Identification and characterization of the limbal epithelial stem cells (LESCs) has proven to be a major accomplishment in anterior ocular surface biology. These cells have been shown to be a subpopulation of limbal epithelial basal cells, which serve as the progenitor population of the corneal epithelium. LESCs have been demonstrated to play an important role in maintaining corneal epithelium homeostasis. Many ocular surface diseases, including intrinsic  or extrinsic  insults, which impair LESCs, can lead to limbal stem cell deficiency (LSCD). LSCD is characterized by an overgrowth of conjunctival-derived epithelial cells, corneal neovascularization, and chronic inflammation, eventually leading to blindness. Treatment of LSCD has been challenging, especially in bilateral total LSCD. Recently, advances in LESC research have led to novel therapeutic approaches for treating LSCD, such as transplantation of the cultured limbal epithelium. These novel therapeutic approaches have demonstrated efficacy for ocular surface reconstruction and restoration of vision in patients with LSCD. However, they all have their own limitations. Here, we describe the current status of LSCD treatment and discuss the advantages and disadvantages of the available therapeutic modalities. The functions of cornea include protecting the delicate internal parts of the eye and allowing proper transmission of light. The corneal epithelium is the outermost layer of cornea, which is a crucial barrier against mechanical, chemical, and pathogenic insults. In fulfilling its barrier function, this self-renewing stratified epithelium turns over every 5–7 days. The self-renewal of the corneal epithelium is governed by the stem cells that reside in the basal layer of the limbal epithelium, adjacent to the corneal epithelium . The firLESCs are morphologically small, have a high nuclear-to-cytoplasm ratio, and are relatively undifferentiated cells with rare cycling and high proliferative capacity , 5. The Limbal epithelial basal cells are relatively undifferentiated and thus lack the expression of differentiation markers such as keratin 3, keratin 12 , and con3H-Tdr) or bromodeoxyuridine (BrdU). After a chasing period , the labeling signal in the rapidly dividing TA cells is diminished due to dilution or by transiting out of the tissue due to differentiation, whereas the slow-cycling stem cells still retain their labeling. Application of this labeling technique to mouse limbal/corneal epithelia revealed that the LRCs were exclusively localized in the basal layer of the limbal epithelium. In contrast, the peripheral and central corneal epithelia contained no LRC, which was compelling evidence that the corneal epithelial stem cells were located in the limbal epithelium [Stem cells are commonly believed to cycle infrequently . This chithelium , 19.LESCs have high proliferative capacity, which is demonstrated in vitro by an ability to generate holoclone colonies . On the µm [The limbal stem cell niche is a specific and highly regulated microenvironment, which is required for harboring and maintaining LESCs –26. It hµm . It is gClinically, LSCD is caused by the depletion or dysfunction of LESCs, which leads to the inability to sustain corneal epithelial homeostasis –35. PatiClinical treatment of the LSCD varies based on the severity and extent of involvement. For those patients with mild and moderate LSCD, treatments involve the control of the symptoms and causes. For patients with severe LSCD, it is necessary to undergo ocular surface reconstruction (OSR). OSR is a series of procedures to reconstitute the anatomic and physiologic ocular surface, including amniotic membrane transplantation (AMT), conjunctival limbal grafting, simple limbal epithelial transplantation (SLET), and cultivated limbal epithelial transplantation (CLET) , 38–41. Amniotic membrane transplantation (AMT) is a method to help recreate the integrity of the ocular surface. The amniotic membrane (AM) consists of an overlying basement membrane with a rich extracellular matrix, including heparin sulfate proteoglycans, laminin, and collagens. These components act as a scaffold for the epithelial cells , 42, 43.Traditional autologous limbal transplantation has a long history. In 1989, Kenyon and Tseng described a large series (26 consecutive cases) of conjunctival limbal autograft (CLAU) in patients with unilateral ocular surface diseases . A six-mFor patients with a total bilateral LSCD, allograft limbal stem cell transplant is one of the approaches to reconstruct the ocular surface . The conIn 2012, Sangwan et al. introduced a simple limbal epithelial transplantation (SLET) . In thisA multicenter study on 68 eyes from patients who underwent SLET for unilateral LSCD reported promising results . ClinicaCell therapy involves tissue engineering techniques and the idea of stem cell plasticity for achieving corneal epithelial regeneration. This approach represents new potential therapeutic modalities. The underlying principal is to use the least amount of tissue to ex vivo expand cells into an epithelial sheet on carriers and to reconstruct severely damaged ocular surfaces.2 limbal biopsy sample were expanded in vitro on a feeder layer consisting of nonproliferating 3T3-J2 feeder cells and a polymerized fibrin matrix. Confluent cultured limbal epithelial sheet was placed on a corneal wound bed. Two-year follow-up showed that the regenerated corneal epithelium was stable. In 2010, Pellegrini et al. reported a long-term clinical investigation of CLET with the large samples (112 LSCD patients) [Transplantation of autologous cultures of limbal epithelial stem cells was first reported by Pellegrini et al. . Two patatients) . In thisatients) , 67. IntA major challenge for CLET-based therapies is to maintain LESC homeostasis and enhance the self-renewal of LESCs in limbal epithelial cultures during ex vivo expansion. MicroRNAs (miRNAs) are emerging as important controllers of stem cell potency, proliferation, and differentiation –74. For It is well established that autophagy is required for stem cell homeostasis in various tissues . ConsistBilateral LSCD patients who have no remaining LESCs can turn to autologous cultivated oral mucosal epithelial transplantation (COMET). It has been shown that COMET is a feasible substitute for allogenic limbal stem cell transplantation without the need for long-term systemic immunosuppression –89.The cultivated oral epithelial cells formed a stratified tissue. The tissue-engineered oral epithelium expressed proliferation and progenitor markers Ki-67 and p63 in the basal layer of the cell sheets, suggesting that the epithelium had regenerative capacity . The traMesenchymal stem cells (MSCs) are a group of fibroblast-like multipotent mesenchymal stromal cells . MSC canBasic science has contributed greatly to our understanding of the location, function, regulation of proliferation, and differentiation of limbal epithelial stem cells. Conventional autogenic and allogenic conjunctival limbal transplantation is an effective method but is limited by tissue availability. To overcome the shortage of donor-based tissues, scientists now focus their attention on cell-based therapy. With the refinement of in vitro culture and expansion techniques, and improved scaffolds and matrices, it is anticipated that a new generation of regenerative procedures will be available for use in the clinic to ultimately resolve the problem of LSCD. Finally, an emerging idea that supplies factors in vivo to activate and preserve the remaining limbal stem cells may lead to a pharmacological therapy which will ultimately replace surgery for treatment of corneal diseases with limbal stem cell deficiency.


Introduction: Aging LGBT experience significant health disparities compared to their heterosexual counterparts. As a sexual minority, they have a high likelihood of acquiring physical and mental illnesses. Methods: This study investigated factors that are associated with reports of depression among LGBT. Data came from Project Gray Pride, a study of aging gay, lesbian, bisexual, and transgender men and women living in a metropolitan area in Texas (N=126). Linear regression analysis was used. Results: Results show a negative association between respondents who self-identified as cisgender female and depression compared to those who self-identified as transgender. However, social vigilance and reports of health symptoms positively correlate with depression. Additionally, the interaction term of gender and race showed that respondents who were non-Whites and female reported higher levels of depression relative to their counterparts who were White males or White transgender. Other factors such as HIV status, age, education were not statistically significant. Conclusion: This study shows that being socially vigilant increases depression among aging LGBT. Also, aging female LGBT who are non-White may have an increased burden of negative social experiences leading to high levels of depression. As the population of older LGBT is increasing, intervention efforts must take into account these important findings in order to devise sound policies and programs to improve mental health among LGBT.


Chronic Kidney Disease (CKD) is a highly prevalent disease with a substantial medical need for new and more efficacious treatments. The Nitric Oxide (NO), soluble guanylyl cyclase (sGC), cyclic guanosine monophosphate (cGMP) signaling cascade regulates various kidney functions. cGMP directly influences renal blood flow, renin secretion, glomerular function, and tubular exchange processes. Downregulation of NO/sGC/cGMP signaling results in severe kidney pathologies such as CKD. Therefore, treatment strategies aiming to maintain or increase cGMP might have beneficial effects for the treatment of progressive kidney diseases. Within this article, we review the NO/sGC/cGMP signaling cascade and its major pharmacological intervention sites. We specifically focus on the currently known effects of cGMP on kidney function parameters. Finally, we summarize the preclinical evidence for kidney protective effects of NO-donors, PDE inhibitors, sGC stimulators, and sGC activators. The number of patients with chronic kidney diseases has dramatically increased in recent years, driven by the constant increase in life expectancy and the steadily increasing prevalence of risk factors such as diabetes, obesity or hypertension. Besides the progressively declining kidney function harboring a poor prognosis for patients, enormous economic costs—especially for hemodialysis and kidney transplantation—cause a huge and constantly growing burden for healthcare systems. Therefore, intense research and clinical development efforts are ongoing to identify signaling pathways which are critical for maintenance of kidney function and to investigate new pharmacological interventions for the treatment of kidney diseases. In recent years, it has become obvious that, along with the natriuretic peptide (NP)/particulate guanylyl cyclase (pGC)/cyclic guanosine monophosphate (cGMP) signaling cascade, the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway plays a pivotal role for the regulation of physiological kidney function. The disruption of the NO/sGC/cGMP pathways results in the decrease of cGMP levels and can cause severe kidney pathologies. Within this review article, we summarize the current understanding how NO/sGC/cGMP systems regulate physiological kidney function by targeting renal blood vessels and vascular smooth muscle cells, but also by having an impact on renal innervation, glomeruli, mesangial cells, podocytes, macula densa and tubular cells. In addition, we discuss how impairment of this pathway in different functional kidney compartments leads to chronic kidney pathologies. Finally, we summarize the current pharmacological approaches, aiming to restore NO/cGMP signaling for the treatment of kidney diseases. Here, we focus on cGMP increase via NO donors, inhibition of cGMP degradation by PDE-inhibitors and direct stimulation of the soluble guanylyl cyclase (sGC) by sGC stimulators and sGC activators.In 1998, Drs. Robert Furchgott, Louis Ignarro, and Ferid Murad were awarded the Nobel Prize in Physiology and Medicine “for their discoveries concerning NO as a signaling molecule in the cardiovascular system”. These researchers unveiled the molecular nature of the “Endothelial Derived Relaxing Factor (EDRF)” to be the gas NO, which is produced by the endothelium and potently dilates blood vessels. Further research demonstrated that NO is of broad importance not only for the regulation of vascular tone via smooth muscle cell relaxation, but this signaling system is also active in a variety of other cells types. Depending on the site of action, it influences nerve function and synaptic transmission, has antifibrotic and antiproliferative effects, influences blood clotting and inflammatory processes and is present in adipocytes with impact on fat metabolism. The signaling cascade—especially for the vasodilatory effects—is very well understood and schematically summarized in l-arginine to l-citrulline, a reaction catalyzed in the presence of the nitric oxide synthase (NOS) enzyme. There are three different NOS isoforms identified in mammals: neuronal (nNOS/NOS1), inducible (iNOS/NOS2), and endothelial (eNOS/NOS3). eNOS-derived NO stimulates soluble guanylyl cyclase (sGC)—an intracellular receptor present in effector cells—which results in the generation of cGMP from guanosine triphosphate (GTP). sGC forms a heterodimer consisting of two subunits, α and β, and NO activates sGC by binding to the heme-group present on the β-subunit. However, the heme in sGC is prone to be oxidized (from Fe2+ to Fe3+), e.g. during oxidative stress, which leads to a less tight binding between the sGC and its cofactor, subsequently leading to a heme-free form of sGC (Apo-sGC)  , and Apo nNOS/NOS, inducib3+-sGC to the native Fe2+-sGC form, which is then again able to respond to NO and produce cGMP [Recently, it has been identified that the enzyme cytochrome b5 reductase 3 (Cyb5R3) is able to reduce Fe1 subunit is expressed in glomerular podocytes, whereas the β2 is found in principal cells of the collecting duct [1 subunit revealed that sGC is expressed largely in the renal vasculature and granular cells thus playing an important role in renal perfusion and renin release, respectively [The differential expression of enzymes isoforms in various cells and tissue types helps to directly regulate the relevant physiological function. In the case of NOS enzymes, as the names suggest, within the kidney, eNOS is expressed predominantly in the renal vascular endothelium but also in the tubuli, nNOS is expressed in the macula densa and in specialized neurons surrounding renal arteries and interstitial cells and although iNOS is quite lowly expressed in the normal kidney, expression increases in response to stimuli such as inflammation . Sullivaing duct . The useectively . While bectively . However+ channels in the tubules and collecting duct and antagonism of vasopressin in the collecting duct [Besides the NO/sGC/cGMP signaling, there is a second major source for cGMP production within the kidney. Renal cGMP levels can also be increased via stimulation of the particulate guanylyl cyclase (pGC) by natriuretic peptides. Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are produced and secreted predominantly from cardiomyocytes and endothelial cells, especially under disease conditions such as pressure overload and heart failure, and are involved in regulating sodium and water excretion and maintaining blood pressure. NPs work in an endocrine manner and bind to membrane-bound pGC which consists of an extracellular ligand-binding domain and cytoplasmic guanylyl cyclase domain. While there are at least seven pGCs identified, some of which are known to be stimulated by multiple ligands, it is well known that ANP and BNP specifically bind to pGC A (GC-A), whereas CNP binds to pGC B (GC-B). Both GC-A and GC-B are expressed in the kidney and have been shown to be of clinical relevance. Similar to cGMP derived from stimulation of sGC by NO, the physiological effects of cGMP derived from pGC stimulation are also mediated through the three target proteins, namely PKG, CNG ion channels and PDEs. Depending on the organ system(s) involved, the binding of ANP/BNP to GC-A induces vasodilation, natriuresis, and diuresis and in addition, it also has antifibrotic, antihypertrophic, and anti-inflammatory effects , whereasing duct . HoweverThe kidney consists of specialized morphological structures with specific functions . These f+, Cl−, K+ and Ca2+), passive or active water reabsorption, and also contribute to pH regulation. Each segment works in concert to maintain homeostasis. Tubules are peculiarly sensitive the effect of diuretics. The macula densa is a specific part of the distal tubule that is located close to the glomeruli arterioles. The macula densa not only regulates flow rate through the nephron but also sodium chloride concentration and osmolality of the tubular fluid. Its function depends ultimately on ATP. Like other organs, kidney function is also controlled by nerves that are widely connected to specific kidney structures  integrity is impaired, that leads to podocyte foot process effacement . Increasl-NAME was reduced by NO donors or cGMP agonist (8-bromo-cGMP) [In glomeruli and tubules of healthy and nephrotic rats, enzymes of cGMP production were found to be involved in the development and progression of kidney disease . In precmo-cGMP) , suggestmo-cGMP) . Improvemo-cGMP) . This efmo-cGMP) .Regulation of salt and water hemostasis is one of the major functions of the kidney and the tubular system. There is body of evidence that tightly regulated cAMP levels influence tubular electrolyte transport but the impact of the NO/sGC system on tubular transport is only partly understood and the available data are widely descriptive.+-reabsorption in the proximal tubule which suggest also a blood pressure lowering mechanism independent from cGMP-mediated vascular smooth muscle relaxation [In proximal tubules, the majority of sodium is reabsorbed, which is mainly mediated by sodium transporters, such as the sodium-hydrogen exchanger (NHE). It was shown that the activity of NHE3 on sodium retention was reduced by cGMP increase mediated by both, the NO/sGC and NP/pGC pathway . More relaxation . The levlaxation . Moreovelaxation .In the distal tubules, it is well established that ANP tightly regulates sodium-reabsorption but the role of the NO/sGC induced cGMP production is not yet characterized. There is indirect evidence that cGMP might play a functional role here since cGMP-regulated cation-channels were identified in the DCTL of the mouse . HoweverIn the collecting duct, cGMP derived by pGC signaling regulates the activity of the epithelial sodium channel (ENaC). However, it was also shown that NO could inhibit sodium reabsorption in isolated collecting duct preparations of rats . These dIn a rat model of hypertensive chronic kidney disease induced by NO synthase inhibition, bilateral renal denervation decreased local Renin-Angiotensin System (RAS) activity . These dBeside the role of cGMP/sGC/NO pathways in the development of CKD, acute kidney injury (AKI) also depends on cGMP/NO production. AKI is a pathology triggered by different types of insults that increase susceptibility to CKD development and progression . MitochoIncreasing circulating and tissue containing cGMP/NO exerts a plethoric role in maintaining capillary relaxation (in vessels beside macula densa as well as intra glomerular), preserving integrity of kidney structures  and reducing interstitial fibrosis and mesangial cells contraction and expansion as inflammation. Increasing NO/cGMP levels is foreseen as an effective treatment approach to oppose the loss of kidney structure integrity and functionality during acute and chronic kidney diseases. Therefore, hypertensive nephropathy, DKD, CKD could potentially be attenuated by such treatments, preventing kidney disease progression and end-stage renal failure.Given the broad importance of the NO/sGC/cGMP pathway for kidney health, pharmacological interventions causing cGMP increase could become an efficacious treatment option for CKD. Currently, a variety of approaches aimed to pharmacologically modify these pathways and increase cGMP are available or in under clinical and preclinical investigation. A prominent increase of cGMP could be established by either NO-supplementation by NO-donors or by direct stimulation of the sGC by using sGC stimulators and sGC activators, or by preventing cGMP degradation by blocking of PDEs. PDE5 and PDE9 are the major enzymes for cGMP-degradation and are both abundantly expressed in the kidney. Already in the 19th century, between 1860 and 1890, first descriptions for the beneficial effects of nitroglycerin (GTN) for the treatment of angina pectoris were published. Therefore, NO-donors and nitrates, which can enzymatically and non-enzymatically release NO, were the first pharmacological tools for influencing this pathway. The nitrate-derived NO binds to the heme group in the beta subunit of the sGC which triggers the formation of cGMP as described above. Nitrates are still in use for treatment of angina pectoris, however could not be used for chronic oral treatment due to physicochemical-properties, which are substantially limiting the use for chronic oral treatment regimens like they are necessary in chronic kidney diseases. In addition, nitrates could also increase reactive oxygen species which should also be avoided in disease with increased oxidative stress and endothelial dysfunction, such as diabetic kidney disease. However, these compounds are still used in preclinical settings to better understand the pathophysiology of renal diseases.In contrast to NO-donors, the sGC stimulators and sGC activators are able to stimulate the sGC even in the absence of NO and have synergistic and additive effects to the endogenous NO, respectively. These compounds also bind directly to the sGC and enhancing the enzymatic activity thereby triggering the formation of cGMP out of GTP. Since cGMP plays a significant role in regulating kidney function, both sGC stimulators and sGC activators may become useful treatments for kidney diseases.sGC stimulators such as riociguat (BAY 63-2521), vericiguat (BAY 1021189) or IW-1973 bind to the sGC heterodimer sGC and stimulate the sGC even in the absence of NO. The binding site is still controversial: Initial findings indicate that the sGC stimulator BAY 41-2272 binds to the alpha-subunit of the enzyme , whereas2+ in the heme group to Fe3+ and the heme group is lost subsequently which makes the enzyme insensitive to NO (sGC can exist in two redox states depending on the level of oxidative stress present in the tissue. Oxidative stress leads to an oxidation of the Feve to NO . The so-Phosphodiesterases (PDEs) cleave the major cyclic nucleotides like cAMP and cGMP in a highly efficient manner and terminate these second messengers signaling within milliseconds. PDEs, of which 11 families were identified, are different in their cellular distribution, in their regulatory domains and in their substrate specificity. Besides PDE6, which is localized in the eye, PDE5 and PDE9 are the main cGMP degrading enzymes. PDE5 and PDE9 are the major enzymes for cGMP-degradation and are both abundantly expressed in the kidney and blocking of PDE5 and PDE9 may effectively target also renal tissues.PDE5 Inhibitors (PDE5i) were introduced into medical therapy for the treatment of erectile dysfunction (ED) in 1998 when sildenafil was launched (Viagra™), followed by vardenafil (Levitra™) and tadalafil  in 2003. In 2007 and 2009, sildenafil and tadalafil were also approved for the treatment of pulmonary arterial hypertension (PAH) as Revatio™ and Adcirca™, respectively. PDE9 Inhibitors are not launched for any indication. However, it was shown preclinical that PDE9 inhibitors increased learning and memory in animal models and had some treatment effects in dementia models. Therefore, currently clinical testing is ongoing in Alzheimer dementia.S-nitrosated human serum albumin (SNO-HSA), displayed a dual beneficial effect, as it was able to increase hematopoiesis as well as effectively reduce renal fibrosis [In recent years, many preclinical studies assessed the physiological effects of cGMP-increasing mechanisms not only on physiological kidney function but investigated the pharmacological and therapeutic impact on kidney diseases. NO-donors, PDE5 inhibitors and sGC stimulators and sGC activators were studied in preclinical models and an overview on these studies is summarized in fibrosis . AlthougAlong these lines, phosphodiesterase inhibitors do show only moderate effects on systemic blood pressure and are well tolerated drugs. While there is almost no preclinical evidence evaluating the therapeutic effects of PDE9 inhibitors in kidney disease, several studies have demonstrated that treatment with PDE5 inhibitors is reno-protective. In a rabbit model of acute kidney injury, sildenafil was shown to reduce renal histopathology and electrolyte derangement while showing an improvement in creatinine clearance. Furthermore, it is well known that a majority of CKD patients also suffer from hypertension, and therefore targeting the antihypertensive effects of PDE5 inhibitors would be additionally beneficial in reducing kidney disease burden. Several studies using rodent models of renovascular hypertension have shown that PDE5 inhibitors such as sildenafil or vardenafil were able to cause significant decreases in blood pressure compared to administration of saline ,89. In aAs mentioned above, the effects of inhibition of cGMP degradation is limited in diseases with impaired endothelial function and low endogenous cGMP production. Therefore, NO-independent sGC stimulators and sGC activators might be even more beneficial. The effect of sGC stimulators and sGC activators on the kidneys was comprehensively summarized in a review by Stasch, J.P. et al., 2015 . In numeTogether, NO/sGC/cGMP plays a pivotal role for the regulation of kidney function by regulation of the kidney blood flow as well as by protective effects on glomerular and tubular compartments. Numerous preclinical studies have shown that targeting the NO/sGC/cGMP signaling cascade and increasing cGMP production are beneficial in various kidney pathologies. In the future, it will become very interesting to see if the compelling preclinical findings with sGC stimulators and sGC activators in kidney disease animal models can be successfully translated into clinical studies in kidney disease patients. First studies, using Nitrates and PDE inhibitors in rather small populations, were not fully conclusive. However, currently, a Phase 2 clinical study with an sGC stimulators in ongoing in CKD patients (NCT03217591) and sGC activators have also shown a promising preclinical profile  and migh


Cognitive impairment following spinal cord injury (SCI) has received considerable attention in recent years. Among the various systemic effects of SCI that contribute towards cognitive decline in this population, cardiovascular dysfunction is arguably one of the most significant. The majority of individuals with a cervical or upper-thoracic SCI commonly experience conditions called orthostatic hypotension and autonomic dysreflexia, which are characterized by dangerous fluctuations in systemic blood pressure (BP). Herein, we review the potential impact of extreme BP lability on vascular cognitive impairment (VCI) in individuals with SCI. Albeit preliminary in the SCI population, there is convincing evidence that chronic hypotension and hypertension in able-bodied individuals results in devastating impairments in cerebrovascular health, leading to VCI. We discuss the pertinent literature, and while drawing mechanistic comparisons between able-bodied cohorts and individuals with SCI, we emphasize the need for additional research to elucidate the mechanisms of cognitive impairment specific to the SCI population. Lastly, we highlight the current and potential future therapies to manage and treat BP instability, thereby possibly mitigating VCI in the SCI population. It is now widely recognized that cognitive impairment is a serious consequence of SCI. The majority of spinal injuries occur in early adulthood and individuals can survive for decades with a potentially permanent impairment . CognitiConsidering the segmentally differentiated autonomic innervation of the heart and vasculature, neurological level (and severity) of SCI determines the extent of subsequent cardiovascular dysfunction . With di2 vasoreactivity, maintains the spatiotemporal adequacy of cerebral perfusion [Across a wide range of systemic BPs , cerebral vasculature maintains a fairly uniform brain perfusion via a process called autoregulation. Furthermore, increased local metabolic demands in the brain (such as during a cognitive task) are typically coupled with an increase in regional cerebral blood flow (CBF). This phenomenon, called neurovascular coupling, ensures adequate substrate delivery and removal of metabolites within the activated brain region. Neurovascular coupling and cerebral autoregulation, along with COerfusion , as wellerfusion . More imerfusion ,9, resulerfusion ,11. Simierfusion . Intereserfusion , highligConversely, several studies have substantiated the association between chronic hypertension and cognitive decline in non-SCI individuals. Linked with impaired vascular tone, enhanced blood brain barrier permeability, and profound structural remodeling, abnormally high BP for a prolonged period of time detrimentally alters both structural and functional properties of arteries . Among tIt is also noteworthy that unlike the able-bodied individuals with either persistent hypotension or hypertension, individuals with cardiovascular impairment secondary to high-level SCI generally experience both extreme ends of the BP spectrum . The penPreventing and/or controlling volatile BP fluctuations to mitigate VCI following SCI can be approached in a number of ways. In terms of preclinical validation, this could be achieved by: (1) restoration of supraspinal control through neural regeneration , (2) preWe are only beginning to explore the interplay between cardiovascular and cognitive impairments following SCI. Given the wealth of research in the non-SCI population, many principles can potentially be extrapolated in order to expedite our understanding of the precise mechanisms involved. Future research is necessary to develop effective strategies to prevent or ameliorate cognitive impairment in persons with SCI. Advances in these areas will significantly impact independence and quality of life in this population.


Immunofluorescence double-labelling and transgene mice were used to determine which cell types express TSPO. [18F]-GE180-PET reliably detected the cuprizone-induced pathology in various white and grey matter regions, including the corpus callosum, cortex, hippocampus, thalamus and caudoputamen. Cuprizone-induced demyelination was paralleled by an increase in TSPO expression, glia activation and axonal injury. Most of the microglia and around one-third of the astrocytes expressed TSPO. TSPO expression induction was more severe in the white matter corpus callosum compared to the grey matter cortex. Although mitochondria accumulate at sites of focal axonal injury, these mitochondria do not express TSPO. In Cup/EAE mice, both microglia and recruited monocytes contribute to the TSPO expressing cell populations. These findings support the notion that TSPO is a valuable marker for the in vivo visualization and quantification of neuropathological changes in the MS brain. The pathological substrate of an increase in TSPO-ligand binding might be diverse including microglia activation, peripheral monocyte recruitment, or astrocytosis, but not axonal injury.Positron emission tomography (PET) ligands targeting the translocator protein (TSPO) represent promising tools to visualize neuroinflammation in multiple sclerosis (MS). Although it is known that TSPO is expressed in the outer mitochondria membrane, its cellular localization in the central nervous system under physiological and pathological conditions is not entirely clear. The purpose of this study was to assess the feasibility of utilizing PET imaging with the TSPO tracer, [18F]-GE180, to detect histopathological changes during experimental demyelination, and to determine which cell types express TSPO. C57BL/6 mice were fed with cuprizone for up to 5 weeks to induce demyelination. Groups of mice were investigated by [18F]-GE180 PET imaging at week 5. Recruitment of peripheral immune cells was triggered by combining cuprizone intoxication with MOG Multiple sclerosis (MS), an inflammatory disorder of the central nervous system (CNS), is associated with the development of demyelinated plaques, oligodendrocyte destruction and axonal degeneration. These pathological processes are paralleled by the activation of astrocytes and microglia, as well as the recruitment of peripheral immune cells into the central nervous system (CNS) ,2. NotwiThe pathogenic cascade(s) underlying neurodegeneration is incompletely understood. Recent data suggest that CNS-resident microglia as well as infiltrating inflammatory monocytes play a key role in neurodegeneration ,11. For + cells -(R)-PK11195 [During the last two decades, the prototypic TSPO radioligand in use has been (11C)-PK11195, with the first human brain studies conducted in patients with Rasmussen’s encephalitis , with gl-PK11195 ,68,  mice with eGFP-expressing microglia and RFP-expressing monocyte-derived macrophages [+ lymphocytes in EAE [High TSPO expression has been demonstrated in various macrophage populations such as in synovial macrophages , myocardrophages  to invess in EAE , as it is in EAE  and in as in EAE . FurtherAxonal degeneration is believed to be a major cause of permanent neurological disability in MS patients. It is assumed that myelinated axons contain two populations of mitochondria: stationary and motile mitochondria. It has been shown that the volume of mitochondrial stationary sites is increased in demyelinated CNS axons . In lighPET ligands targeting TSPO represent promising tools to visualize neuroinflammation in MS patients. This may provide information beyond contrast-enhanced MRI and is readily applicable to all patients. This study illustrates that the pathological substrate of an increase in TSPO ligand binding might be diverse including microglia activation, peripheral monocyte recruitment, and astrocytosis.


Diversity is crucial for the immune system to efficiently combat infections. Natural killer (NK) cells are innate cytotoxic lymphocytes that contribute to the control of viral infections. NK cells were for long thought to be a homogeneous population of cells. However, recent work has instead revealed NK cells to represent a highly diverse population of immune cells where a vast number of subpopulations with distinct characteristics exist across tissues. However, the degree to which a chronic viral infection affects NK cell diversity remains elusive. Hepatitis C virus (HCV) is effective in establishing chronic infection in humans. During the last years, new direct-acting antiviral drugs (DAA) have revolutionized treatment of chronic hepatitis C, enabling rapid cure in the majority of patients. This allows us to study the influence of a chronic viral infection and its subsequent elimination on the NK cell compartment with a focus on NK cell diversity. In our recent study , we show that chronic HCV infection irreversibly impacts human NK cell repertoire diversity. Due to the fact that infection with HCV nearly always becomes chronic, we first investigated the imprint of the persisting infection on the NK cell compartment. To overcome shortcomings of prior studies, where typically a limited number of phenotypic parameters had been assessed, we applied stochastic neighbor embedding (SNE) analysis to complex flow cytometry data. This analysis revealed that the NK cell receptor repertoire showed distinct patterns in HCV patients as compared to healthy controls. The phenotype was primarily driven by reduced expression levels of activating receptors in the patients. Interestingly, we also noted a high spread of the expressed markers in-between the HCV infected patients while healthy controls clustered closely together. Hence, NK cells from HCV-infected patients displayed a greater variation in how surface receptors were expressed.Next, we aimed to grasp the altered expression variation on the inter-individual level. Therefore, we implemented donor-to-donor expression variation (DEV) as a novel immune metric that more globally estimates immune population variation between individuals. We also assessed intra-individual variation within the NK cell compartment of chronic HCV patients as compared to healthy controls. To this end, inverse Simpson diversity index (SDI) was employed. SDI is a measure of population diversity originating from ecology, which has previous been used to study NK cell receptor repertoire diversity. We based our SDI-assessment on known NK cell differentiation markers. Taken together, with these measurements, it was possible to determine both inter- and intra-individual NK cell diversity and to estimate the impact of chronic HCV on the measures.DEV and SDI were first validated on a large cohort of 202 healthy controls in relation to CMV status and secondly in comparing peripheral blood and liver samples. As expected, the inter-individual diversity, DEV, was increased for NKG2C and KIR on NK cells in CMV-positive individuals. These two receptors are often found on CMV-driven adaptive-like NK cell expansions. On intrahepatic NK cells, CD69 had increased DEV as compared to peripheral blood. This was also expected since CD69 rarely is expressed on circulating NK cells but found on liver-resident NK cells. Studying the intra-individual diversity (SDI), we detected significantly lower SDI for NK cells in CMV-positive as compared to CMV-negative individuals. Again, this was anticipated since the CMV-driven adaptive-like NK cell population has a fairly fixed surface receptor phenotype. Due to the specific phenotype of intrahepatic NK cells, we found reduced SDI compared to peripheral blood NK cells in this setting as well. Taken together, these analyses validated DEV and SDI as relevant immune metrics to gauge global NK cell diversity.Figure 1). Of note, increased DEV was associated with severity of liver fibrosis. Strikingly, chronic HCV infection furthermore caused a significant reduction of NK cell SDI . This was independent from CMV serostatus and other clinical parameters such as age, gender, stage of liver fibrosis, and treatment outcome. Distinct from these differences in NK cell receptor repertoire diversity, examination of the NK cells’ ability to react by natural cytotoxicity, respond to cytokine stimulation, or mediate antibody-dependent cell-mediated cytotoxicity revealed no major differences between the chronic HCV patients and healthy controls. Yet, grouping the patients according to their SDI values, into either normal  or reduced SDI, revealed that patients with reduced SDI displayed impaired cytokine production upon K562 stimulation.Next, we applied the DEV and SDI measurements to the chronic HCV cohort. The previously observed higher spread in the expression level of individual NK cell receptors among the patients led to an elevated DEV as compared to healthy controls (Finally, patients were followed longitudinally during DAA treatment and up to 48 weeks after treatment completion. In this setting, we addressed if the observed impact of chronic HCV on NK cell diversity normalized after clearance of virus, or if the alterations were irreversible. SNE analysis comparing the NK cell phenotype at baseline and follow-up time-points revealed only few signs of normalization during therapy and after viral clearance. This involved reduction of Granzyme B expression and increase of NKG2D expression levels. Yet, expression of the majority of the examined markers remained unaltered. In line with this, NK cell SDI stayed reduced following viral elimination as well. Thus, the imprint on the intra-individual NK cell diversity appeared to be long-lasting. In contrast, the elevated DEV levels observed during chronic HCV infection decreased one year after successful viral clearance for many of the parameters. This could be attributed to the improvement of liver inflammation and reduced liver stiffness.In conclusion, our study was one of the first studies to examine the influence of a chronic infection on NK cell receptor repertoire diversity and we also studied this at very late time points after viral clearance. With new immune metrics gauging inter- and intra-individual NK cell diversity, we could identify a previously unrecognized imprint on the NK cell compartment caused by the virus. This was evident by elevated DEV, associated with the severity of liver fibrosis, as well as by reduced SDI in the HCV patients as compared to healthy controls. DEV normalized in parallel with improvement of liver stiffness upon viral clearance. In contrast, no reinvigoration of NK cell SDI was observed even up to one year after successful viral clearance. Thus, a chronic infection is able to leave a permanent imprint on the composition of human NK cells, an imprint that might persist for years after the pathogen has been successfully eliminated.


Elder family financial exploitation (EFFE) has attracted the attention of scholars and professionals across disciplines. This qualitative study examines the experiences of help seeking by non-perpetrator family members with a focus on the role of social services. 15 in-depth interviews were examined in which social services were mentioned as being involved. Findings provide insight into the role and involvement of social services, whether wishes expressed by victims to participants made a difference in help-seeking, and gaps experienced. Participants described social services professionals as those who (1) received reports of exploitation; (2) provided education and served as a liaison with families; (3) conducted assessments, including cognitive assessment of elders; and (4) acted as connectors to other systems. In some cases, when elders were assertive about their wishes, they had results such as reporting exploitation or transferring power of attorney to non-perpetrator family members. In other cases, elders were prevented from taking such action because of undue influence by perpetrators, disregard of their wishes, due to being uninformed, or opposing helpful family members. Participants explained experienced gaps in two ways: by attributing responsibility to social services in terms of failure to believe victims, do meaningful cognitive assessments, and navigate family dynamics. On the other hand, participants were not able to clearly ascribe responsibility, and questioned whose fault it was, suggesting opportunities for improved systems functioning. Recommendations for improving the role of social services in addressing the help-seeking needs of concerned family members coping with EFFE follow.


Gene editing strategies, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9), are revolutionizing biology. However, quantitative and sensitive detection of targeted mutations are required to evaluate and quantify the genome editing outcomes. Here we present AlleleProfileR, a new analysis tool, written in a combination of R and C++, with the ability to batch process the sequence analysis of large and complex genome editing experiments, including the recently developed base editing technologies. Gene editing technologies are revolutionizing the way genetic experiments are conducted. It is now feasible to generate genome edited cell lines or animals in record time. Multiple designer nucleases have been developed for gene editing, including customized zinc-finger nucleases (ZFNs) , transcrin vitro and in vivo models across various species [in vivo NHEJ. Indeed, we recently generated a quadruple knock-out mouse embryo lacking all four Id (Inhibitor of DNA binding) genes (Id1-4) in the mouse by injecting eight sgRNAs and Cas9 mRNA into fertilized mouse oocytes [Genome editing using the HDR pathway enables the precise insertion of new or the repair of existing mutations. For example, induced pluripotent stem cells (iPSCs) paired with genome editing strategies are revolutionizing how inherited diseases are studied, including cardiac diseases , by enab species –14. Impo species , 9. Mult oocytes . Two gui oocytes .Id gene KO study were analyzed manually [The potential applications of gene editing are numerous but are hindered by the limitations of existing quantitative technologies to quantify NHEJ/HDR or base editing efficiency and profile the generated mutants. Multiple tools have been developed in the recent years to analyze NHEJ/HDR outcomes and base editing experiments, including Cas-Analyzer , CRISPR-manually . Anticiphttps://github.com/abruyneel/AlleleProfileR). Alternatively, AlleleProfileR can be utilized using the Docker container platform . The Docker image provides a standalone environment to run AlleleProfileR which includes several external bioinformatics tools, this avoids dependency issues and helps users to get going immediately. A step-by-step guide on how to conduct analyses can be found in AlleleProfileR is provided as open-source software and can be installed locally from source  reads, gene-of-interest loci and configurations. The reference genome needs to be supplied in fasta format, but it is not necessary to provide the entire genome or chromosome, a large enough region spanning the sequenced region is sufficient. As only BAM files are accepted as input sequencing files, raw reads need to be preprocessed first which involves preprocessing, merging, and aligning the reads to the reference. Users can execute these preprocessing steps using their own favorite tools or processing pipeline, alternatively AlleleProfileR can apply these instructions to batches of files directly by linking to external software, such as fastp  for FAST# load the location of the fastq files in the input folders# directory structure should be provided as files/input/sample.name/.samples <- AlleleProfileR.read.folders(type = "fastq")# preprocess the files, this step will perform quality control using fastp, merge # paired-end reads using PEAR, and map the reads using BWA.AlleleProfileR.preprocesssamples, genes, and index. The samples parameter supplies the samples for analysis, the gene table contains the coordinates of the genes of interest, and index is the reference genome. Optional parameters include among others cutoff, the minimum percentage occurrence required for reporting, and ignore.snp and ignore.single, Booleans indicating whether SNPs or alleles with only a single occurrence should be ignored, respectively.Finally, AlleleProfileR requires configuration specifics with respect to the genomic regions of interest  as well as specifics as to how AlleleProfileR should process the results. These configuration settings can be supplied using the AlleleProfileR.setup command, which accepts many parameters. Essential configuration settings are # load the location of the bam files in the input folderssamplestable <- AlleleProfileR.read.folders(type = "bam")# set configurationcrispr_config <- AlleleProfileR.setupin silico experiments demonstrating the performance of the algorithm, such as deletions ranging from 5 to 120 bases resulting in chimeric reads matched bases are indicated as ‘M’, whereas deleted or inserted bases are represented as ‘D’ or ‘I’, respectively, which allows to pinpoint and extract the gene-editing events. However, chimeric reads or split reads, which occur when a sequencing read aligns to two distinct portions of the genome and are indicative of structural variation, require a more complex analysis procedure, and may arise when DSB-mediated NHEJ produces large indels with the same reads being mapped both downstream and upstream of the event site. AlleleProfileR deals with these reads by reassembling the individual reads, based on the truncation annotations performed by the aligner. The aligner will designate non-aligned parts of chimeric reads as clipped; soft (S) or hard (H), depending on whether these bases were removed from the sequence in the alignment record. In one pair of the chimeric read alignments, part of the sequence will be mapped, part will be clipped, in the other alignment of the pair, the clipped section will be mapped, absent any further indels, enabling the deconvolution of the chimera inducing event. The tutorial in Fig D in  and moreBased on the gene region information in the configuration, and on the variant calling output, AlleleProfileR will also assess the impact of the variant by assessing various metrics, such as start codon presence, frameshifts, indels within the coding sequence, premature stop codons, etc. Following code snippet will process all data using the aforementioned configuration:# process files and determine allelic variantsAlleleProfileR.batchAll analyses, both at the level of individual reads as well as identified alleles is written to csv files in the output folder. AlleleProfileR can generate various summary statistics to summarize the experimental output, such as the incidence of WT, NHEJ, HDR reads. HDR is quantified as proportion of reads that perfectly match the HDR template. If other variants in addition to the HDR variant are present in a given read, it will be flagged as a combination of HDR and NHEJ (HDR-NHEJ). All non-HDR or HDR-NHEJ reads different from WT are considered NHEJ. Base edit efficiency is quantified at the level of base conversion of individual bases rather than alleles, as neighboring off-target bases may also be edited to a certain extend. AlleleProfileR incorporates various plotting options such as alignments for NHEJ/HDR experiments and sequence logo plots for base editing experiments  to aid iAlleleProfileR is a tool to determine gene edit (NHEJ or HDR) or base edit efficiency from NGS data. The software development hypothesis was to resolve the issue of chimeric reads resulting from large deletions when using multiple guides targeting a small region in the genome and enabling batch operation whilst simultaneously making the tool broadly applicable and flexible. The algorithm was implemented in a combination of R and C++ using Rcpp  and is aTNNT2 mutation in human induced pluripotent stem cells et al. [Fet al.  which prin vitro or in vivo genome editing experiments. The advantages of AlleleProfileR over currently established tools is that it operates in batch mode and efficiently deals with chimeric reads caused by using two guides and provides multiple visualization tools for displaying the results, in contrast to most current tools that allow the analysis of single samples, small indels, or single guides.In summary, AlleleProfileR offers a robust workflow for processing S1 FileThe tutorial additionally incorporates validation experiments and comparisons with other tools.(PDF)Click here for additional data file.


Clinical success of immunotherapy is driving the need for new prognostic and predictive assays to inform patient selection and stratification. This requirement can be met by a combination of computational pathology and artificial intelligence. Here, we critically assess computational approaches supporting the development of a standardized methodology in the assessment of immune-oncology biomarkers, such as PD-L1 and immune cell infiltrates. We examine immunoprofiling through spatial analysis of tumor-immune cell interactions and multiplexing technologies as a predictor of patient response to cancer treatment. Further, we discuss how integrated bioinformatics can enable the amalgamation of complex morphological phenotypes with the multiomics datasets that drive precision medicine. We provide an outline to machine learning (ML) and artificial intelligence tools and illustrate fields of application in immune-oncology, such as pattern-recognition in large and complex datasets and deep learning approaches for survival analysis. Synergies of surgical pathology and computational analyses are expected to improve patient stratification in immuno-oncology. We propose that future clinical demands will be best met by (1) dedicated research at the interface of pathology and bioinformatics, supported by professional societies, and (2) the integration of data sciences and digital image analysis in the professional education of pathologists. A picture is worth a thousand words. This is the essence of the technological transition from macropathology to micropathology as captured in Virchow’s core principle “omnis cellula et cellulae” first popularized in 1858 . One hunImmuno-oncology requires a detailed understanding of the tumor microenvironment, including the identification and quantification of different immune cell subsets, their spatial context, and the expression of immune checkpoint markers. Changes in immune cell infiltration and biomarker expression before and after therapeutic intervention are critical parameters for clinical development . Image aIn recent decades, electronic data processing has transformed medical radiology, molecular diagnostics, and genetic testing. At the same time, the practice of pathology which forms a crucial link between these clinical disciplines has not changed significantly. The current convergence of new imaging technologies with tissue-based multiplexed immunohistochemistry (IHC)  enter the space by applying their expertise in big data science and artificial intelligence to data analysis and integrated decision making. Pharmaceutical companies have also recognized the importance of advanced pathology to their own work. Roche has already received FDA clearance for the VENTANA MMR IHC Panel for patients diagnosed with colorectal cancer (CRC) . DevelopThis paper provides a comprehensive outline of image analysis and machine learning (ML) applications for precision immunoprofiling. We will demonstrate how digital tools can facilitate pathology workflows in the assessment of established immune biomarkers and enable the deep characterization of the tumor microenvironment through spatial analysis and multiplexing. Further, we will identify computational methods driving morpho-molecular integration and deep learning methodologies for the discovery of novel therapeutic targets.cytotoxic T lymphocyte-associated protein 4, CD152) and PD-1/PD-L1 checkpoints  has driven rapid regulatory approval for treatment of patients with both solid and hematologic malignancies [Immuno-oncology has been revolutionized by the introduction of immune checkpoint inhibitors (ICI). ICI are monoclonal antibodies targeting immuno-regulatory molecules on the surface of T cells, antigen-presenting cells, and neoplastic cell populations . Clinicagnancies . Assessmgnancies , 13, urognancies , and rengnancies . Howevergnancies . Inter-ognancies , 77. Thi6–107 cells, which can be rapidly and reproducibly scored using purpose-built image analysis algorithms [Digital scoring of PD-L1 can assist the pathologist to overcome these barriers by providing standardized metrics for biomarker assessment at single cell resolution across whole tissue sections , 44, 55 gorithms . PatholoStandardized biomarker assessment protocols are essential to accelerate clinical development of immuno-oncology therapeutics . Co-deve2 of tumor tissue to form the Immunoscore® (IS) as marketed by HalioDx [The identification, localization, and spatial relationships of specific immune cells—before, during, or after therapy—have significant prognostic and predictive potential . Both co HalioDx . This ap HalioDx . A world HalioDx . Recent  HalioDx . However HalioDx .x-y location of each individual marker positive cell on a histological slide for precise nearest neighbor and infiltration analyses is already feasible  is among the most recent and innovative approaches for high-dimensional imaging of antibody-tagged epitopes in FFPE tissue . CODEX sCombining spatial imaging approaches with mass spectrometry may serve to reach yet another dimension in precision profiling of the tumor microenvironment. Multiplexed ion beam imaging (MIBI) uses antibodies labeled with isotopically pure elemental metal reporters (mass tags) to simultaneously detect up to 100 targets on FFPE tissue sections . ConventThe recently proposed “Cancer Immunogram” suggests seven categories including tumor foreignness, general immune status, immune cell infiltration, absence of checkpoints, absence of soluble inhibitors, absence of inhibitory tumor metabolism, and tumor sensitivity to immune effectors as the most important predictors of immunotherapy response . As suchTumors with a high mutational load frequently exhibit an immunologically activated phenotype . Recent T lymphocytes). PD1T cells were reproducibly detected and quantified in pretreatment biopsies of lung cancer patients and correlated strongly with treatment response to ICI in two independent clinical cohorts. This translational approach highlights how digital image analysis can represent a powerful companion diagnostic for cancer immunotherapy applications.Incorporating digital information on immune cell infiltration with molecular data is a powerful approach to inform the “Cancer Immunogram.” Conde and colleagues recently provided a proof of principle for the association of specific genetic alterations with defined immunophenotypes by combining digital assessment of CD8+ T cell infiltration in lung squamous-cell carcinoma with manual scoring of PD-L1 and targeted next generation sequencing . JoiningML will transform the field of immuno-oncology. We expect that ML will drive a paradigm shift in the way data are collected and analyzed to discover new prognostic markers or to construct more rigorous risk classification to empower stratified medicine. ML is essentially a set of computer algorithms that learn generic rules to perform any given task directly from data, without requirements of predefined knowledge or domain expertise. This, in many ways, is similar to how pathologists have previously gained knowledge and expertise through continuous practice that has led to new diagnostic classifications or prognostic factors in the clinical routine.Since ML is heavily data-driven, it enables a means to derive unbiased statistics from data. Accurate and continuous variables are more informative and can provide more biologically relevant information than the semi-quantitative scores presently implemented in diagnostic practice. Although several classical regression models exist to facilitate the discovery process of predictive or prognostic factors, the power of ML surpasses that of traditional tools when data is large and complex. ML can crunch through a vast amount of data and learn complex relationships between parameters and outcomes without the need to prespecify the relationships as normally required in traditional regression models. Nonetheless, data-driven approaches have critical downsides if not used with caution. ML can learn meaningless, biologically uninterpretable features that correlate to outcomes if the data has not been carefully preprocessed to remove any spurious features. Training datasets must have been generated in a highly standardized manner; otherwise, AI algorithms are likely to “misinterpret” sampling differences and artifacts between sets as distinctive biological characteristics. This phenomenon is known as “garbage in, garbage out.” Expert pathologist input in the training of image analysis algorithms is therefore of critical importance. Furthermore, ML tends to “overfit” to discovery data which results in an over-optimistic estimate of the performance of a model, while, in fact, it cannot be generalized well to new unseen datasets. It is, therefore, necessary that ML models are rigorously validated on new datasets that are independent of those used during the model development. Originating from the field of computer vision, deep convolutional neural network (DCNN) is a ML approach that is specialized in image analysis tasks , 50, 51.In immuno-oncology, ML as a pattern recognition tool enables an accurate and reproducible means for the unbiased assessment of regularities in the expression of immunohistochemical markers, tumor morphology, and the spatial distribution of tumor infiltrating lymphocytes (TILs). The ability of ML tools to detect key features in complex immunophenotypic datasets underlines their potential importance for the development of novel predictive models in cancer research. Initial studies underline the potential of ML methods for clinical translation:Yuan et al. computationally profiled cells of triple-negative breast cancer cohorts on H&E-stained sections and were able to uncover three categories of lymphocytes  with an unsupervised clustering method based on the proximities of lymphocytes to tumor cells . InteresHeindl et al. demonstrated that quantifying the number of spatial clusters or hotspots of immune cells and cancer cells is prognostic in ER+ breast cancer using a fully automated H&E stained image analysis algorithm . The spaIn medical research, survival analysis is a traditional means to assess the prognostic significance of each candidate covariate. The most widely used survival model is the Cox proportional hazards model. It assumes that the risk of an event of interest to occur (failure) is not time-dependent, is determined by a linear combination of all covariates, and might also include interaction terms between the covariates. This somewhat too simplistic assumption may not be able to capture the precise effects of the observed medical covariates on the risk of failure. This is particularly true in immuno-oncology where variable effects of immune infiltrates and immune-related gene signatures are observed depending on the tumor type and host immune status . DNN appIt has been shown that DNN performs better than the Cox proportional hazard model in various medical applications , 58, 86.diagnostic medical devices directive [Regulatory issues in the use of digital pathology devices have been broadly recognized by national and international bodies. Pioneering companies have recently received CE certification for routine pathology applications in the European Union under the In vitro irective . Comprehirective , 65. Theirective , 65. BotConcerns have been raised that inaccurate study design, statistical analysis, and reporting of research lead to a significant waste of research funding and inaccurate scientific conclusions . A majorAdvanced multiparametric imaging applications and ML have the potential to translate our evolving understanding of tumor-host interaction into better patient stratification and new treatment strategies. Computational pathology will help to derive complete, standardized, and reproducible datasets to facilitate the individualized prediction of immunotherapy response. Upskilling the pathology workforce through professional education and the recognition of computational pathology by professional societies will be essential to meet future clinical demands for optimal patient care.


Gender differences play a pivotal role in the pathophysiology and treatment of major depressive disorder. This is strongly supported by a mean 2:1 female-male ratio of depression consistently observed throughout studies in developed nations. Considering the urgent need to tailor individualized treatment strategies to fight depression more efficiently, a more precise understanding of gender-specific aspects in the pathophysiology and treatment of depressive disorders is fundamental. However, current treatment guidelines almost entirely neglect gender as a potentially relevant factor. Similarly, the vast majority of animal experiments analysing antidepressant treatment in rodent models exclusively uses male animals and does not consider gender-specific effects. Based on the growing interest in innovative and rapid-acting treatment approaches in depression, such as the administration of ketamine, its metabolites or electroconvulsive therapy, this review article summarizes the evidence supporting the importance of gender in modulating response to rapid acting antidepressant treatment. We provide an overview on the current state of knowledge and propose a framework for rodent experiments to ultimately decode gender-dependent differences in molecular and behavioural mechanisms involved in shaping treatment response. MDD poses a serious threat to global mental health. It is the second leading cause of disability worldwide , with moOestrogens influence synaptic plasticity, neurotransmission, neurodegeneration and cognitive function . In addiThe sexually dimorphic anatomy and function of the brain is strongly influenced by a broad variety of parameters, such as hormonal status, variance in body fat, liver metabolism, the transcription machinery, gene accessibility via epigenetic modifications and others ,12,13,14Dusp6 for female and Emx1 for male gender . . 67]. Different molecular pathways are activated by ketamine in a gender specific manner. Indeed male chronic isolated rats showed an increased spine density after ketamine treatment in medial prefrontal cortex via restoration of synapsin 1, PSD95 and GluR1 levels while those proteins were not altered in female rats after ketamine treatment . However, the literature reported as well studies showing no effects of gender in the effects of ketamine. Indeed, a recent work revealed no effects of gender in the acute and chronic effects of ketamine in ICR mice .Recently, the major ketamine metabolites have been drawn into focus of international research studies. In 2016, Zanos and colleagues showed that the ketamine metabolite -hydroxynorketamine (HNK) produced antidepressant-like effects in mice similarly to those of ketamine but without the ketamine-associated side effects . They weChou and colleagues applied the learned helplessness paradigm in male and female rats as a depression model in rodents . They coOn the other hand, some publications show a lack of response to HNK in rodents. Shirayama et al. reported that they could not find an effect of HNK in a rat model of learned helplessness  using thTo our knowledge, there is only one paper  focusingRapid-acting antidepressant agents share some key neurobiological pathways, which probably mediate their antidepressant-like effects . By alteIn line with the glutamate hypothesis of depression and depression treatment, pharmacological research focuses on the role of NMDA and AMPA receptors. Based on the evidence showing that the NMDA receptor antagonist ketamine produced sustained antidepressant effects in both humans and rodents, other ways to manipulate glutamate transmission were subsequently examined. Scopolamine, a muscarinic acetylcholine receptor (mAchR) antagonist, produced rapid, antidepressant effects even in treatment resistant depressed patients . Rodent A receptor signalling reduces glutamate transmission, which explains why GABA receptor antagonists and modulators are also important in the search for rapid-acting antidepressant drugs. Negative allosteric modulation of the alpha 5 subunit of the type A GABA receptor provides cortex- and hippocampus specific [GLYX-13 which is also known as Rapastinel—targets the outer surface of the NMDA receptor without occupying its ion pore and acts as a partial agonist ,80. Thatspecific  antideprspecific  in an anspecific .Cannabis sativa. Anxiolytic [1A) is important for the effects of cannabidiol. In a study with rats [1A and cannabinoid receptor CB1 signalling. Sales et al. have shown that i.p. injection of cannabidiol also led to an acute (30 min) antidepressant response in rodents [Cannabidiol (CBD) is the non-psychotomimetic compound of xiolytic  and antixiolytic  have beeith rats , cannabi rodents . They we rodents ,93.Inducing structural and functional plasticity in the PFC is a key process of antidepressant action. It has been shown that psychedelics such as lysergic acid diethylamide (LSD) and others see  are alsoBrain stimulation techniques represent an important part of last-line treatment options for MDD therapy. They are usually considered for patients with severe depression and treatment-resistant MDD courses. By applying electricity, all brain stimulation approaches interfere with activation and/or inactivation of certain brain circuits, brain regions and molecular pathways. Electroconvulsive therapy (ECT) is one of the oldest treatment paradigms in psychiatry, which is effective and approved for treatment-resistant depression, depression with psychotic symptoms, mania, catatonia and treatment-resistant schizophrenia . Under aNeuroinflammation, reduced levels of monoaminergic neurotransmitters and altered HPA axis activity have been identified as key mechanisms in depression pathophysiology . SimilarECT increased several monoamines after long-term treatment in patients . In addiInterestingly, although ECT is a very old technique, there is not much evidence for gender-specific aspects of ECT treatment reported in literature. A retrospective comparison from 2005 analysed patients with MDD, bipolar disorder and schizophrenia and found that women with MDD and schizophrenia received their first ECT session earlier meaning they had less previous antidepressant drug trials than men . In addiThere is a considerable amount of evidence collected from both animal and human studies, highlighting the central role of gender in depression pathophysiology and treatment. A better understanding of the molecular mechanism activated by hormones both in health and psychiatric disorders combined with a precise knowledge of the pharmacological interaction between hormones and antidepressant drugs, would be suited to redefine treatment guideline and possible identify molecular targets relevant for drug discovery and for gender personalized therapy.


In this review, we will dissect the manifold influences that antibiotics have on the cardiovascular system and their effects on thromboinflammation.The gut microbiota influence host vascular physiology locally in the intestine, but also evoke remote effects that impact distant organ functions. Amongst others, the microbiota affect intestinal vascular remodeling, lymphatic development, cardiac output and vascular function, myelopoiesis, prothrombotic platelet function, and immunovigilance of the host. Experimentally, host-microbiota interactions are investigated by working with animals devoid of symbiotic bacteria, i.e., by the decimation of gut commensals by antibiotic administration, or by taking advantage of germ-free mouse isolator technology. Remarkably, some of the vascular effects that were unraveled following antibiotic treatment were not observed in the germ-free animal models and  During the past decade, microbiome research has started to explore how the densely colonized gut resident ecosystem (microbiota) affects the host's vascular physiology , 2. ThisIndeed, mounting evidence is linking the gut microbiota to the onset of cardiovascular disease and arterial thrombosis –12. AmonApoe)-deficient mice starting at weaning (4 weeks of age) resulted in a reduced development of aortic root lesions at 20 weeks of age, when the mice were fed with a 1.0% choline-rich chow diet  control mice   . InteresWhile the obvious question for the fundamental differences between antibiotic treatment and GF housing state with regard to hematopoietic functions is apparent, these important studies clearly demonstrate that antibiotic intervention has manifold effects on innate immune cell types, which may lead to inconclusive mechanistic data in hematologic research. Therefore, gnotobiotic models based on GF knock-out mouse strains are essential to control for the validity of results obtained by antibiotic treatment protocols.The lipophilic essential vitamin K has two sources: it can be found in green leafed plants as phylloquinone (vitamin K1) and as the microbial metabolite menaquinone (vitamin K2), which is produced by several bacterial species –48. SyntIn this context, since 1980s, N-methylthiotetrazole (NMTT) cephalosporins  administration has been frequently reported to result in impaired hemostasis , 50–52. 2+ is necessary for vancomycin to cause these effects. They hypothesized that the cell scrambling induces an accelerated clearance of platelets from the blood, resulting in the observed thrombocytopenia. Importantly, all the described side effects are induced by exploiting vancomycin concentrations normally reached during standard protocols of antibiotic treatments  are increased platelet consumption/destruction or reduced platelet production. Linezolid, an antibiotic commonly used to treat vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus infections, is known to induce myelosuppression, but is more frequently associated with thrombo- than pancytopenia  and (b), direct interference with Ca2+ influx by blocking L-type voltage-dependent Ca2+ channels (2+ levels of rat ventricular myocytes are decreased, leading to reduced contractility (2+-influx in the vascular endothelium (Since the 1950s, aminoglycoside antibiotics are known to have hypotensive effects and a negative inotropic effect on the heart . Gentami of dogs . All fou of dogs . Two mecchannels . Belus eactility . Furtheractility . Not onlothelium . This inGF mouse models and antibiotic treatment protocols are frequently used as comparable methods to investigate the effect of gut microbiota in triggering inflammatory vascular phenotypes. In times of spreading antimicrobial resistance, every unnecessary use of antibiotics should be carefully weighed up with the expected findings . As poinAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pneumococcal vaccine development is driven by the achievement of high activity in a single gatekeeper assay: the bacterial opsonophagocytic killing (OPK) assay. New evidence challenges the dogma that anti-capsular antibodies have only a single function that predicts success. The emerging concept of multi-modal protection presents an array of questions that are fundamental to adopting a new vaccine design process. If antibodies have hidden non-opsonic functions that are protective, should these be optimized for better vaccines? What would protein antigens add to protective activity? Are cellular immune functions additive to antibodies for success? Do different organs benefit from different modes of protection? Can vaccine activities beyond OPK protect the immunocompromised host? This commentary raises these issues at a time when capsule-only OPK assay-based vaccines are increasingly seen as a limiting strategy. The development of vaccines has historically placed great emphasis on three gold standard functional assays of antibody activity: the opsonophagocytic killing (OPK) and serum bactericidal assays for bacteria and the neutralization assay for viruses. The pneumococcal vaccine is no exception, and design has principally included only capsular antigens, which are held only to the OPK assay standard. This approach presents a narrow vision of both immunogen and antibody function. A growing body of evidence indicates that this single-minded approach is passé, and that not only can anti-polysaccharide antibodies do much more than enable OPK, but new protein antigens can also be protective even in the absence of neutrophils. This commentary addresses the question, “In an era recognizing a need for broader anti-pneumococcal vaccines that protect against old and newly recognized aspects of disease, should measuring vaccine efficacy be modified by adding other assays of functions of both capsules and proteins as immunogens?” We posit that capsular polysaccharide and protein immunogens have much broader bioactivities in terms of both quantity and quality of immune functions, which can be targeted and harnessed as better vaccines are developed. In a striking demonstration of the limitations of a single assay approach to predicting vaccine efficacy, Doyle and Pirofsky described two mouse monoclonal antibodies, 7A9 and 1E2, raised against a pneumococcal capsular polysaccharide [In 1937, Harry Eagle described two dissociable activities of anti-pneumococcal horse serum: aggregation of bacteria vs. protection in animal models of sepsis . As earlWhat else do capsules do that can be harnessed for vaccines? Although not tested as yet, a short list of possible modes of protection by anti-capsular antibodies can be generated by focusing on the multiple modes of bacterial clearance . AntibodIntroduction of protein antigens to the vaccine design discussion opens up a wide array of bioactivities that could be folded into standalone or existing vaccines to potentially improve prevention of disease . ProteinA major hurdle for protein-based vaccines is the definition of an assay related to the protein’s function that would correlate to protection and predict improved vaccine efficacy. Particularly attractive would be activities in sites in which capsule-based vaccines show deficient protection: pneumonia, otitis media, or nasopharyngeal carriage. Many in vitro and in vivo assays have shown the bioactivities of many pneumococcal proteins, but adding them into vaccines requires assays that accurately correlate a functional assay with protection, often independent of prevention of sepsis. The difficulty of implementing this requirement was illustrated by a recent Phase II clinical trial of a combination of 10 polysaccharides with the histidine triad protein PhtD and a pneumolysin toxoid . PhtD isHaemophilus, and meningococcal diseases [Noncapsular antigens can clearly protect against disease. For instance, virtually all respiratory bacterial pathogens display phosphorylcholine on their surfaces, and this decoration has been shown to enable several pathogens to interact with host cells via the platelet activating factor receptor ,17. C-rediseases ,20,21,22The process of shepherding protein-based vaccines through clinical trials will require reworking or replacing the OPK assay with functional assays for many other modes of defense. Revamping the OPK assay would be appropriate for testing whether blocking bacterial complement-binding proteins protects against activation of the complement cascade in phagocytosis. Complement-binding proteins of pneumococcus include the surface adhesin CbpA/PspC, which has been demonstrated to recruit Factor H from serum to inhibit amplification of C3b activation. Antibodies to CbpA/PspC block the interaction with Factor H, and thus allow complement fixation to continue ,24,25.For most activities of proteins in pathogenesis, new assays will need to be deployed in the clinic. Similarly to neutralization standards for viral vaccines, pneumolysin toxoid vaccines will need to be measured not only for the ability to neutralize pneumolysin-induced hemolysis, but also for prevention of the oligomerization required for pore formation . Matrix-Approximately 30% of adults hospitalized for pneumococcal pneumonia go on to develop bacteremia . CriticaStreptococcus pneumoniae to bind polymeric immunoglobulin receptor, its ligand on mucosal epithelial cells, and 37 kDa laminin, its ligand on vascular endothelial cells [Antibodies against a variety of pneumococcal adhesins have been demonstrated to reduce the ability to adhere to host cells and thus attenuate disease severity in murine models. Perhaps the best characterized, antibodies specific for CbpA/PspC have been shown to inhibit the ability of al cells ,42. As ial cells ,43. TargS. pneumoniae [S. pneumoniae infection and pneumolysin toxicity are most likely already ongoing at the time of initial antimicrobial therapy in a patient care setting. Moreover, cell-wall-acting antimicrobials may worsen tissue injury, given the bolus of pneumolysin that would be released with bacterial death. Thus, immunization to protect against invasive disease caused by all serotypes of S. pneumoniae and to neutralize pneumolysin toxicity would be the protective method of choice.Pneumolysin is a pore-forming toxin produced by all clinical strains of eumoniae . It is teumoniae . Being aeumoniae ,46,47. Peumoniae . Thus, aeumoniae . Pneumoleumoniae ,51. PresCritically, molecular genetics allow the construction of hybrid molecules that contain both adhesin and toxin neutralizing epitopes. Multivalent pneumococcal protein vaccines that are composed of pneumolysin and fragments of key adhesins have been tested and shown to elicit strong and broad protection . Such anS. pneumoniae, secretory immunoglobulins play a pivotal role in pathogen control before the development of disease. While S. pneumoniae expresses an IgA protease that limits the impact of anti-pneumococcal IgA, previous work has shown that secretory IgA (sIgA), the polymeric form of the antibody, is capable of contributing to pathogen control through agglutination, as well as by facilitation of complement-mediated killing [For opportunistic mucosal pathogens like  killing ,71,72. T+ T cells specific to various pneumococcal protein antigens are vital in the initiation of a robust response within the respiratory tract and at other mucosal sites . CD4+ ctrophils ,79 as wetrophils ,81. The antibody , as wellantibody ,84. The nization ,86. Re-sS. pneumoniae, we suggest that the current standard of evaluating vaccine efficacy must also evolve beyond the OPK assay. For S. pneumoniae, a tremendous amount of data has shown that control and eradication of the bacteria during infection is reliant on many mechanisms, not simply the generation of opsonizing antibody. Antibodies that aggregate bacteria, impair bacterial–host cell binding, promote complement- mediated killing, disrupt bacterial signaling, enhance killing by NETs, and modulate immune cell responses are all potent mechanisms of potential vaccine-induced pathogen control which the OPK assay ignores. Knowing how each of these defense mechanisms applies to the many organs damaged by pneumococcal infection will also provide expanded avenues for designing better multi-modal vaccines.As we begin to recognize the clinical limitations of current pneumococcal vaccines and consider approaches towards the rational design of an improved vaccine against


The injured spinal cord is difficult to repair and regenerate. Traditional treatments are not effective. Stem cells are a type of cells that have the potential to differentiate into various cells, including neurons. They exert a therapeutic effect by safely and effectively differentiating into neurons or replacing damaged cells, secreting neurotrophic factors, and inhibiting the inflammatory response. Many types of stem cells have been used for transplantation, and each has its own advantages and disadvantages. This review discusses the possible mechanisms of stem cell therapy for spinal cord injury, and the types of stem cells commonly used in experiments, to provide a reference for basic and clinical research on stem cell therapy for spinal cord injury. Spinal cord injury (SCI) is currently the most difficult traumatic neurological condition to treat in the clinic. Following the primary injury, which causes immediate structural damage, a series of secondary injuries, including hemorrhage, edema, demyelination, and axonal and neuronal necrosis, are involved in the pathological process after SCI , 2. AfteThe pathological period of SCI is divided into three phases: acute phase < 48 h), sub-acute phase (48 h to 14 days), and chronic phase > 6 months) . Since e h, sub-a months .n = 31; NCT02163876) and thoracic  injury showed that the sensory function of some patients with a mild injury was restored [Neural stem cells (NSCs) are stem cells located in the lateral ventricle of the brain, the dentate gyrus of the hippocampus, and the central canal of the spinal cord. As a barrier to the brain and spinal cord parenchyma, they are closely related to cerebrospinal fluid circulation . The mairestored , and no Mesenchymal stem cells (MSCs) have become the favorite seed cells in the preclinical and clinical practice of regenerative medicine due to their readily obtainable source, wide biological effects, lack of ethical problems, and low immunogenicity . A largeBone marrow mesenchymal stem cells (BM-MSCs) are derived from bone marrow but are different from bone marrow hematopoietic cells, which grow in an adherent manner and differentiate into mesoderm cells. Currently recognized markers that are specific for BM-MSCs include CD29, CD90, and CD44 . InitialAdipose-derived mesenchymal stem cells (A-MSCs) are derived from adipose tissue and can be obtained from liposuction and liposuction. The advantages of A-MSCs are that large numbers of cells are easy to obtain, result in fewer traumatic injuries, are not associated with ethical problems, and can be autologously transplanted. The mechanism by which A-MSCs treat SCI and promote regeneration is by secreting a large number of neurotrophic factors, such as BDNF and GDNF, regulating activated immune cells and promoting nerve regeneration and anti-apoptotic effects –78. In aAmniotic epithelial mesenchymal stem cells (AE-MSCs) are obtained from the amniotic membranes and amniotic fluid derived from embryos. The traditional hypothesis is that the amniotic membrane protects embryos and maintains the normal development of organs. It is often discarded as biological waste after maternal delivery. A large number of studies conducted in recent years have shown that AE-MSCs are also very effective MSCs for the treatment of SCI in regenerative medicine. They have many characteristics of MSCs and have therefore been used as seed cells for SCI regeneration . The useUmbilical cord mesenchymal stem cells (UC-MSCs) are MSCs derived from the umbilical cord or cord blood, which are easy to obtain and expand in vitro. The absolute advantage compared with other types of stem cells is their low immunogenicity. Many preclinical studies have found that transplantation of UC-MSCs into rodents with SCI significantly improve functional defects . The theEmbryonic stem cells (ESCs) are highly undifferentiated, pluripotent cells that are able to differentiate into the cells of various tissues in adult animals. Because of their high degree of undifferentiation, these cells have been used as an in vitro cell differentiation and regulation model. Since Evans and Kaufman  first isAdult endogenous stem cells (AESCs) are stem cells located in the adult nervous system. In the spinal cord tissue, ependymal cells, radial glial cells, and strip cells near the central canal of the spinal cord have stem cell characteristics . NormallSpermatogonial stem cells (SSCs) are a subtype of spermatogonia  that maiAs mentioned above, although ESCs might substantially improve the treatment of SCI, their application has many limitations. Therefore, a suitable alternative source is an urgent unmet medical need. Induced pluripotent stem cells (iPSCs) are considered effective alternative cell sources for ESCs that can be obtained from cell reprogramming by introducing transcription factors into somatic cells, as described by Yamanaka. Currently used methods include virus-mediated gene transfection, targeted insertion, gene transposon insertion and protein transfection . MoreoveNSCs derived from transplanted iPSC promote remyelination, axonal regeneration, and the secretion of neurotrophic factors, while reducing inflammation . AccordiStem cell therapy has shown great potential in reconstructing the injured spinal cord and promoting functional recovery Fig. , but man


Eukaryotes have cell-autonomous defenses against environmental stress and pathogens. Autophagy is one of the main cellular defenses against intracellular bacteria. In turn, bacteria employ diverse mechanisms to interfere with autophagy initiation and progression to avoid elimination and even to subvert autophagy for their benefit. This review aims to discuss recent findings regarding the autophagic responses regulated by bacterial effectors. Effectors manipulate autophagy at different stages by using versatile strategies, such as interfering with autophagy-initiating signaling, preventing the recognition of autophagy-involved proteins, subverting autophagy component homeostasis, manipulating the autophagy process, and impacting other biological processes. We describe the barriers for intracellular bacteria in host cells and highlight the role of autophagy in the host-microbial interactions. Understanding the mechanisms through which bacterial effectors manipulate host responses will provide new insights into therapeutic approaches for prevention and treatment of chronic inflammation and infectious diseases. Autophagy, which literally means “self-eating,” is an intrinsic process of eukaryotes that delivers cytoplasmic material to lysosomes for degradation. During this process, cytoplasmic material is enclosed by phagophores. Then, the phagophores elongate to form autophagosomes that fuse with lysosomes to form autolysosomes where the cargoes are degraded . AutophaThere are three forms of autophagy that are commonly described: chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy. Macroautophagy is then divided into non-selective and selective autophagy. Various cargoes, such as defective mitochondria, defective endoplasmic reticulum (ER), lipids, and foreign organisms, are targeted by selective autophagy for degradation. When autophagy engulfs microorganisms for clearance, this pathway is called “xenophagy,” which plays a central role in cellular defense .To survive in host cells, bacteria employ multiple mechanisms to protect against cellular defenses. Effectors, one type of weapons used by bacteria, are proteins translocated from the bacterial cytoplasm to the host cell cytoplasm by a series of secretion systems (T1SS–T8SS) .Bacterial effectors have the capacity to influence host cellular biological processes, including signaling pathways, tight junctions, phagocytosis, apoptosis, and autophagy , 6. ThisThere is a constant battle between bacterial evasion mechanisms and host cellular defenses, and the fate of intracellular bacteria is determined by the outcome of this battle. Intracellular pathogens can be internalized by either phagocytic or non-phagocytic cells. After entry into host cells, bacteria are localized to internalization vacuoles, which are designated as phagosomes . To survMycobacterium tuberculosis), or disrupt the vacuoles to escape from the phagosomes   that recruits the autophagy marker protein LC3 to pathogen-containing phagosomes, and the subsequent fusion of these phagosomes with lysosomes results in pathogen digestion . To previmurium) . Xenophaimurium) , 10. HerBacteria can be eliminated by autophagy, thus, Gram-negative bacteria use T3SS effectors to suppress or subvert this process. We summarized all related research work in Shigella flexneri effector IcsB, was recently found to repress the early recruitment of LC3 during infection  T3SS is responsible for suppressing autophagy by actively manipulating the recruitment of focal adhesion kinase (FAK) to Salmonella-containing vacuoles (SCVs) and then stimulating the Akt-mTORC1 signaling pathway. However, the effector(s) that are responsible for this process remain unclear , thereby masking the bacteria from recognition by autophagy mechanisms. Therefore, IcsB mutants are targeted for autophagy during multiplication in host cells infected with S. flexneri -mediated autophagy pathway for its survival  induces autophagy by targeting the initial events of autophagic signaling  are highly fusogenic with each other and with other organelles of the endocytic pathway; therefore, good-sized vacuole formation is promoted can decrease LC3 recruitment around L. pneumophila strains in macrophage cells , and even two secretion systems (Vibrio parahaemolyticus), to mediate autophagy. Meanwhile, some effectors are versatile in manipulating autophagy (IcsB). Moreover, in the study of the effector LpSpl, the author found that the effector RavZ is not present in all strains of L. pneumophila, suggesting that this strain employs other effector, namely LpSpl  deleting entire secretion systems or single genes and then analyzing the changes in autophagic responses in host cells infected with WT/mutant strains; (b) similarity searches to seek effectors that exhibit similar structures to host proteins, such as RavZ  and LpSpLpSpl ; and (c)LpSpl  and Etf-LpSpl . Most ofractions . In vivolike LAP .o domain, which appears to play a key role in the regulation of autophagy through amino acid sensing, and even more directly, autophagosome-lysosome membrane fusion. Though the details is unclear, this study enlighten us to further elucidate the role of V-ATPase in autophagosome fusion with the lysosome  use various methods to determine multiple related proteins in autophagy, including morphological observations and monitoring autophagic flux to support accurate information about autophagic responses. Please refer to a comprehensive review of this subject ; (b) undStudies on effectors-autophagy interactions in host cells will provide new insights into the pathogenic mechanisms of infections and inflammation. A better understanding of the mechanisms used by bacterial effectors to manipulate autophagy will help the study of mechanisms in immunity, drug design, and novel therapeutic approaches for infectious diseases and chronic inflammation.JS designed the theme and topic and obtained research funds. YJ drafted the manuscript and organized the figure and tables. YJ and JS finalized the manuscript.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.


Articular cartilage lesions generated by trauma or osteoarthritis are the most common causes of pain and disability in patients.The development of photopolymerizable hydrogels has allowed for significant advances in cartilage repair procedures. Such three-dimensional (3D) networks of polymers that carry large amounts of water can be created to resemble the physical characteristics of the articular cartilage and be delivered into ill-defined cartilage defects as a liquid solution prior to polymerization in vivo for perfect fit with the surrounding native tissue. These hydrogels offer an adapted environment to encapsulate and propagate regenerative cells in 3D cultures for cartilage repair. Among them, mesenchymal stem cells and chondrocytes may represent the most adapted sources for implantation. They also represent platforms to deliver therapeutic, biologically active factors that promote 3D cell differentiation and maintenance for in vivo repair.This review presents the benefits of photopolymerization of hydrogels and describes the photoinitiators and materials in current use for enhanced cartilage repair. Articular cartilage lesions remain a critical, unsolved problem in orthopaedics due to the inadequate capacity of this particular tissue for self-repair , which drive them to seeking treatment to relieve the secondary symptoms of joint disability , daily activity modification (e.g. weight reduction and non-weightbearing strengthening), bracing, orthotics, and non-irritating aerobic conditioning  and subsequent chondrogenesis. The techniques include microfracture  versus microfracture after 5 years  technique extracts and cultivates articular chondrocytes ex vivo for transplantation to sites of cartilage lesions . Recent evidence underlined the importance of selecting donor sites to optimize the contour of autograft and minimize the potential morbidity of harvest sites  data in a computerized tool system, it may be also envisaged to precisely print a combination of resources, resulting in a 3D construction mimicking the native articular cartilage tissue.The most commonly used natural materials in cartilage research include agarose, alginate, chitosan, collagen, fibrin, and hyaluronan  for optimal transport of oxygen, nutrients, and waste products, making them ideal for medical applications  showed that chondrocytes encapsulated in a PEO diacrylate (PEODA) hydrogel exhibited increased proteoglycan and collagen contents and equilibrium moduli, dynamic stiffness, and streaming potentials. Hayami et al.  noted that chondrocytes in styrenated gelatin using camphorquinone as a photoinitiator displayed steady expression of type-II collagen and aggrecan core protein mRNAs. Levett et al.  observed that the use of PEG-based photopolymerizable hydrogels increased sulfated glycosaminoglycans (sGAGs) and collagens in the newly formed tissue. Ramaswamy et al.  noted that MSCs undergo effective chondrogenesis in a cartilage-mimetic hydrogel using PEG with matrix metalloproteinase 2 (PEG/MMP-2) that can be delivered in vivo and photopolymerized intra-operatively in situ. Ramaswamy et al.  showed that a photopolymerized mGL hydrogel was capable of supporting MSC growth and TGF-β3-induced chondrogenesis. Williams et al.  is a classical factor used in cartilage engineering studies, leading to the stimulation of chondrogenesis and proliferation  is a less extensively studied molecule that is expressed almost exclusively in cartilage  agents, potentially protecting such “cargo” from physiological degradation. Photopolymerizable hydrogel-guided delivery of gene therapy vectors may thus be also envisaged as a potential tool to promote the effective, long-lasting healing of damaged articular cartilage  photopolymerizable hydrogels, nevertheless, very limited work has been performed thus far with hybrid systems for applications in cartilage repair.Cutting-edge evidence has advanced our general knowledge on the feasibility of using photopolymerizable hydrogels for cartilage research. Yet, more work is needed to demonstrate the potential benefits of these systems as convenient, adapted, and safe systems for the treatment of cartilage defects. First, current experimental work both in vitro and in clinically relevant animal models in vivo has to be expanded in order to define optimal conditions for effective therapy , and/or gene vector). Also, the initiation of clinical trials where various, optimized photopolymerizable hydrogels will be applied in patients is of the utmost importance to provide effective systems suitable for improved cartilage repair in translational approaches in a close future.