family history of late-onset AD, demonstrate emergence of AD endophenotypes in women of peri-menopausal age . AD endoLongitudinal evaluations showed progressive AD biomarker abnormalities in the menopause transition, including chiefly declines in hippocampal and temporal lobe GM volumes, CMRglc declines in temporal regions and PCC, and increased Aβ deposition in frontal cortex , 2021. F31Phosphorus Magnetic Resonance Spectroscopy (31P-MRS), reflecting higher ATP synthesis  includes oral and transdermal preparations thought to have systemic effects, and localized administrations  that do not have systemic effects. Herein, we focus on systemic MHT. The treatment of choice for women with a uterus consists of combined (opposed) estrogens and progestins. The treatment of choice for women without a uterus is unopposed, or estrogen-only therapy. Estrogens can be estradiol or conjugated equine estrogens (CEE). Progestins vary in their hormone derivatives, as in hormonal contraceptives. The most commonly used are medroxyprogesterone acetate (MPA) and micronized progesterone.There is a relatively large literature on MHT effects on brain and cognitive aging. In spite of this, results of whether MHT is viable for support of cognitive function and AD risk reduction are mixed. The hypothesis that MHT might protect against AD arose in large part from early observational studies and small clinical trials demonstrating a protective effect of MHT on cognitive function and AD risk among MHT users compared with never-users , especiaRandomized, placebo-controlled clinical trials of MHT for AD prevention have also provided conflicting results. The first large trial to test MHT for dementia prevention was the Women’s Health Initiative (WHI). The WHI included two studies, the WHI Estrogen-plus-Progestin Study, in which women with a uterus were randomly assigned to receive either combined MHT (Prempro) or a placebo; and the WHI Estrogen-Alone Study, in which women without a uterus were randomly assigned to receive either estrogen-alone therapy (Premarin) or a placebo. Cumulatively, the WHI showed some benefits related to use of MHT, including one-third fewer hip and vertebral fractures, and one-third lower risk of colorectal cancer relative to placebo . Howevera priori primary outcome of interest, all-cause dementia became the default primary outcome because of the lack of a sufficient number of AD cases at follow-up. In a sample of 2,947 post-menopausal women with prior hysterectomy, there was no evidence that CEE lowered the risk of all-cause dementia , which examined the impact of MHT for dementia prevention among women ages 65 and older, thus in late post-menopause. These studies focused on oral CEEs in women with prior hysterectomy, and CEE/MPA in naturally post-menopausal women . Althougdementia . Howeverdementia . Thus, tIn terms of MHT effects on cognition, the WHIMS trial found that both opposed and unopposed therapy were associated with slightly worse mean scores in global cognitive function compared to placebo . These eHowever, the newer Early versus Late Intervention Trial with Estradiol (ELITE)-cog and Kronos Early Estrogen Prevention Study (KEEPS) trials have reported no beneficial or adverse effects of MHT on cognition among recently post-menopausal women within 6 years of the menopause diagnosis . NonetheTo date, eight meta-analysis have examined the neuroprotective effects of MHT on AD risk . Early mThere is some evidence that MHT may facilitate maintenance of some aspects of cognition when initiated in early post-menopause or prior. Verbal memory is consistently seen to be maintained or sometimes enhanced with estrogen-alone treatment. A review of randomized, placebo-controlled trials of MHT and verbal memory indicate a beneficial effect of estrogen alone therapy in women younger than age 65, especially surgically post-menopausal cases . AdditioClinical trials using brain scans as endpoints lend support to the hypothesis that both age at treatment initiation and type of MHT are important factors to consider. As summarized in Structural MRI studies reported less consistent evidence of protective effects of MHT. Some report greater GM volumes in MHT users versus non-users  or versuThere are also reports of increased white matter hyperintensities with MHT use  althoughIn addition, randomized controlled trials that incorporated fMRI indicated a higher activation of fronto-cingulate regions and hippocampus during verbal, non-verbal and spatial working memory tasks, although results are not always consistent . Since tOverall, brain imaging studies of MHT suggest a putative positive role of estrogen against regional cerebral atrophy and metabolic decline, with an advantage of unopposed over combined MHT , and of In conclusion, active debate remains on whether MHT has value for neuroprotection. Natural history studies and some clinical trials suggest that MHT may support cognition and brain function in peri-menopausal and recently post-menopausal women. However, most studies demonstrating benefits are based on observational studies, or studies of younger women which may have better captured the critical window for MHT action vs. larger clinical trials of older post-menopausal women. Observational studies are subject to bias as women who choose to use MHT have in general higher education, and tend to have healthier lifestyles and better overall health before and after taking MHT than women who do not . Taking Understanding sex-driven effects of ovarian hormones on dementia risk is a crucial step toward development of precision medicine strategies for AD prevention. In recent years, significant progress has been made in discovering how ovarian steroid hormones influence cognitive aging, prompted in part by advancements in the research on sex differences in AD. Across the female lifespan, there is compelling evidence that estradiol levels influence brain structure, function, and biochemistry in many regions affected by AD. There are also increasing indications of complex interactions of estradiol with other sex hormones, chiefly progesterone and androgens.In this review we examined the effects of puberty, the menstrual cycle, hormonal contraceptives, menopause, and MHT on cognitive aging and neuroimaging biomarkers of AD. While this field is still in its infancy, there is increasing evidence for associations between indicators of estrogen exposure, such as pubertal timing, menstrual cycle frequency, number of pregnancies, and OC use, and cognitive function over the course of a woman’s life .More work has been done to investigate changes in cognition and AD biomarkers during the transition to menopause, and more so as due to MHT use. Clinical studies indicate a dip in cognitive performance, mostly verbal memory, during peri-menopause, possibly followed by a rebound post-menopause. Significant heterogeneity has been noted as related to age, menopause status, use of MHT, and genetic risk factors. Hardly any clinical studies analyzed data in relation to women’s existing genetic predisposition to AD or other neurological conditions. On the other hand, neuroimaging studies of midlife women at genetic risk for AD have provided robust evidence for emergence of AD endophenotypes with onset in peri-menopause among natural cyclers . SurgicaMenopause hormone therapy use has been heavily scrutinized due to the disparity between basic science, observational studies, and large randomized clinical trials. Overall, MHT action on brain is dependent on multiple factors, including chronological age, stage of reproductive aging, duration of hypogonadism, and presence of symptoms, as well as the formulation of MHT, route of administration, and the health status of the brain. Currently, MHT is not indicated to alleviate cognitive complaints or for AD prevention. However, some argue that MHT given to healthy peri-menopausal and early post-menopausal women under age 60 for about 5 years may be recommended for support of cognitive function with careful consideration of other risks . There iin vitro and in vivo, ApoE expression can be differentially regulated either by 17-beta-estradiol or specific agonists, depending on activation of ER subtypes  demonstrated safety and established the pharmacokinetics profile of PhytoSERM (Previous work has shown that, subtypes . These dsubtypes , for AD subtypes . The inihytoSERM . ResultsIn conclusion, ovarian steroid hormones are long overlooked but critical contributors to brain aging and AD risk. While the neurobiological consequences of hormonal activity have only begun to be understood, converging evidence supports a role for cumulative estrogen exposure in reducing risk of developing AD later in life. This strongly argues for continued examination of sex hormones and reproductive history factors in AD prevention strategies for women. There is an urgent need for prospective epidemiological, clinical and biomarkers studies with data taken at several time-points starting at midlife that examine the associations between lifetime estrogen exposure and neurological function in later life. Understanding the dynamic interplay between sex, chronological aging, endocrine aging, and additional AD risk factors is crucial to inform and justify primary precision-medicine strategies for AD prevention.LM and SJ discussed the concepts and wrote the manuscript. ES, GJ, CB, JD, SP, and RD reviewed the literature and provided critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Sport-related concussion (SRC) is a major concern among athletes and clinicians around the world. Research into fluid biomarkers of SRC has made significant progress in understanding the complex underlying pathophysiology of concussion. However, little headway has been made toward clinically validating any biomarkers to improve the clinical management of SRC. A major obstacle toward clinical translation of any fluid biomarker is the heterogeneity of SRC overlapping with multiple physiological systems involved in pathology and recovery. Neuroinflammation post-SRC is one such system that may confound fluid biomarker data on many fronts. Neuroinflammatory processes consist of cell mediators, both within the central nervous system and the periphery, that play vital roles in regulating the response to brain injury. Further, neuroinflammation is influenced by many biopsychosocial variables present in most athletic populations. In this commentary, we propose that future fluid biomarker research should take a systems biology approach in the context of the neuroinflammatory response to SRC. We highlight how biological variables, such as age, sex, immune challenges, and hypothalamic-pituitary-adrenal (HPA)-axis responses to stress, may alter neuroinflammation. Further, we underscore the importance of accounting for health and lifestyle variables, such as diet, exercise, sleep, and pre-morbid medical factors, when measuring inflammatory markers of SRC. To successfully move toward clinical translation, fluid biomarker research should take a more holistic approach in study design and data interpretation, collecting information on hidden variables that may be influencing the neuroinflammatory response to SRC. These biomarkers are then measured in their respective fluid mediums and assumed to be representative of the physiological state of the brain after injury. However, this approach ignores basic principles of systems biology, where multiple networks of physiological processes may influence levels of a particular biomarker or confound the relationship between biomarker and brain damage. For instance, ongoing secondary injury processes may alter the concentration of biomarkers that do not directly measure neuroinflammation  over time, which are further compounded by fluctuating blood–brain barrier (BBB) permeability throughout recovery.8Considerable scientific progress has been made toward the discovery, optimization, and validation of fluid biomarkers of sport-related concussion (SRC). Leading candidate biomarkers include glial fibrillary acidic protein, ubiquitin-C-terminal hydrolase 1 (UCH-L1), Tau, neurofilament light (NF-L), and inflammatory cytokines .The neuroinflammatory response to TBI, including SRC, is highly complex, incorporating multiple biopsychosocial variables that should serve as a contextual framework for future SRC biomarker research. This commentary aims to highlight key variables that may confound much of the research currently being done on both brain injury and neuroinflammatory markers of SRC.9 Last, there are many lifestyle factors that can influence inflammatory processes like exercise, diet, sleep, and medications. All these confounding factors  that make it a difficult field to study in heterogenous conditions like SRC. Age and sex are variables that must be considered for almost every aspect of SRC pathology and recovery in terms of immunological changes throughout development or sexually dimorphic inflammatory responses to injury. Moreover, many hormones exhibit regulatory functions over immunological processes. The effects of stress  must also be considered given the bidirectional communication between the hypothalamic-pituitary-adrenal (HPA) axis and the immune system.tors see  contribu10–12 and anatomical vulnerabilities .13 Various mediators of neuroinflammation  show age-related changes that could influence measures of inflammation and subsequent markers of brain damage. For example, microglia have shown age-dependent regional functional and morphological heterogeneity in addition to more dominant immune-vigilant phenotypes in the developing brain.14 Peripheral immune cell composition changes throughout development, too. For instance, healthy leukocyte counts have been shown to peak in children <9 years of age, whereas neutrophil counts were highest between the ages of 15 and 18 years.15Age is an important variable in SRC and neuroinflammation, especially considering the physiological differences in the developing brain 16 Inflammatory responses also change throughout adulthood given that both healthy aging and age-related diseases are associated with dysregulation of nuclear factor-κB (NF-κB) pathways and cytokine networks.17 These age-related differences in immune function likely result in altered contributions to the neuroinflammatory response to SRC that should be considered in all biomarker studies. In the context of adolescent athletes , investigators may consider measuring puberty status using physical exams, hormonal measures, picture-based interviews, or self-report tools  to examine associations between neurodevelopment and biomarker outcomes.18In addition, reduction in monocyte chemotaxis has been reported in children compared to adults.19 Estradiol and progesterone also interact with cells of the CNS, mediating many effects through activation of estrogen and progesterone receptors and downregulation of inflammasome activity (a proinflammatory process driven by NF-κB activation).20–22Biological sex is another variable that has frequently been reported as a modifier of recovery post-TBI, but has been largely ignored in the literature concerning neuroinflammation post-SRC. Pre-clinical models are often used to study inflammatory responses to mild TBI given the ability to maintain homogenous conditions in a model organism. To that effect, there has been a major bias toward the male sex given the layer of complexity added by the female menstrual cycle. The menstrual cycle introduces variations in circulating estrogen and progesterone, which can have many anti-inflammatory effects, both centrally and peripherally.19 In addition to female hormone cycles, testosterone levels also fluctuate in males23; however, the influence of testosterone has not been taken into consideration in SRC biomarker studies to date. Peripheral immune cell composition may be partially regulated by circulating hormone levels given the expression of estrogen, progesterone, and androgen receptors found on many immune cells and the sexually dimorphic responses to immune challenges (including brain injury).24–27 Whereas sampling sex hormone levels on all study participants would be ideal, a feasible alternative for females could be to implement self-reported menstrual cycle surveys to investigate associations between menstrual regularity and timing and the inflammatory variables in question.In animal TBI studies, estrogen and progesterone have been shown to suppress proinflammatory cytokine production  in microglia and overall astrogliosis, increase levels of anti-inflammatory cytokines , and reduce levels of cerebral edema.29 GCs regulate inflammation with both pro- and anti-inflammatory actions depending on the context and chronicity of the stressor. In response to acute stressors, GCs display anti-inflammatory actions mediated through binding of glucocorticoid receptors (GRs), which are then translocated to the nucleus where they interact with GC responsive elements to block transcription of proinflammatory factors .9 GRs are expressed all over the body, including many cell mediators of systemic and neuroinflammation. Microglia exhibit high GR expression and are prime targets for GC-induced immunosuppression.30Physical stress in the form of injury  and psychological stress  activate the HPA axis, resulting in a release of glucocorticoids  throughout the body to restore homeostasis.31 GCs can also act on endothelial cells of the BBB to increase expression of tight junctions, thus decreasing permeability to peripheral immune cells and dampening neuroinflammation.33 In the context of SRC, potential HPA-axis dysfunction could lead to decreased GC secretion, unchecked neuroinflammation, and an impaired stress response.35 Additionally, animal TBI studies have shown how associated peripheral injuries may influence inflammation and recovery after brain injury, which may be relevant in clinical SRC studies.37Peripheral immune cells also express GRs, and T cells have even been reported to undergo GC-mediated apoptosis to help reduce inflammatory actions.9 Similar events are thought to occur as a result of psychological stressors priming and increasing microglial reactivity.38 Acute psychological stress has been shown to increase levels of circulating IL-1β, TNFα, and IL-6 for up to 2 h in humans.39 Chronic stress  can influence systemic inflammation and the response to a traumatic event as well. For example, social determinants of health  have been associated with chronic inflammation.40The opposite effect can occur where the HPA axis is excessively activated, priming microglial reactivity in the presence of excessive GC levels, resulting in maladaptive inflammation and increased neuronal death.41 and elevated proinflammatory cytokines in circulation of those with chronic mental health disorders like post-traumatic stress disorders or major depression.43 Given the overlap of non-specific symptoms between mental health disorders and SRC, it is reasonable to assume that psychological distress influences neuroinflammation through HPA-axis activity, which may result in altered biomarker concentrations and clinical outcomes. Considering the complex biological response to stress, researchers may benefit from evaluating cortisol levels, asking questions pertaining to various social determinants of health, and implementing psychological screening tools in their assessments.Studies examining the consequences of chronic stress have also shown increased monocyte mobilization from bone marrow44 High-calorie diets have also been linked to increased neuroinflammation, thought to be one of the main drivers of various cognitive and mood disorders.45 Conversely, diets like the Mediterranean or ketogenic diet are associated with anti-inflammatory pathways, potentially attenuating neuroinflammation and positively influencing cognition.46 Alcohol and caffeine consumption have also been shown to influence both peripheral and central levels of inflammation.48 A novel, yet highly understudied, field in SRC research is beginning to discover links between nutrition, metabolism, and SRC outcomes, which might be partially attributable to changes in the gut microbiome and systemic inflammatory states.49–52Lifestyle factors that influence inflammation may be easily controlled in pre-clinical studies; however they are more difficult to account for in clinical SRC populations beyond self-reported measures. Examples of lifestyle variables include diet, exercise, and sleep quality. Many dietary variables have been shown to influence inflammation. For example, diets rich in trans-fats and high glycemic index carbohydrates lead to increased levels of proinflammatory cytokines in the general circulation.53 For example, IL-6 levels are released from muscle tissue after acute exercise whereas levels of TNFα and IL-1β are actively suppressed, all of which have been recently associated with SRC.54 Further, exercise has been shown to suppress microglial activation and downregulate the expression of many proinflammatory cytokines in the brain, mechanistically explaining (at least in part) the neuroprotective and therapeutic effects of aerobic exercise for SRC.56 Last, exercise influences regional macrophage function in: 1) skeletal muscle during physical activity; 2) adipose tissue during energy mobilization; and 3) circulation concerning the development/progression of atherosclerosis.53 These influences of exercise on various immunological and cytokine networks may obscure the use of inflammatory molecules as potential diagnostic or prognostic biomarkers for SRC and need to be taken into consideration in these studies.Given the athletic context in which SRCs occur, the influence of exercise on measures of inflammation requires careful consideration. Exercise can produce both pro- and anti-inflammatory effects that stem from localized muscle tissue and peripheral immune cells.57 Interestingly, sleep and immune function have their own bidirectional effects. Animal and human studies have shown sleep quality to change in response to infections, injury, and neurodegenerative disorders.58 Sleep deprivation has also been reported to increase the expression of IL-1β, IL-6, TNFα, and C-reactive protein.59 A potential mechanistic explanation (particularly relevant for SRC) lies in the intimate relationship between sleep and the HPA axis given that regulation of sleep onset and completion depends on the circadian release of cortisol.9 Conversely, sleep disruption impacts HPA-axis function, which may lead to hyperactivity in the stress response, which as outlined above, could exacerbate inflammation. Overall, many lifestyle factors are insufficiently documented and incorporated into current biomarker analyses, which will continue to limit their clinical translation to wider athletic and general populations. Investigators may consider asking targeted questions toward recent diet habits, exercise activity, and sleep quality at the time of sample collection.Sleep quality can also have a major impact on inflammation, especially given that sleep disturbance is a common symptom post-SRC.61 Exposure to natural allergens  may lead to increased inflammation and activated immune cells in persons with a pre-disposition to environmental allergies.62 Bacterial or viral infection would also activate a host immune response. Considering the current state of the COVID-19 pandemic and recent vaccination efforts, clinical SRC studies will need to document the type and recency of vaccine administration given that this will influence cytokine levels during data collection.64Last, there are multiple health-related factors that can influence inflammation. The use of non-steroidal anti-inflammatory drugs , exogenous corticosteroids , and even oral hormonal contraceptives may confound inflammatory data and must therefore be considered in clinical studies.Despite enormous progress over the past decade, research into fluid biomarkers of SRC remains largely in its infancy given a large focus on a handful of biomarkers at single, variable time points post-injury. Studies in general for neurological disorders (including SRC) have yet to successfully identify candidate markers of underlying pathophysiology that are associated with standard clinical measures of recovery. This remains one of the largest barriers to the clinical translation and validation of the leading candidate biomarkers of SRC. However, we may not surpass this obstacle without fully understanding the complex interaction between the neuroinflammatory response to SRC and fluid biomarker measures. Many covariates, such as age, sex, stress, and lifestyle factors, may seem trivial at first glance; however, they each introduce an abundance of hidden physiological variation in different contexts, which must be considered when designing and interpreting biomarker studies. Many questions remain around how, when, and to what degree these factors may influence aspects of neuroinflammation. Further, the methods of capturing these data should be carefully selected when assessing their impact on biological outcomes, and SRC biomarker investigators may benefit from collaboration with experts across multiple domains of inflammation. A more holistic approach, rather than reductionist approach, to studying fluid biomarkers of SRC may be more optimal in moving the field toward clinical translation given that the neuroinflammatory response to SRC is consistently shown to be greater than the sum of its parts.


Cellular metabolism is important for determining cell function and shaping immune responses. Studies have shown a crucial role for stromal cells in steering proper immune responses in the lymph node microenvironment. These lymph node stromal cells (LNSCs) tightly regulate immune tolerance. We hypothesize that malfunctioning LNSCs create a microenvironment in which normal immune responses are not properly controlled, possibly leading to the development of autoimmune diseases such as rheumatoid arthritis (RA). Therefore, we set out to determine their metabolic profile during health and systemic autoimmunity. We included autoantibody positive individuals at risk of developing RA , RA patients and healthy volunteers. All study subjects underwent lymph node biopsy sampling. Mitochondrial function in cultured LNSCs was assessed by quantitative PCR, flow cytometry, Seahorse and oleate oxidation assays. Overall, mitochondrial respiration was lower in RA(-risk) LNSCs compared with healthy LNSCs, while metabolic potential was only lower in RA LNSCs. To maintain basal mitochondrial respiration, all LNSCs were mostly dependent on fatty acid oxidation. However, RA(-risk) LNSCs were also dependent on glutamine oxidation. Finally, we showed that RA LNSCs have impaired metabolic flexibility. Our results show that the metabolic landscape of LNSCs is not only altered during established disease, but partly already in individuals at risk of developing RA. Future studies are needed to investigate the impact of restoring metabolic capacity in LNSC-mediated immunomodulation and disease progression. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by infiltration of immune cells into the synovium. RA-specific autoantibodies such as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs) are a hallmark of RA . These aDEAF1 transcription, which can induce autoreactive immune cell activation -oleic acid was used as a measure to determine the specific activity of the mitochondrial fatty acid β-oxidation flux in human cultured LNSCs, as described previously -oleic acid. However, we did not detect any significant differences in long-chain fatty acid β-oxidation between donor groups  LNSCs, we aimed to explore metabolic substrate used in these cells in more detail. Therefore, a Seahorse Fuel Flex assay was used which measures the use and preference of three main metabolic pathways sustaining basal mitochondrial respiration. This assay measures the oxidation dependency and flexibility of one of the metabolic substrates glucose, fatty acids or glutamine relative to the other two. Fuel dependency represents the reliance of LNSCs on a metabolic pathway to maintain basal mitochondrial respiration. RA-risk and RA LNSCs were non-significantly more dependence on glucose oxidation compared with healthy LNSCs A,B. FattIn addition to the Fuel Flex assay, we performed so-called Substrate Oxidation Stress assays which give an indication of fuel use during homeostasis and mitochondrial stress. Glucose, fatty acids and glutamine oxidation were individually inhibited to measure relative substrate demand of cultured LNSCs sustaining basal and maximum mitochondrial respiration. These measurements showed that basal mitochondrial respiration was significantly lower when glutamine oxidation was inhibited in healthy and RA LNSCs , indicatFinally, to investigate if a specific step in one of the main metabolic pathways is impaired and potentially causing the observed differences in the metabolic profile of RA (-risk) LNSCs, we measured mRNA levels of genes related to glucose, fatty acid and glutamine oxidation. However, under homeostatic conditions, no significant differences were found between the donor groups, partly due to high inter-individual variation . Cellular metabolism is crucial in modulating cell fate and function, thus also for the proper management of immune responses. Inflammation urges cells to enhance their energy production to sustain proliferation and the production of pro- and anti-inflammatory molecules. This suggests that altered cellular metabolism can contribute to dysfunctional immune responses, which is generally also associated with autoimmunity . MetabolTo investigate the metabolic profile of LNSCs during health and autoimmunity we compared LNSCs collected from patients with RA with LNSCs from autoantibody positive individuals at risk of developing RA and seronegative healthy volunteers. Of interest, we found that several metabolic alterations identified in RA LNSCs could also be detected in LNSCs from individuals at risk of developing RA.Metabolic alterations related to oxidative phosphorylation, such as basal and maximum mitochondrial respiration and ATP production, were already significantly lower in RA-risk LNSCs compared with healthy LNSCs and did not further decrease in RA LNSCs, suggesting these metabolic alterations take place before onset of clinical disease. We assume that the proliferation of LNSCs did not influence the detected metabolic alterations, since it has been shown that the proliferation phase of cultured LNSCs starts approximately 27 h after seeding . AlthougEnsuing the distinct differences found in mitochondrial respiration, we investigated whether mitochondrial characteristics such as mitochondrial mass, membrane potential, ROS production and mtDNA content are linked to these differences. The mitochondrial membrane potential has a window in which effects are desirable for providing energy reserves, as low mitochondrial membrane potential might reflect mitochondrial stress and lead to apoptosis, whereas high mitochondrial membrane potential might lead to excessive ROS production through the mitochondrial respiratory chain . We founMetabolic pathway oxidation assessment showed that compared to glucose and glutamine, LNSCs, similarly to tolerogenic DCs ,23, are Even though the real-time Seahorse measurements show a noticeable drop in total OCR after each of these three metabolic pathways is individually inhibited, not all mitochondrial respiration is inhibited. This might indicate that other metabolic processes contribute to basal and maximum mitochondrial respiration of cultured LNSCs. Other metabolic processes contributing to mitochondrial respiration could be, for instance, short- and medium-chain fatty acids, which do not rely on the carnitine shuttle that is inhibited by etomoxir, or amino acids other than glutamine. Additionally, non-mitochondrial oxygen consumption by other biochemical processes in LNSCs such as very long-chain fatty acids that are oxidized in peroxisomes and other cellular enzymatic processes that consume oxygen could contribute to basal respiration.To our knowledge, this study is the first to investigate mitochondrial function in cultured human LNSCs during health and disease. Unfortunately, the relatively low number of study subjects included in this study makes it hard to reach statistical significance. However, these unique human lymph node biopsy studies in the context of autoimmunity are extremely challenging to collect and analyze. Moreover, it is challenging to find seronegative healthy volunteers who would like to participate in such a study and LNSCs are typically slow growing cells, thus making these experiments quite labor intensive and time consuming. A limitation of our study setup is that we cannot inhibit all cellular metabolic pathways in parallel, so it is difficult to define the specific contribution of all active metabolic pathways to mitochondrial respiration.In conclusion, our results show that the metabolic landscape in LNSCs is already altered in autoantibody positive individuals at risk of developing RA, providing an interesting target for future therapies aimed at disease prevention. Mitochondrial dysfunction of LNSCs could disrupt homeostasis and induce loss of tolerance, chronic inflammation and ultimately joint damage. Future studies are needed to investigate how these metabolic alterations of the local stromal microenvironment regulate the immune response in the context of autoimmunity.