Heart failure, a leading cause of hospitalizations and deaths, is a major clinical problem. In recent years, the increasing incidence of heart failure with preserved ejection fraction (HFpEF) has been observed. Despite extensive research, there is no efficient treatment for HFpEF available. However, a growing body of evidence suggests stem cell transplantation, due to its immunomodulatory effect, may decrease fibrosis and improve microcirculation and therefore, could be the first etiology-based therapy of the disease. In this review, we explain the complex pathogenesis of HFpEF, delineate the beneficial effects of stem cells in cardiovascular therapy, and summarize the current knowledge concerning cell therapy in diastolic dysfunction. Furthermore, we identify outstanding knowledge gaps that may indicate directions for future clinical studies. Heart failure is one of the main issues of global healthcare affecting more than 64 million patients worldwide . The disPatients presenting signs and symptoms of heart failure should undergo routine laboratory tests, including natriuretic peptides and echocardiography. According to the ejection fraction, heart failure is classified into two main phenotypes—heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). According to the European Society of Cardiology and the American Heart Association/American College of Cardiology, HFrEF is diagnosed in patients with EF ≤40%, whereas HFpEF is diagnosed in patients with EF ≥50%. HFmrEF (heart failure with mildly reduced ejection fraction) is characterized by an ejection fraction between 40% and 50% ,4.Today, there is a marked discrepancy in the prevalence of HFrEF and HFpEF. While the incidence of HFrEF significantly decreases, there is an upward trend in the incidence of HFpEF . As the β-blockers, or mineralocorticoid receptor antagonists (MRAs) [HFpEF poses one of the most significant challenges of modern medicine. Within the past three decades, there was no improvement in its prognosis. Although it usually raises fewer concerns, the mortality rate of HFpEF is comparable with that of HFrEF . Recentls (MRAs) . HoweverFor instance, in the CHARM-preserved trial, the implementation of candesartan only reduced admissions to hospital for HF . The PARDue to their proven efficacy, the sodium-glucose co-transporter-2 inhibitors (SGLT2i) are already accepted for HFrEF treatment. Furthermore, new indications for its use have appeared. As insulin resistance is one of the major risk factors for HFpEF, researchers hope SLGT2i is effective in the context of the treatment of the disease.The EMPEROR-preserved trial was the first large-cohort double-blind, placebo-controlled study investigating an SGLT2i for HFpEF, namely empagliflozin. The study achieved its primary endpoint: a composite of cardiovascular death or heart failure hospitalization, although it was related mainly to the decreased number of heart failure hospitalizations. The difference in the overall death rate and the number of hospitalizations between both groups was statistically non-significant .Another SGLT2i, dapagliflozin intake, is associated with favorable outcomes in patients with HFpEF and HFmrEF. It improves the quality of life as well as exercise tolerance . The druTo summarize, to date, there are few available drugs that alleviate the symptoms of HFpEF, and none has a proven efficacy in mortality improvement. Accordingly, a significant breakthrough is still awaited. Therefore, it is essential to develop etiology-based treatment strategies to improve the prognosis.Due to their numerous salutary traits, stem cells have attracted significant attention from researchers in recent years. Their application may be highly beneficial in a multitude of various diseases, including cardiovascular diseases, such as: coronary artery disease, dilated cardiomyopathy, and pulmonary hypertension. There is also a growing body of evidence concerning its possible efficacy in the treatment of HFpEF. The purpose of this review is to comprehensively describe the presumed beneficial effects of stem cells and to summarize the current state of knowledge regarding their use in HFpEF.To better understand the role of stem cells in the treatment of HFpEF, it is crucial to know its multifactorial pathophysiological background. Among multiple components, it is assumed that immunological reactions, fibrosis, and microvascular dysfunction may play a central role in the development of the disease.Macroscopically, HFpEF is characterized by myocardial stiffness and impaired relaxation ,15. The The common histopathological findings of HFpEF are interstitial fibrosis, hypertrophy, decreased density, inflammatory infiltrate, and impaired autoregulation of microcirculation ,18,19,20Today, the inflammatory hypothesis of HFpEF is becoming more relevant. The immunological dysfunction may be a stand-alone condition. However, as the epidemiological data shows an association of HFpEF with systemic inflammation due to the previously mentioned comorbidities and aging, it may be an organ manifestation of a systemic pathology.(1)Dysfunction caused by microvasculature defects due to comorbidities;(2)Dysfunction caused by inflammatory activity within the heart as a response to mechanical stress;(3)Dysfunction caused by intrinsic pro-inflammatory activity due to clonal hematopoiesis.The pathogenesis of HFpEF has been explored only to a certain degree. The knowledge about the development of the disease is mainly derived from animal models. Nevertheless, it has partially enabled researchers to fill the existing knowledge gaps to complete the mechanistic theory. Based on the available research, we divided the pathophysiological mechanisms into three large groups, namely:Several chronic diseases may increase the risk of HFpEF by inducing a hyperinflammatory state characterized by the elevated serum concentration of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), soluble suppression of tumorigenicity 2 (sST2), and pentraxin 3. It may impair the endothelial function and increase the expression of endothelial adhesion molecules, such as the vascular cell adhesion molecule (VCAM) and the intercellular adhesion molecule (ICAM), on endothelial cells . These p(1)Directly by interleukin 10 (IL-10) and transforming growth factor β (TGF-β) production, which stimulates fibroblast activation and collagen deposition;(2)Indirectly by antigen presentation and T cell activation.Subsequently, the inflammation is aggravated by macrophage infiltration. Cardiac macrophages may be divided into two subsets playing distinct roles. The circulatory macrophages may be distinguished from the resident cardiac macrophages by C-C chemokine receptor type 2 (CCR2) positivity . ResidenThere is a positive feedback loop between T lymphocytes and macrophages. As mentioned above, the VCAM and ICAM proteins facilitate T lymphocyte infiltration. The T cells release IFN-γ, which attracts macrophages. Macrophages thereafter present antigens to the T lymphocytes. T cells, in turn, enhance the expression of adhesion molecules on the endothelium, leading to the massive influx of immune cells into the perivascular space . This coPro-inflammatory cytokines can also activate nicotinamide adenine dinucleotide phosphate oxidase, increasing reactive oxygen species (ROS) production, which stimulates the proliferation and activation of collagen-producing myofibroblasts . ROS, duNO is also crucial for the formation of cyclic guanosine-3,5-monophosphate (cGMP), a molecule indispensable for activating cGMP-induced protein kinase (PKG). The NO-cGMP-PKG signaling not only triggers vascular smooth muscle cell relaxation but also via titin phosphorylation and may have a protective effect in HFpEF. Titin is one of the main cytoskeletal proteins and is responsible for the recoil of the sarcomere early after a contraction is completed, which translates into the diastolic distensibility of the cardiomyocytes. PKG phosphorylates the N2B segment of titin and in this way, increases its activity and compliance. If the process is interrupted, the cardiomyocyte resting tension increases, leading to diastolic dysfunction ,27,28.(1)Inhibition of platelet aggregation and thrombus formation by the decrease of P-selectin expression on platelets and Willebrand factor-mediated platelet aggregation adhesion ,30;(2)Immunomodulatory properties due to the decrease in the production of pro-inflammatory type 1 T helper cell cytokines (IFN-γ and IL-2) ,32;(3)Prevention of the deleterious proliferation of vascular smooth muscle cells by ubiquitin-conjugating enzyme H10 degradation ;(4)Inhibition of the cytokine-induced expression of VCAM-1 and monocyte chemoattractant protein-1 (MCP-1) ,34.The importance of this mechanism may be emphasized by the fact that coronary infusions of NO donors could diminish the diastolic left ventricular stiffness in patients with aortic stenosis or dilated cardiomyopathy . NO secrThe next hypothesis does not undermine the previous one, although it identifies a different leading cause underlying the whole cascade of immunological activation within the myocardium. However, the final steps, i.e., the influx of immune cells into the myocardium, remain common for both mechanisms.As HFpEF is strongly associated with hypertension, it has long been recognized that the pathophysiology of this condition consists of the myocardial response to increased mechanical stress, i.e., pressure overload. However, the effect of elevated blood pressure on the heart is much more complex and encompasses many immunological reactions.It has been detected that, due to mechanical stress, CCR2 ligands, such as MCP-1, MCP-2, chemokine (C-C motif) ligand 12 (CCL12), and stromal cell-derived factor 1α (SDF-1α) are overexpressed ,22,35. TThe stress induced by hypertension is not limited solely to the cardiovascular system. It should be regarded rather as a systemic phenomenon. Importantly, there is a correlation between diastolic dysfunction and increased bone marrow and splenic signal in 18F-FDG PET/CET . Thus, hIn response to structural damage, the heart releases alarmins, i.e., endogenous molecules that activate immune cells in response to cellular damage. They are subsequently transported into the spleen where they activate pro-inflammatory macrophage populations. The released macrophages subsequently infiltrate the myocardium, which intensifies the inflammation. In the murine model, the alarmins released by the damaged cardiomyocytes cause splenic morphological changes  as well as a shift in the population of immune cells within the organ over the course of heart failure, which reflects the splenic involvement in this condition. Additionally, an increase in the number of subtypes of dendritic cells in the spleen, blood, and the heart was observed. In the same study, a splenectomy reversed cardiac remodeling and decreased macrophage and dendritic cell infiltration of the myocardium .This demonstrates the importance of the so-called cardiosplenic axis, which is a bidirectional route of the immune cells. It may be relevant in maintaining homeostasis; however, if dysregulated, it may become pathological and lead to maladaptive cardiac remodeling. It may also constitute a possible future therapeutic target in HFpEF.Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by multiple mutations in hematopoietic stem cells, resulting in the clonal expansion of one subpopulation originating from a single founding cell sharing its DNA alterations . The effThese processes tend to accumulate with age, with CHIP mutations being detected in 1% of individuals under the age of 40, but approximately 10–20% of those over the age of 70 . A retroIt has been hypothesized that a similar link exists between CHIP mutations and HFpEF. The silenced TET2 gene does not inhibit the pro-inflammatory activity of macrophages, which then stimulate the production of molecules, such as IL-1, IL-6, chemokines , and platelet factor 4 . Il-6 isStem cells are undifferentiated cells that can differentiate into specialized tissue cells. Their other unique property is self-renewal, i.e., the ability to replicate and generate new stem cells. We can differentiate embryonic and adult stem cells. Embryonic cells are pluripotent, i.e., they can differentiate into any mature somatic cells. They are abundantly present only during embryonic development; hence, it is almost unfeasible to exploit them in clinical practice. Adult stem cells are present in various tissues and show promising potential. They are considered unipotent, oligopotent, or multipotent, i.e., they have the capability to differentiate into a specific cell type, a few cell types, or multiple cell types of one lineage.In clinical studies on cardiovascular diseases, researchers mostly use endothelial progenitor cells , mesenchymal stem cells , and cardiac stem cells . Interestingly, it also became possible to obtain so-called induced pluripotent stem cells (iPSCs) directly from any type of somatic cells. All these types of stem cells exert enormous reparative and regenerative effects that could be utilized in the treatment of HFpEF.EPCs originate from the bone marrow and are characterized by expressing CD133, CD34, and the vascular endothelial growth factor receptor-2 . Their uIn clinical studies, the properties of EPCs were mostly examined in patients with coronary artery disease. This feasible and safe treatment modality seems to be highly efficient in preventing complications due to acute myocardial infarction. The EPC injection may attenuate deleterious post-infarction remodeling by enhanced neovascularization . ThereafIt was also hypothesized that EPC-capture stents, i.e., stents covered with CD34 antibodies that bind EPC from the peripheral blood, might facilitate healing by promoting neointima formation. However, the results so far of the implementation of EPC-capture stents do not support this thesis .Additionally, the vasodilatory potential of EPCs may be exploited in clinical practice. In patients with pulmonary arterial hypertension, there is a significant decrease in pulmonary resistance observed after the delivery of EPCs overexpressing eNOS. This is associated with an increase in the quality of life and in exercise capacity .In individuals with HFpEF, a decrease in circulating EPCs is observed. It is postulated that microvasculature alterations are one of the most relevant components inducing HFpEF ,54; therMSCs are usually obtained from bone marrow or fat tissue. Today, their isolation from the peripheral blood has also become possible, which appears to be a promising method . They arMSCs, by the increased expression of B-cell lymphoma 2 gene (BCL2), survivin, and HIF-1, may have a strong antiapoptotic potential. This effect is particularly intensified in hypoxic conditions ,60. MSC Another favorable effect of MSCs is they also display immunomodulatory activity, which may be beneficial and is particularly relevant in HFpEF. Experimental studies revealed MSC therapy may diminish the inflammatory infiltrate of the myocardium. Moreover, they have the ability to modify the level of multiple cytokines, which may establish conditions facilitating myocardial regeneration ,64.The angiogenic capacity of MSCs associated with the release of VEGF, which is especially aggravated in hypoxia and inflammation, may result in increased vascular density ,66. MoreThe following unique feature of MSCs that may be used in cardiac diseases is their antifibrotic activity. By stimulating the activity of several metalloproteinases (MMP), especially MMP-2 and MMP-9, MSCs may significantly reduce the deposition of collagen fibers . FurtherThus far, the effects of MSCs on cardiovascular diseases have been extensively examined in clinical surveys, and the results have been summed up in meta-analyses. MSC treatment yields a significant hemodynamic improvement in acute myocardial infarction, especially if the transplantation is performed within the first week. MSCs are beneficial regardless of the cell delivery route, i.e., trans-endocardial injection shows a similar efficiency to intracoronary infusion ,71.Pooled data analysis demonstrated MSC treatment is also highly effective in patients with HFrEF. It results in a decrease in the incidence of readmission and improved exercise capacity as well as an improvement on the in New York Heart Association (NYHA) scale. Although MSCs appear to be a promising etiology-based treatment strategy for HFpEF, there are no clinical studies to date concerning their application in the treatment of the disease .Cardiospheres are multicellular clusters generated from endomyocardial biopsy specimens. They are composed of CSCs and myofibroblasts in the center, surrounded by differentiated supporting cells, such as vascular smooth muscle and endothelial cells. The outer layer protects the stem cells against oxidative stress and is crucial for maintaining their self-renewal ability . CardiosDue to the release of various microRNAs (miRNAs) and extracellular vesicles, CDCs have a strong protective effect. Among those factors, the most relevant are miRNA-146a and the pregnancy-associated plasma protein-A (PAPP-A) containing exosomes exerting potent anti-apoptotic activity .Cardiac progenitor cells, similar to the two previously mentioned stem cells, may also promote angiogenesis by the release of VEGF and insulin growth factor-1 (IGF-1) . An incrCDC treatment may also exert an immunomodulatory effect. It is considered that they can switch the pro-inflammatory M1 macrophage phenotype secreting cytokines, such as TNF-α, IL-1, and IL-6, into the anti-inflammatory M2 phenotype, ensuring favorable conditions for tissue repair. The CDCs also exert an immunosuppressive effect by decreasing neutrophil recruitment .Based on experimental studies, CDC therapy may be associated with the greatest benefits in the treatment of cardiovascular diseases . HoweverPSCs were first generated in 2006 by Yamanaka et al., from fibroblasts. The researchers induced pluripotency of the differentiated somatic cells by using four transcriptional factors, including Oct3/4, Sox2, Klf4, and c-Myc . This inBy using specific protocols, iPSCs may be subsequently differentiated into any cell type, including cardiomyocytes, which potentially means a biopsy would not be necessary to generate CSCs in the future. This could be especially advantageous in conditions associated with myocyte loss  to reverse or repair heart damage . iPSCs mEven though iPSCs represent a very promising method of cardiac regenerative medicine, it is still in its infancy. More experimental studies are required to develop efficient and safe methods for obtaining iPSCs before their use in clinical trials.Due to their numerous unique properties, researchers noticed stem cells might also be utilized in the treatment of HFpEF. In experimental studies, different models of HFpEF have been used together with various kinds of stem cells  with success , which is a source of both EPCs and MSCs, may be efficient in HFpEF. The SVF treatment significantly ameliorated diastolic dysfunction in rats of an advanced age. Additionally, it was associated with favorable alterations in coronary arteries, i.e., greater coronary flow reserve (CFR) and decreased left wall thickness .In mice, in the Van Linthout et al., study, HFpEF was induced by diabetes due to the application of streptozotocin. Then, placenta-expanded MSC-like cells were injected intravenously. The study demonstrated its immunosuppressive as well as antifibrotic properties. There was a significant decrease in the cardiac expression of the pro-inflammatory factors (TGF-ß1 and IFN-γ) and an increased anti-inflammatory Treg cell count. There was also a decrease in cardiac fibrosis via enhanced titin phosphorylation and an increase in arteriole density. A limitation of this study was echocardiography was not performed. However, it was concluded the treatment resulted in diastolic improvement as an in vitro analysis showed a lower passive force of cardiomyocytes isolated from mice receiving placenta-expanded MSC-like cells .HFpEF can also be obtained in rats receiving a high-salt diet due to pressure overload. In the Gallet et al., study, animals were fed a high-salt diet for 6–7 weeks. They then received CDC intracoronary or a placebo. As a result, significant hemodynamic improvements were observed, i.e., normalization of the left ventricle relaxation and left ventricle diastolic pressure in comparison with the placebo-treated counterparts. In the CDC-treated group, a significant fibrosis reduction was demonstrated due to a decrease in collagens 1 and 3 content.Furthermore, the treatment was associated with decreased pro-inflammatory cytokines and reduced myocardial macrophage and leukocyte infiltration. CDC therapy also enhanced cardiomyocyte proliferation and due to increased microvascular density, ensured a favorable microenvironment for their survival and optimal function. These benefits resulted in a decrease in lung congestion and increased survival .These findings were confirmed by the de Couto et al., preclinical trial. In this study, an analogous model was used, and due to CDC treatment, consistent results were obtained. However, the in-depth investigation revealed some additional benefits of CDC treatment. Due to an improvement in eNOS activity and a reduction in ROS, vasodilatation was restored. The therapy also reduced oxidative stress and VCAM-1 expression, resulting in decreased macrophage attachment in-vitro .Rieger et al., carried out the first study concerning stem cell therapy in HFpEF in a novel large-animal model. In this randomized, placebo-controlled, blind trial, HFpEF was induced in swine by embolization-mediated nephrectomy. Then, the 26 Yorkshire pigs were subdivided into four groups. The animals recruited to three of the groups received different types of stem cells, whereas the last group was a control group. The stem cells were injected into the renal artery by an angiogram and MRI.Promising outcomes were obtained in the porcine group receiving a combination of stem cells, i.e., MSCs and kidney-derived stem cells (KSCs). In this group, a decrease in end diastolic pressure-volume relationship (EDPVR) was detected, which reflects a reduction in distensibility and diastolic function improvement.Additionally, a beneficial effect on chronic kidney disease was observed. There was an improvement in the glomerular filtration rate (GFR) in the group receiving both MSCs and KSCs, presumably due to the downregulation in genes associated with fibrosis, apoptosis, and inflammatory responses .Promising results from a few experimental studies and the improvement of the diastolic parameters in diseases, such as chronic myocardial ischemia, HFrEF, and dilated cardiomyopathy due to cell therapy, have prompted researchers to examine the efficiency of stem cells in HFpEF ,94,95.The first results of the application of stem cell therapy in HFpEF were published by Frljak et al. In this groundbreaking prospective crossover research, 30 patients were treated with the standard medical treatment for HFpEF for six months and then underwent a transendocardial CD34+ cell transplantation. The CD34+ cells were injected transendocardially into the areas of diastolic dysfunction . The sixBased on experimental studies, stem cell transplantation has a potent immunomodulatory effect, which may be particularly beneficial in the treatment of HFpEF. It has been detected that stem cell therapy may suppress the inflammatory state by downregulation of pro-inflammatory factors, such as TNF-α, IL-1β, IL-6, and MCP-1 . This maThe immunomodulatory effect, especially a decrease in TGF-ß and in the macrophage infiltration, may also explain the antifibrotic properties of stem cell therapy. Moreover, MSCs, because they promote MMP-2 and MMP-9 secretion, can inhibit extracellular matrix remodeling associated with collagen and fibronectin accumulation . The antExperimental studies showed stem cell therapy may promote angiogenesis, which appears to be particularly relevant in HFpEF ,102. It Stem cells can also exert a cardioprotective potential via the overexpression of BCL2, survivin, and HIF-1 ,60. It hTherefore, stem cell therapy may become the first highly efficient etiology-based therapy due to its complex mechanism of action, as summarized in Experimental studies have provided promising information concerning stem cell therapy in cardiovascular diseases. It has been shown its immunomodulatory effect may improve vasculogenesis and decrease fibrosis, ameliorating diastolic dysfunction and improving prognosis. To date, only one study investigating stem cells in HFpEF has revealed substantial clinical benefits. Further follow-up studies are necessary to assess the long-term results and possible side effects. The results of the ongoing regress-HFpEF clinical trial concerning CDC in HFpEF as well as further studies are needed to prove the efficiency and provide information concerning the safety and long-term effects of stem cell therapy of HFpEF.As mentioned above, strategies of stem cell administration are beneficial in the treatment of coronary artery disease. Nevertheless, in HFpEF as the function of the whole left ventricle is impaired, intracoronary infusion might be more efficient than transendocardial injection. Furthermore, this significantly less invasive delivery method may presumably diminish the risk of arrhythmogenic complications by preventing local tissue injury ,108.We also suggest future studies in this field should focus on performing allogeneic stem cell transplantations, which may give better therapeutic effects than autologous transplantations. It is worth noting that patients with HFpEF are very frequently elderly patients, which limits the regeneration potential of stem cells in the case of their autologous origin. It is also worth noting that allogeneic stem cell transplantation may also make it possible to avoid CHIP-related mutations.Due to insufficient data, many aspects of stem cell therapy of HFpEF still remain obscure. For instance, there is insufficient information to establish a specific treatment schedule. In cardiovascular diseases, mostly single injections of stem cells were examined. However, sequential treatment may possibly result in better outcomes.Similarly, there is no consensus on which kind of stem cells should be administered in HFpEF. We propose tailored therapy may be considered in subsequent studies. For instance, patients with fibrosis and inflammation within the myocardium may achieve significant benefits from MSC or CDC treatment, whereas patients with a decrease in microvascular density and endothelial dysfunction are more likely to benefit from EPC treatment. Presumably, combined therapy composed of different kinds of stem cells, due to their synergistic properties, may be the gold standard of HFpEF treatment in the future. Hopefully, subsequent studies will provide comprehensive information to advance this treatment to obtain the most beneficial breakthrough stem cell therapy for HFpEF.Today, there is a growing tendency in the incidence of HFpEF. However, there is no therapeutic option to modify the course of the disease, and it is still characterized by a high mortality rate. Hypotheses explaining the complex pathogenesis of HFpEF associate the development of the disease mostly with inflammation. Importantly, stem cells exert immunomodulatory properties, which as a consequence, may improve the diastolic function. There are promising results of a few experimental studies of stem cell therapy in HFpEF. However, further clinical studies are crucial to determine stem cell therapy effectiveness and safety and to develop specific clinical treatment guidelines.


Permanent transseptal left bundle branch area pacing (LBBAP) is a promising technique developed to avoid the detrimental effects of pacing‐induced dyssynchrony with right ventricular (RV) pacing, by offering more physiologic activation of the heart. Lesions to tributary veins of the coronary sinus have been increasingly reported, mostly associated with venous fistula or venous septal system infringement. Despite being mostly benign, venous complications may be related to the maneuver of contrast injection through the sheath and failure to follow simple but essential steps. Permanent transseptal left bundle branch area pacing (LBBAP) is a promising technique developed to avoid the detrimental effects of pacing‐induced dyssynchrony.We present two cases with venous system infringement after LBBAP. Both were made during contrast injection through the sheath after screwing the pacing lead deep into IVS. We discuss important steps that could prevent such issues. We also challenge the practice of contrast injection during septography to confirm lead position in cases where electrocardiographic parameters reveal good LBBAP.In the first case, an 89‐year‐old woman presented for consultation due to recent‐onset fatigue. Her heart rate was 32 beats/min with blood pressure of 142/86 mmHg. The electrocardiogram revealed type 2 second‐degree AVB. There were no signs of coronary artery disease on angiogram. Her echocardiogram showed left ventricular ejection fraction (LVEF) of 63%. The electrophysiologic study confirmed second‐degree infra‐Hisian AVB. Left bundle branch area pacing was performed with a 5.6‐F stylet‐driven pacing lead with extendable helix  delivered through a preshaped sheath . Nonselective LBBAP was obtained (threshold 0.7 V at 0.4 ms) and stimulation to peak left ventricular activation time (LVAT) of 62 ms Figure . UnipolaIn the second case, a 72‐year‐old man with Chagas cardiomyopathy (LVEF 17%) was admitted due to recurrent implantable‐cardioverter defibrillator (ICD) shocks. He had class III NYHA functional class despite optimal medical therapy. He also received a dual‐chamber ICD implantation years ago. Device interrogation showed multiple episodes of ventricular tachycardia (VT) treated with appropriate shocks.There was a ventricular pacing burden >80% because of baseline intermittent 2:1 AVB. Baseline ECG registered atrial paced activity, right bundle branch block, native QRS width of 150 ms, and first‐degree AVB Figure . Given aSeptal coronary vein infringement during LBBAP is considered rare, although probably underdiagnosed. With rapid adoption of LBBAP, case reports of cardiac venous infringement have been increasing. Batul et al. reported two cases of venous drainage through cardiac venous system during LBBAP, using both stylet‐driven and lumenless fixed helix leads.The common aspect of the reports of coronary venous penetration is the absence of serious complications or pacing parameters. This is probably related to the small size of the fistulas. Despite some operators opting to reposition the lead, one should question whether this step is necessary. Some authors have hypothesized that small‐diameter lumenless leads might reduce the risk, which was not confirmed. It seems that lead penetration, as well as the inappropriate contrast injection technique may precipitate venous infringement.Operators performing LBBAP should consider some steps before contrast injection to confirm the lead position in the interventricular septum. We avoid venous septum injury by (1) pulling the sheath slightly backward from the IVS and confirm sheath position in fluoroscopy; (2) using a small volume of nondiluted contrast (2 mL) injected gently and slowly. Following this stepwise approach, we may reduce the chance of high contrast pressure toward the IVS. Operators should also question whether is necessary to perform septography in cases with good electrocardiographic parameters signaling LBBAP. However, if they consider that contrast injection is essential, they should perform it in a stepwise approach.There was no funding source.Authors declare no conflict of interests for this article. Patients provided full written consent.Video S1.Click here for additional data file.Video S2.Click here for additional data file.Video S3.Click here for additional data file.


Main shoot total leaf number (TLN) is a key determinant of plant leaf area and crop adaptation. Environmental factors other than photoperiod can affect TLN in sorghum, implying that leaf appearance rate (LAR) and development rate can differ in response to temperature. The objectives of this study were to determine (1) if temperature effects on TLN can be explained as a consequence of differences in temperature responses across phenological processes and (2) if genotypic differences in these responses can be linked to agroecological adaptation.Nineteen sorghum genotypes were sown on 12 dates at two locations in Ethiopia with contrasting altitude, creating temperature differences independent of photoperiod. TLN and temperature were recorded in all experiments and LAR for six sowing dates.Tbase) for LAR than for pre-anthesis development rate (DR). In contrast, genotypes with no effect of temperature on TLN had similar Tbase for LAR and DR. Across genotypes, Tbase for LAR and DR were highly correlated, but genotypes with low Tbase had the greatest difference in Tbase between the two processes. Genotypic differences were associated with racial grouping.Eleven of the genotypes showed a temperature effect on TLN, which was associated with a significantly higher base temperature (Tbase, can affect specific adaptation to agroecological zones, as these differences can affect TLN in response to temperature and hence canopy size and the duration of the pre-anthesis period. These can both affect the amount of water used and radiation intercepted pre-anthesis. A multi-disciplinary approach is required to identify genotype × environment × management combinations that can best capture the ensuing specific adaptation.Genotypic and racial differences in responses of phenological processes to temperature, in particular in  Sorghum bicolor), which is usually grown under rainfed conditions in semi-arid tropical and sub-tropical environments, where the timing and severity of drought stress can be highly variable in both space and time (−−1 °C−1), which in turn depends on temperature and photoperiod.Phenology is an important aspect of crop adaptation, as the timing of anthesis can play a significant role in minimizing the adverse effects of end-of-season drought stress on crop yields (−1 °C−1) and the duration of the period between plant emergence and PI (°Cd), which in turn depends on the pre-anthesis phenological development rate (DR). LIR, LAR and DR (and thus PI) each depend on temperature, and the response is such that rates will be zero when temperature is below a base temperature (Tbase) or above a maximum temperature (Tmax) and rates will be highest at the optimum temperature (Topt)   . Such a As sorghum is a short-day crop, the duration (°Cd) from emergence to PI will be extended for photoperiod-sensitive genotypes if the photoperiod prior to PI exceeds a threshold , althougTbase) that were linked to racial grouping adds veracity to these results. In addition, significant genotypic differences in LAR and in the effect of temperature on TLN have been reported for sorghum genotypes that included germplasm of Ethiopian background (A previous study (Sorghum bicolor (L.) Moench] genotypes were sown on 12 different dates in 2013 and 2014, with six sowing dates per year per location. Sowings were conducted at ~3- week intervals, ranging from 24 March to 8 July in 2013 and 12 April to 25 July in 2014. The difference in sowing dates between locations was generally no more than 2 d, with only one occurrence of a 4-d difference  and Kulumsa , which represent lowland and highland altitudes respectively. The sorghum [fference . Combinefference . During fference .−1 P2O5) in the form of di-ammonium phosphate and nitrogen fertilizer (23 kg ha−1 nitrogen as urea) were applied at sowing and at 35 d after sowing, respectively. All experiments were well watered.Each experiment (location × sowing date) was set up as a randomized complete block design with two replications, with a different randomization for each experiment. Plots consisted of a single row of 5 m length with a row spacing of 0.75 m and 1.5 m distance between blocks. Seeds were manually drilled into the rows and seedlings were thinned to 0.15 m between plants at ~20 d after emergence. Phosphorus fertilizer , Birmash (94.1 % kafir), Jamiyu (91.2 % Ethiopian highland durra) and Geremew (80.7 % kafir). The last also contained 11.0 % Ethiopian highland durra and 7.1 % caudatum (caudatum/guinea mixed race: Adukura (50 % caudatum and 22 % guinea) and Bobe red (63 % caudatum and 22 % guinea). For ESH-2, the racial classification was not available. Racial classification was closely associated with the four major sorghum agroecological growing areas in Ethiopia. These areas differ in altitude , rainfall (mm) and duration of the growing period (d), and comprise the highlands , intermediate zone , wet lowlands  and dry lowlands   . The foucaudatum .In each experiment, observations were conducted on three plants per plot that were tagged when about five leaves had fully expanded. Main shoot TLN was recorded in all experiments once the flag leaf was fully expanded. In addition, the number of fully expanded main shoot leaves throughout the vegetative period was counted at regular intervals in all experiments in 2014. A leaf was considered as fully expanded when its ligule became visible above the enclosing sheath of the previous leaf  for TLN was conducted to determine if there were any effects of location and genotype on TLN across pairs of experiments with common sowing dates. Hence, the 24 experiments (2 locations × 12 sowing dates across two seasons) were analysed in terms of sowing date (11 d.f.) and location within sowing date (12 d.f.). The presence of significant genotype and location effects on TLN was subsequently analysed in more detail using a paired-sample uide 9.4 .−1). Because temperatures were relatively constant for large periods of time during the growing season (−1). For some early-flowering genotypes, the number of observations was too limited in some experiments to reliably determine LAR. For situations with LAR data, weather data were then used to calculate the average daily temperature for the period during which leaf number was linearly related to DAS. The data for LAR and mean daily temperature that were thus obtained for each genotype in each experiment were then plotted against each other in order to determine the response of LAR (leaves d−1) to mean daily temperature. Each regression had up to 12 data points (2 locations × 6 sowing dates). The intercept of the regression with the x-axis was considered to be the base temperature (Tbase) for LAR and represented the average daily temperature at which LAR is zero. The slope of the regression (leaves d−1 °C−1) represents the increase in LAR (leaves d−1) for each degree increase in average daily temperature. To determine if genotypes differed in Tbase, the slope of the regression or both, the following fixed effect non-linear model was fitted:For each genotype in each of the 12 experiments in 2014, the average number of fully expanded leaves was plotted against the number of days after sowing (DAS). Leaf number was the average value across the two replications for each experiment (six plants per genotype). For the period during which leaf number was linearly related to DAS, the slope of the regression was taken as the LAR . Analyses were done in R version 4.05 (nls) function  genotype × experiment interaction and in particular the highly significant (P < 0.0001) genotype × location (sowing date) interaction  on average producing >3 more leaves at Melkassa than at Kulumsa (kafir genotypes showed such a response. The difference in the location effect on TLN between the durra and kafir groups was highly significant (P < 0.01) according to a t-test. However, the seven caudatum and caudatum/guinea genotypes were more evenly distributed between the two groups . Across all experiments, the average daily temperature ranged from ~16.0 to 24.1 °C, although the range was slightly lower for Adukara (16.0–22.9 °C), where only data for the second slope  gave a significantly (P < 0.0001) better fit than a common regression across all genotypes. Similar results were obtained with a model that assumed a genotype-specific Tbase but a common slope across genotypes. Importantly, however, in both cases, the model improved significantly (P < 0.05) if genotype-specific values for both slope and Tbase were used. Moreover, a common Tbase of 11 °C, commonly used as the Tbase of phenological processes for Australian sorghum (P < 0.0001) than that of using genotype-specific values. This indicates that it was appropriate to fit separate slopes and Tbase in eqn (1) for each genotype  to average daily temperature was strongly linear across experiments , such that genotypes with high Tbase increased the LAR quickest with increasing temperature. Hence, the three genotypes with the greatest slope all belonged to the kafir race with high Tbase. Nonetheless, there were no significant differences in the average slope among racial groups (−1 (Adukara) to 0.315 leaves d−1 . The two caudatum/guinea genotypes both had low LAR at this temperature and the average of this group was significantly lower than that of the highland durra genotypes  and DR (P < 0.001) than the eight with no temperature effect on TLN (Tbase for LAR was on average 2.2 °C higher than Tbase for DR (P < 0.05) greater than zero based on a t-test for pairwise comparisons. In contrast, for the other eight genotypes Tbase for LAR was, with the exception of Macia , with Ma3 having greater phyllochron (lower LAR) than ma3 if ma2 is recessive, but not if Ma2 is dominant, whereas Ma1 had greater phyllochron than ma1 if Ma2 is dominant, but not if ma2 is recessive. For maize, Tbase, but no such information is available for sorghum. The current analyses indicated that genotypic differences in the response of LAR to temperature were associated with genotypic differences in both Tbase and the slope of the response, even though the two factors were highly correlated. The analyses also indicated that the Tbase of 11 °C that has been derived in Australia  for a particular genotype, then an increase in average daily temperature will result in a relatively larger change in thermal units (°Cd) for LIR compared with DR. This will increase LIR to a relatively larger extent than DR, which in turn will increase the number of leaves that are initiated at the apical meristem prior to PI, resulting in greater TLN.Significantly greater TLN under the high temperatures at Melkassa compared to lower temperatures at Kulumsa for similar sowing dates indicated a temperature effect on TLN that was independent of photoperiod. Similar increases in TLN under higher temperatures during the period following emergence have been observed previously in maize  and sorgTbase for LAR, rather than for LIR, genotypic differences in the response of LAR and LIR to temperature are highly coordinated prior to PI, as observed for maize (pla1 gene that accelerates LIR was phenotypically identified through accelerated LAR (Although the current study estimated or maize . SimilarTbase across phenological processes, as the average difference in Tbase between LAR and DR of 2.2 °C for the 11 genotypes with a significant temperature effect on TLN was significantly greater than zero. This implies that at low temperatures, development will progress more rapidly than leaf appearance and leaf initiation. This would reduce the number of leaf primordia initiated prior to PI, which would explain the significantly lower TLN of these genotypes at Kulumsa compared to Melkassa (Tbase for LAR exceeds Tbase for DR.The current results  showed sMelkassa ; Fig. 6.Tbase across phenological processes to the emergent consequence of a temperature effect on TLN. Significantly, this response was linked to racial grouping. Compared with kafir genotypes, highland durra genotypes on average had significantly lower Tbase for both LAR and DR, significantly greater difference in Tbase, and significantly greater response of TLN to temperature (durra landrace from Ethiopia (Tbase on the response of TLN to temperature was not uniform, as caudatum genotypes on average had a Tbase for both LAR and DR that was comparable with that of highland durra genotypes (caudatum genotypes were consistently located on or below the regression line and highland durra genotypes on or above the regression line. The reason for this distinct behaviour is unclear. The consistent racial effect on Tbase for LAR and DR, and on the difference in Tbase between these two processes (To our knowledge, this is the first report for sorghum that links significant genotypic differences in perature  and 4. Tenotypes , but theenotypes . As a corocesses , contradThe analyses showed that significant genotypic differences in the response of LAR to temperature exist for sorghum, and that individual genotypes can have different temperature responses across processes, resulting in a response of TLN to the temperature environment. Although the study was conducted with Ethiopian germplasm, the association between temperature response and racial groups indicates generic associations that would facilitate incorporation of the current insights into any sorghum breeding programme through selection of targeted racial groups.Tbase for LAR and pre-anthesis DR can potentially affect adaptation to post-anthesis drought. A lower Tbase for LAR would accelerate leaf appearance. Increased LAR can increase early vigour of the main shoot, and the resulting shift in carbon supply–demand balance of the plant can reduce tillering (Tbase for DR could accelerate progress to anthesis, which can also reduce pre-anthesis water use (Tbase for either LAR or DR could thus be beneficial to adaptation to post-anthesis drought stress.Genotypic differences in Tbase values had the greatest difference in Tbase between processes (Tbase on adaptation to drought, particularly under high temperatures. Because of the association between LAR and LIR (Tbase for LAR will probably result in the initiation of more leaves prior to PI, although the extent of that will depend on the slope of the response of LAR to temperature. Any effect on TLN will be exacerbated under high temperatures, as the genotypes with low Tbase for LAR and DR tend to have the greatest difference in Tbase between these two processes and are thus most likely to increase TLN if temperatures increase (Tbase per se. Interestingly, there appeared to be some racial differences in this response, as caudatum genotypes generally had a smaller increase in TLN for a given difference in Tbase between LAR and DR than highland durra and caudatum/guinea genotypes (Tbase. However, the consequences of such changes in agronomy on grain yield, particularly across cropping regions with variable timing and intensity of drought and heat stress, can be complex and would require targeted simulation studies to unravel any interactions (However, the observation that genotypes with low rocesses  can comp and LIR , a low Tincrease ; Fig. 6.


Rheumatoid arthritis, RA, is a chronic autoimmune disease characterized by joint pain, tenderness, swelling, and stiffness. This disease affects nearly 1% of the world population. RA predominates in females and typically develops between the ages of 30 and 50 years. Common therapeutics for the treatment of RA include immune system suppressants such as tumor necrosis factor, or TNF, inhibitors. There is growing concern related to multiple clinical cases reporting an unexpected onset of psoriasis following the use of TNF inhibitors. This adverse event is counterintuitive since some tumor necrosis factor inhibitors are indicated for the treatment of plaque psoriasis. In this study, we analyzed over 880 thousand postmarketing safety reports from patients being treated for RA with a single therapeutic and provided evidence for a statistically significant association of psoriasis adverse events with TNF inhibitor use as compared to methotrexate. Additionally, we quantified the reported odds ratios and their 95% confidence intervals between four individual TNF inhibitors and found that the degree of association with psoriasis was variable among the drugs studied, with certolizumab pegol exhibiting the highest reported risk. RA is characterized by joint pain, tenderness, swelling, and joint stiffness2. While the cause of RA is not certain, risk factors include genetics, smoking, obesity, sex, and age2. Similar to other autoimmune diseases, RA predominantly affects females, with a 2:1 to 3:1 female to male ratio1. While RA can develop at any age, typical onset occurs between 30 and 50 years3. RA is commonly treated with immunosuppressants such as methotrexate, hydroxychloroquine, sulfasalazine, or leflunomide, T-cell costimulatory inhibitors such as abatacept, interleukin-6 receptor (IL-6R) antibodies such as tocilizumab, Janus kinase (JAK) inhibitors, and tumor necrosis factor (TNF) inhibitors such as certolizumab pegol, adalimumab, golimumab, infliximab, and etanercept4.Rheumatoid arthritis (RA) is a common chronic autoimmune disorder primarily attacking the joints, affecting between 0.5 and 1% of the world population5. Another important class of therapeutics in treating RA are IL-6 pathway inhibitors, which either inhibit IL-6 directly or block its receptor IL-6R6. Another class of therapeutics, JAK inhibitors, suppress cytokine-mediated signals by the JAK-STAT pathway7.T-cell costimulatory inhibitors prevent the activation of T-cells, which are believed to play a key role in the pathogenesis of RA. Abatacept, containing an extracellular domain of CTLA-4, accomplishes this by competitively binding the proteins CD80 and CD86, blocking their interaction with CD288. Recently, there has been growing concern over TNF inhibitor-induced psoriasis, with many case reports detailing the onset of psoriasis in patients using TNF inhibitors, such as certolizumab pegol11. This relationship is seemingly paradoxical given that four of the five TNF inhibitors are indicated as a treatment for a type of psoriasis, plaque psoriasis: certolizumab pegol12, adalimumab13, infliximab14, and etanercept, which is a fusion of the TNF receptor and the IgG1-Fc part of an antibody15.Anti-TNF treatments suppress the inflammatory response which results from the binding of TNF to its receptors15. These effects may be related to the cross-regulation of TNF and type-I interferons, including interferon alpha (IFN-α)16. TNF downregulates IRF7 and NF-κB pathways, which are responsible for IFN-α and TNF-α production in plasmacytoid dendritic cells17. Blocking the activity of TNF allows the production of IFN-α to increase10. Increased IFN-α has been associated with an increased risk of psoriasis18, possibly explaining the association between the use of TNF inhibitors and an increased risk of psoriasis. In this study, we analyzed over 880 thousand RA reports from FDA Adverse Event Reporting System (FAERS) and quantifed the evidence using the latest population scale dataset for a statistically significant association of psoriasis with TNF inhibitor use. Furthermore, we quantified the difference in the reported risk of developing psoriasis in RA patients between the TNF inhibitor cohorts.There have been postmarketing reports of incidents of new and worsening psoriasis in patients administered any of the four of the aforementioned TNF inhibitorshttps://www.fda.gov/drugs/questions-and-answers-fdas-adverse-event-reporting-system-faers/fda-adverse-event-reporting-system-faers-latest-quarterly-data-files.The United States Food and Drug Administration Adverse Event Reporting System (FAERS) is a data repository which collects voluntary drug-related reports from healthcare professionals, consumers, and legal representatives. In cases where the adverse event (AE) is reported to the manufacturer, the manufacturer is required to forward the report to FAERS. At the time of the study, FAERS contained 17,392,666 AE reports collected from the first quarter of 2004  to the second quarter of 2022. The reports are available online: 21.FAERS reports are added and stored quarterly in a set of text files. Subsets of data are organized by specific report fields  and their respective case identifiers. The data format is not uniform and has been modified several times since its inception. Therefore, appropriate modifications have been introduced. Additionally, as the AE reports are collected from all over the world, the respective drug brand names are translated into the generic equivalentspsoriasis in the analysis: erythrodermic psoriasis, guttate psoriasis, nail psoriasis, dermatitis psoriasiform, pustular psoriasis, and psoriasis.The MedDRA dictionary version 25.1 was searched to define the measured study outcomes by higher level terms such as “immune associated conditions not elsewhere classified (NEC)” and “psoriatic conditions”. All of the psoriasis-associated preferred terms (PT) were used in the query. In order to avoid any indication-related confounding effects, psoriatic conditions associated with RA, such as psoriatic arthropathy, were excluded from the MedDRA PT list. The following PTs were used to define psoriasis AE report numbers were calculated for each drug cohort: certolizumab pegol (n = 98), adalimumab (n = 107), golimumab (n = 20), tocilizumab (n = 29), abatacept (n = 40), infliximab (n = 29), rituximab (n = 11), etanercept (n = 260), tofacitinib (n = 24), and methotrexate (n = 7). Disproportionality analysis was performed using reported AE frequencies to calculate reporting odds ratios (RORs). These numbers were used to calculate psoriasis reported frequencies. Methotrexate was selected as the control cohort due to its unique mechanism of action (MOA) as an immunosuppressant which inhibits the conversion of folic acid to folate cofactors, and common use as a monotherapy in RA.Out of the total of 17,392,666 AE reports in FAERS, there were a total of 881,182 RA indication-containing reports, and for 663,922 of them, RA was listed as the only indication. Those records were further split by monotherapies and only reports by physicians, pharmacists and other healthcare professionals were included to avoid bias and increase clinical relevance. The final monoindication + monotherapy sets were the following: certolizumab pegol (n = 5168), adalimumab (n = 9221), golimumab (n = 2899), tocilizumab (n = 4819), abatacept (n = 7574), infliximab (n = 5579), rituximab (n = 2519), etanercept (n = 89543), tofacitinib (n = 10686), and methotrexate (n = 6142). Demographic analysis was performed for TNF inhibitors and methotrexate RA AE cohorts  analysis for Figs. Standard Error of Log Reporting Odds Ratio;95% Confidence Interval;22. Methotrexate monotherapy exhibited the lowest reported frequency of psoriasis at 0.11% out of all of the monotherapies studied. Methotrexate is a first-line therapeutic for RA in a clinical setting, making it a suitable control choice.Methotrexate, an immunosuppressant drug, was selected as the control RA treatment based on its unique MOA as a competitive dihydrofolate reductase inhibitorpsoriasis AEs were calculated for each TNF inhibitor monotherapy. The frequencies of psoriasis reports between the TNF inhibitors ranged from 0.29 to 1.90%: certolizumab pegol 1.90%, adalimumab 1.16%, golimumab 0.69%, infliximab 0.52%, and etanercept 0.29% , whereas all of the other monotherapy cohorts had 95% CIs that were greater than 1.00 in both the lower and upper bounds.psoriasis reports in the methotrexate-treated patient cases allowed for overlapping 95% CIs between the individual TNF inhibitors. In order to clarify the difference between the TNF inhibitors, we derived the ROR + 95% CI values by doing a pairwise comparison without methotrexate as a common control.The small number of psoriasis AEs when compared directly with other RA drugs used as a control cohort instead of methotrexate. Certolizumab pegol compared to adalimumab yielded ROR 1.65 and 95% CI , compared to golimumab 2.78 , compared to tocilizumab 3.19 , compared to abatacept 3.64 , compared to infliximab 3.70 , compared to rituximab 4.41 , compared to etanercept 6.64 , compared to tofacitinib 8.59 , and compared to methotrexate 16.94   not all of the non-TNF inhibitor treatments were associated with psoriasis compared to the single methotrexate control group, with some overlapping with the TNF inhibitor 95% CI ranges, (iii) pairwise comparisons of certolizumab pegol with both other TNF inhibitors and non-TNF inhibitor therapeutics show a greater and statistically significant association with psoriasis.In summary, we arrived at three sets of conclusions: (i) all TNF inhibitors have a statistically significant association with psoriasis AE when compared to all the other RA monotherapy cohorts studied.Thus, certolizumab pegol was found to have a particularly high reported risk of psoriasis. In addition, we quantified the risk of psoriasis in RA patients undergoing other types of therapeutic treatments such as IL-6 inhibitors (tocilizumab) and JAK inhibitors (tofacitinib), among others. We discovered that all TNF inhibitor treatments have a statistically significant association with psoriasis in RA patients. We also discovered that all of the non-TNF inhibitor cohorts, except tofacitinib, were statistically significantly associated with psoriasis, though to a lesser extent than certolizumab pegol. Tofacitinib was the only non-TNF monotherapy that was not statistically significantly associated with psoriasis within the 95% CI.To our knowledge, this is the first study to analyze over 660 thousand AE reports for RA indication in postmarketing data to quantify the association between TNF inhibitor monotherapy use and psoriasis for particular pairs of drugs. To address this issue, pairwise ROR + 95% CI analyses were performed. These results revealed that certolizumab pegol exhibited the highest reported risk, with 95% CI statistically separating it from all of the other studied monotherapies ) were calculated for each TNF inhibitor monotherapy cohort and compared with the respective cohort’s frequency of psoriasis reports (Table Of the monotherapies studied, certolizumab pegol exhibited the highest reported frequency and ROR of ts Table , Fig. 6.psoriasis AEs were in line with current understanding of TNF inhibitors’ risk of psoriasis, as stated in the FDA package inserts of four of the five TNF inhibitors: certolizumab pegol12, adalimumab13, infliximab14, and etanercept15. However, given that golimumab’s package insert lacks a warning for psoriasis24, we underscore its statistically significant association with psoriasis as an adverse event.The statistically significant associations of TNF inhibitors with psoriasis AEs.Here we established and quantified an association of the use of TNF inhibitors, as well as the non-TNF inhibitors studied with the exception of tofacitinib, with the risk of psoriasis when compared to methotrexate. Out of the TNF inhibitors studied, certolizumab pegol exhibited the highest reported frequency of psoriasis.We established and quantified an association of TNF inhibitor monotherapies use by RA patients with psoriasis as an AE. All of the TNF inhibitor therapies approved for the treatment of RA and three out of the four non-TNF inhibitor drugs studied had a statistically significant association with 26. The individual cases were not adjudicated for causality assessment. Further studies may be required to establish the molecular mechanism behind this counterintuitive adverse event to better understand the uses and risks of TNF inhibitor therapies as it relates to RA and psoriasis. Most individual reports do not contain details on the progression of the AEs over time.Due to the voluntary reporting nature of the FAERS system, the sample of reports in the FAERS database may be incomplete compared to real population data. FAERS reporting exhibits biases related to its newsworthiness and legal and scientific variables. Due to potential underreporting, some real-world data may be missing from the FAERS database which this study used to conduct its analyses. The changing criteria for AE diagnosis may lead to under or overreporting and affect the reporting frequencies. The frequencies and reporting odds ratios calculated are based on the sample of reports in the FAERS database and may not correspond to real population frequencies


The extracellular domain of tumor necrosis factor receptors (TNFR) generally require assembly into a homotrimeric quaternary structure as a prerequisite for initiation of signaling via the cytoplasmic domains. TNF receptor homotrimers are natively activated by similarly homo-trimerized TNF ligands, but can also be activated by synthetic agonists including engineered antibodies and Fc-ligand fusion proteins. A large body of literature from pre-clinical models supports the hypothesis that synthetic agonists targeting a diverse range of TNF receptors  could amplify immune responses to provide clinical benefit in patients with infectious diseases or cancer. Unfortunately, however, the pre-clinical attributes of synthetic TNF receptor agonists have not translated well in human clinical studies, and have instead raised fundamental questions regarding the intrinsic biology of TNF receptors. Clinical observations of bell-shaped dose response curves have led some to hypothesize that TNF receptor overstimulation is possible and can lead to anergy and/or activation induced cell death of target cells. Safety issues including liver toxicity and cytokine release syndrome have also been observed in humans, raising questions as to whether those toxicities are driven by overstimulation of the targeted TNF receptor, a non-TNF receptor related attribute of the synthetic agonist, or both. Together, these clinical findings have limited the development of many TNF receptor agonists, and may have prevented generation of clinical data which reflects the full potential of TNF receptor agonism. A number of recent studies have provided structural insights into how different TNF receptor agonists bind and cluster TNF receptors, and these insights aid in deconvoluting the intrinsic biology of TNF receptors with the mechanistic underpinnings of synthetic TNF receptor agonist therapeutics. The tumor necrosis factor (TNF) superfamily (TNFSF) of 19 ligands and 29 receptors serve as critical regulators of human immunity, and modulating the activity of individual receptors and ligands for therapeutic benefit in autoimmunity and cancer has been studied for over 40 years . ActivatThe focus of this review is to highlight the structural hypotheses underlying TNF receptor trimerization and subsequent activation of various cytoplasmic signaling cascades, both following activation via native TNF ligands and also with synthetic receptor agonists. A particular emphasis is placed upon areas where the pharmacodynamic activity of a TNF receptor agonist differed between pre-clinical mouse studies and human clinical trials. Several different TNF receptor agonists and TNF receptor targets, are included in this discussion, however the analysis is focused on how the available data inform on the magnitude and specificity of receptor engagement, rather than on the cellular and mechanistic differences between individual TNF receptors themselves. For example, this review focuses on whether the cytokine release syndrome (CRS) observed in human cancer patients treated with a 41BB or CD40 agonist antibody was likely a consequence of the underlying structural features of those antibody therapeutics rather than a deep dive into the specific differences in 41BB mediated costimulation of CD8 positive T cells versus CD40 mediated costimulation of antigen presenting cells –8.Many patients, patient investors and drug developers have dedicated decades of effort to translating the powerful biology of TNF receptor agonists for the benefit of human disease. Most of these efforts have not lived up to the potential shown by the pre-clinical biology, yet many important lessons have been learned along the way. An improved understanding of the structural basis of TNF receptor activation has the potential to guide future development of improved therapeutic agonists.All twenty nine TNF receptors, with the exception of DcR3, are single-pass type 1 membrane proteins, oriented with a cytoplasmic carboxy terminus and an extracellular amino terminus . DcR3 is2+ ion, however it is unknown whether other TNF ligand trimers are similarly dependent upon cation coordination  achieve activation with soluble ligand trimers, and are referred to as Category 1 TNFR. Clinical data are available for GITR agonist antibodies, but not for any of the others. For the so-called Category 2 TNF receptors , soluble ligand trimers fail to activate downstream receptor signaling, unless those ligand trimers are cross-linked either via an Fc domain fused to the amino terminus of the TNF ligand extracellular domain, or if the soluble ligand trimers are cross-linked by an anti-TNF ligand antibody , 22. Thain vivo activity has proven to be an elusive goal, however. Over the years the number of synthetic agonist compounds has expanded, and now includes: monoclonal IgG antibodies, bispecific antibodies, tetravalent antibodies, hexavalent antibodies, Fc-fusion proteins, anticalin fusion proteins, bispecific Fc-fusion proteins, and IgM antibodies  binding activity, as this was shown to be a pre-requisite for agonist activity in most cases . The mosist mAbs .The FcγR-dependent mechanism of TNFR agonist antibodies requires sub-saturating receptor occupancy on both the TNFR target and Fcγ receptors. As illustrated in Amongst the clearest examples of bell-shaped dose response effects in humans treated with TNF receptor agonist antibodies include data from cancer patients treated with BMS-986178 (anti-OX40 mAb), PF-04518600 (anti-OX40 mAb) or mitazalimab (anti-CD40 mAb). Each of these antibodies are dependent on FcγR mediated cross-linking for agonist activity. The main pharmacodynamic marker reported from patients treated with the two OX40 agonist mAbs was proliferation (as indicated by Ki67 expression) of specific T cell subsets , 45. In 6 cells per mL, of which 80% are lymphocytes, 85% of the lymphocytes are T cells, 60% of the T cells are CD4+ T cells and 20% of those CD4+ T cells express OX40. Also consider ‘Patient B’, who has a PBMC count of 0.8x106 cells per mL, of which 70% are lymphocytes, 70% of the lymphocytes are T cells, 50% of the T cells are CD4+ T cells and 5% of the CD4+ T cells express OX40. In both patients, these example lymphocyte/T cell percentages fall within the ‘normal’ range for healthy adults (the magnitude of variability can have a wider range in oncology patients who have received prior chemotherapy), yet Patient A will have approximately 105 CD4+OX40+ T cells per mL of peripheral blood, and Patient B will have approximately 104 CD4+OX40+ T cells per mL of peripheral blood. If the dose of an OX40 agonist antibody is modeled on the basis of achieving 50% receptor occupancy, the appropriate doses for Patient A and Patient B would differ by 10-fold. In a phase 1 clinical trial of SL-279252, we observed even wider variation, with the number of CD4+ T cells ranging from 2.2x103 to 1.1x106, and the percentage of CD4+OX40+ cells ranging from 5.1-48.6%  in late stage cancer patients did not report any clear dose-dependent pharmacodynamic activity following infusion, either alone or in combination with nivolumab. In a phase 1 clinical trial with an FcγR non-binding GITR agonist, TRX518, some evidence reductions in regulatory T cells were reported both in the peripheral blood and within the tumor. The dose-dependence of this effect was not clear from the study given the limited sample size, nor was the potential mechanism of action given that GITR lacks a death domain.Another variable to consider in selecting an optimal dose for a TNF receptor agonist antibody - based on sub-maximal receptor occupancy - involves the potential competition for Fcγ receptor binding. Two pre-clinical studies concluded that the sequencing of an OX40 agonist antibody and a PD-1 inhibitory antibody determined whether or not the combination was efficacious, despite not controlling for the fact that both antibodies were the same isotype, and thus competed with one another for Fcγ receptor binding , 17. Thein vitro. In either model, the low-affinity & high off-rate properties likely endowed the candidate antibody with ‘toggling’ characteristics, wherein receptor occupancy was never fully saturated because the antibodies were constantly associating and dissociating between membrane-proximal TNFR . Examples of antibodies and antibody domain containing molecules with this structure for which clinical data are available include GEN1042 (CD40x41BB bsAb), GEN1046 (PDL1x41BB), FS222 (PDL1x41BB bsAb), PRS-343 , NM21-1480 (PDL1x41BBxHAS trispecific Ab) and MP0317 (FAPxCD40xHSA trispecific Ab). This class of agents tends to lack FcγR binding, since the underlying mechanism is proposed to rely upon antigen-specific clustering of the TNFR binding domain from multiple individual bsAbs in close proximity to one another Figure 4In general, this class of agents lacks clinical evidence of cytokine release syndrome, and liver enzyme elevations are mild and sporadic in comparison to clinical data for bivalent antibodies directed to the same TNFR. This observation strengthens the hypothesis that the CRS and liver tox observed with TNFR agonist bivalent antibodies is largely due to FcγR driven mechanisms. GEN1042 and GEN1046 were tested across a wide dose range , and PK/PD models predicted a bell-shaped dose response curve, as is expected for these agents . DespiteDevelopment of agonist agents which contain three or more TNFR binding domains has progressed more recently, in part due to the clinical experience obtained with the mono- and bi- valent agents described above. For category 1 TNFR, including BaffR, DR3, GITR, LTβR and TNFR1, a hexavalent agonist is likely the minimal valency to exert potent agonism , 61. ForTheoretically, agonists that minimally contain a trimeric TNFR binding domain should lead to receptor activation in a soluble phase, without a requirement for cross-linking. It is tempting to speculate that these agents may not exhibit the bell-shaped dose response curves observed with monovalent and bivalent mAb formats, however emerging clinical data are suggestive of greater nuance. Of the multivalent agonists included in this discussion, some contain pre-formed ligand trimers (RO7227166 & RO7122290), some contain pre-formed ligand hexamers , and some contain a tetravalent, hexavalent or decavalent array of antibody-based TNFR binding domains. Because the pharmacodynamic activity is likely distinct between the antibody based multivalent agents, and those that contain one or more pre-formed TNF ligand trimers, the discussion between both is divided in the following sections.A common characteristic to agents that utilize antibody derived binding domains is that those domains are capable of binding to a TNFR regardless of whether it has pre-assembled into a trimer or hexamer in a cell membrane Figure 5Unlike multivalent TNFR agonists derived from a series of antibody-derived binding domains, agonists which contain pre-formed TNF ligand trimers are predicted to interact with TNF receptors in a unique manner, which potentially better reflects the native physiology of TNF ligand and receptor interactions. As described above, each individual TNFR binding domain in a multivalent TNFR targeted antibody can interact independently with a TNFR target, regardless of whether it is pre-assembled into a trimer or not. As such, there is no guarantee that each TNFR binding domain within an individual antibody molecule will become saturated before the TNFR itself becomes saturated, because there will be competition for free TNFR both between and within individual multivalent antibodies. Agents which contain pre-formed TNF ligand trimers, on the other hand, are expected to stimulate ligand-induced TNFR trimerization, and the stoichiometry of interaction is more likely to be 1:1 between individual ligand and receptor trimers. Emerging pharmacodynamic data from clinical trials supports this assertion, and is described below.Englumafusp alfa (RO7227166) is a fusion construct comprised of a CD19-specific antibody domain fused to a trimerized extracellular domains of human 41BBL. This agent is being developed in conjunction with a CD3xCD20 T cell engager, for patients with relapsed or refractory B cell non-Hodgkin lymphoma. In a phase 1 study, RO7227166 had an acceptable safety profile across a dose range of 0.36 to 33 mg, without reaching a maximum tolerated dose (MTD). CRS was attributed to RO7227166 in just 4.8% of patients, and all of those events were Grade 1 in severity. Importantly, expansion of primed and activated T cell subsets occurred in a dose-dependent manner, without strong evidence of a bell-shaped dose response .RG7827 RO7122290) has a similar structure to RO7227166, but anchors trimerized 41BBL to FAP instead of CD19 and is being developed for patients with advanced solid tumors in combination with atezolizumab (PD-L1 mAb). A phase 1 dose escalation study evaluated RO7227166 across a dose range of 5-2000 mg, without reaching an MTD. A single case of CRS was encountered as a dose limiting toxicity, and no evidence of liver enzyme elevations were observed in the RO7122290 monotherapy arm. The pharmacodynamic changes observed in humans for RO7227166 are substantially more pronounced than those reported for urelumab or utomilumab, and include not only increases in the proportion of T cells that express Ki67 (a marker of proliferation), but also increases in the absolute numbers of T cells following treatment in a dose-dependent manner 0 has a s. FurtherTwo other classes of agonist therapeutics that have been tested in humans containing pre-formed TNF ligand trimers are single- and dual-side Fc fusion proteins. TNF ligands are type II membrane proteins, and fusion of the extracellular domain of a TNF ligand to an Fc domain requires a hinge-CH2-CH3-TNF ligand configuration to ensure unhindered folding and activity of the TNF ligand domain. When expressed, the quaternary structure of an Fc-TNF ligand fusion protein is influenced both by the interchain disulfide bonds in the Fc region leading to covalent dimer formation, and by non-covalent interactions in the TNF ligand domains facilitating trimer formation. The resulting structure is a hexamer consisting of a ‘dimer of trimers’ as illustrated in At least two OX40L-containing Fc fusion proteins entered clinical trials, including a single-sided Fc-OX40L (MEDI-6383) fusion and a PD1-Fc-OX40L (SL-279252) dual-sided fusion protein. Unfortunately, clinical data from a phase 1 clinical trial with MEDI-6383 has not been published. A phase 1 clinical trial testing SL-279252 in patients with a mixture of advanced solid tumors, primarily PD-1 resistant, was completed in 2023. This study examined SL-279252 across a wide dose range of 0.001 through 24 mg/kg, and was well tolerated without any treatment related grade 3 or higher adverse events and no MTD was reached. The primary pharmacodynamic finding was immediate post-dose reductions in the number of peripheral blood CD4+OX40+ T cells following each infusion, which was dose-dependent and believed to be due to migration of OX40+ T cells from the blood into tissues after activation . This fiSL-172154 is a dual-sided Fc fusion protein adjoining the extracellular domains of human SIRPα and human CD40L via a mutated IgG4-derived Fc domain lacking FcγR binding. A phase 1 monotherapy dose-escalation trial was completed in patients with platinum resistant ovarian cancer, and tested SL-172154 across a dose range of 0.1 to 10 mg/kg. In contrast to prior CD40 agonist mAbs, SL-172154 had an acceptable safety profile across the dose range, with a single incidence of grade 3 LFT elevation at the 10 mg/kg dose level, and no MTD was reached. Dose-dependent infusion related reactions were common, primarily grade 1/2, but were not consistent with typical cytokine release syndrome and no elevations in IL-6 and TNFα were observed. CD40 receptor occupancy was approximately 60-80% at the 0.1 mg/kg starting dose, and full receptor occupancy and saturation was observed by the 3 mg/kg dose. The agonist activity of SL-172154 was evident post infusion with near immediate migration of CD40+ B cells and monocytes from the peripheral blood into tissues. This pharmacodynamic effect was concurrent with rapid release of cytokines and chemokines into the serum, including: IL-12, CXCL10, CCL2, CCL3, CCL4, CCL22, IL-8, IL-10 and others . These pOver the past thirty years, a tremendous level of effort, investment, innovation and hope has supported the testing of many types of TNF receptor agonists in human clinical trials. Unfortunately, the resulting clinical data did not closely resemble the biology of the TNF receptor agonism predicted by pre-clinical studies, which prompted the question of whether the failure in translation was more likely a result of the intrinsic biology of TNF receptors, or of the therapeutics used to target those receptors in patients.in vivo are balanced. For example, in vivo is influenced by multiple factors, including the binding affinity and abundance of each target. There are not any publicly available clinical data which demonstrate the relative receptor occupancy kinetics of TNFR agonist antibodies in this manner, so the models should be interpreted in a qualitative manner with these assumptions in mind.Some generalizations on the necessity for TNFRs to trimerize in order to signal were made throughout this review. As with most rules, these generalizations are acknowledged to have limits and special cases where they may not apply. As an example, NGF is a TNF receptor which can be activated by neurotrophin ligands, which are dimeric. In addition, the models proposed in the figures to summarize clinical data from various TNF receptor agonist agents assume that the distribution of a particular agonist agent to its potential binding partners A review of the clinical data across different TNF receptor agonist modalities reveals common themes that should be considered in advancing future agents to the clinic. These themes include the following:1) High-affinity TNF receptor agonist antibodies which bind to Fcγ receptors have a higher likelihood of causing toxicities including cytokine release syndrome and/or liver enzyme elevation than bispecific antibodies which lack Fcγ receptor binding function.2) Both bivalent antibodies and bispecific antibodies show bell-shaped dose response curves in humans, which likely limits the agonist potential of the modality, and creates risk that a ‘recommended phase 2 dose’ may not cause reproducible agonist effects due to variable starting frequencies of the TNF receptor expressing cells between patients, and to dynamic expression of the TNF receptor target within individual patients over time.3) Antibody therapeutics containing three or more domains each capable of binding a TNF receptor target have a higher probability of agonist activity than mono- or bivalent antibody therapeutics, and do not require FcγR binding for function.4) Antibody therapeutics containing three or more domains each capable of binding a TNF receptor target may still encounter bell-shaped dose response curves, similar to bispecific and bivalent antibodies, because TNF receptor saturation can occur in the absence of saturating the cross-linking potential of each antibody.5) Agents containing pre-formed TNF ligand trimers demonstrate more potent evidence of agonist activity than antibody derived agents, potentially because those agents can facilitate ligand-dependent trimerization of TNF receptors.6) Agents which contain multiple TNF ligand trimers demonstrate more sustained dose-dependent pharmacodynamic effects than those which require clustering by another mechanism to facilitate hexameric or higher-order TNF receptor network formation.The consequences of the themes above in terms of safety and efficacy likely vary based on the specific TNF receptor being targeted. For 41BB and CD40 directed agents, cytokine release syndrome may prove to be more problematic than it ever will be for OX40 or GITR directed agents. Regardless, development of agents which require cross-linking for activity (either by FcγR or a target antigen) and thus exhibit bell-shaped dose response curves will be impractical because of the variable and dynamic nature of expression of TNF receptors between and within patients. Whether pre-clinical studies suggesting that this issue can be overcome by reducing the affinity of a TNF receptor agonist antibody will translate to clinical trials is unclear. The observation that tetravalent and hexavalent antibody agonists also exhibit bell-shaped dose response kinetics in humans should raise similar concerns about the ultimate agonist potential of these agents.in vivo, risk of immunogenicity, and potential for decreased tissue penetration. Thus far, these issues have not limited the development of englumafusp alpha, RG7827, SL-279252 or SL-172154, however more data are needed from these and other agents to gain further confidence.The pharmacodynamic effects of agents which contain at least one TNF ligand trimer demonstrate improved translation of pre-clinical to clinical findings in comparison to any of the antibody-based agonists. The ability of TNF ligand containing agonists to trigger ligand-induced trimerization of the target TNF receptor is a likely reason for this observation. The observed advantages of ligand-containing agonists are balanced by the potential drawbacks of a larger molecular format, including manufacturing efficiency, stability Drug developers should carefully review the structural lessons that are now available after over thirty years of clinical experimentation with different TNF receptor agonist agents. While pre-clinical studies may be very important for selecting which TNF receptor to target, the structure of the TNF receptor agonist advanced into the clinic should be made on the basis of clinical data gathered across the class of TNF receptors, rather than on the activity of a particular agonist agent in pre-clinical models.TS, GF and SD wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.


The occurrence of significant Alzheimer’s disease (AD) pathology was described in approximately 30% of normal pressure hydrocephalus (NPH) cases, leading to the distinction between neurodegenerative and idiopathic forms of this disorder. Whether or not there is a specific MRI signature of NPH remains a matter of debate. The present study focuses on asymptomatic cases at risk for NPH as defined with automatic machine learning tools and combines automatic MRI assessment of cortical and white matter volumetry, risk of AD (AD-RAI), and brain age gap estimation (BrainAge). Our hypothesis was that brain aging and AD process-independent volumetric changes occur in asymptomatic NPH-positive cases. We explored the volumetric changes in normal aging-sensitive  and AD-signature areas (hippocampus), four control cortical areas , and cerebral and cerebellar white matter in 30 asymptomatic cases at risk for NPH (NPH probability >30) compared to 30 NPH-negative cases (NPH probability <5) with preserved cognition. In univariate regression models, NPH positivity was associated with decreased volumes in the hippocampus, parahippocampal gyrus (PHG), and entorhinal cortex bilaterally. The strongest negative association was found in the left hippocampus that persisted when adjusting for AD-RAI and Brain Age values. A combined model including the three parameters explained 36.5% of the variance, left hippocampal volumes, and BrainAge values, which remained independent predictors of the NPH status. Bilateral PHG and entorhinal cortex volumes were negatively associated with NPH-positive status in univariate models but this relationship did not persist when adjusting for BrainAge, the latter remaining the only predictor of the NPH status. We also found a negative association between bilateral cerebral and cerebellar white matter volumes and NPH status that persisted after controlling for AD-RAI or Brain Age values, explaining between 50 and 65% of its variance. These observations support the idea that in cases at risk for NPH, as defined by support vector machine assessment of NPH-related MRI markers, brain aging-related and brain aging and AD-independent volumetric changes coexist. The latter concerns volume loss in restricted hippocampal and white matter areas that could be considered as the MRI signature of idiopathic forms of NPH. The relSeveral MRI markers have been proposed to identify NPH-positive cases in elderly cohorts. Early studies proposed traditional 2-dimensional linear measures such as Evan’s, bicaudate index, and callosal angle . The disPrevious studies using automatic machine learning tools focused on the distinction between NPH patients, AD, and other forms of dementia and healthy controls . VolumetThe development of NPH is a long process in old age, and radiographic features show volumetric changes 3 or more years before the onset of clinical symptoms . As the 2.2.1.The study was approved by the local Ethics Committee, and all participants gave written informed consent prior to inclusion. The selection of cases among participants of a still ongoing cohort study aiming to identify predictive biomarkers of subtle cognitive decline among healthy elders was described in detail elsewhere . Briefly2.2.At baseline, all individuals were evaluated with an extensive neuropsychological battery, including the Mini-Mental State Examination MMSE; , the Hos2.3.Imaging data were acquired on a 3 T MRI scanner . The structural high-resolution T1-weighted anatomical scan was performed with the following fundamental parameters: 256 × 256 matrix, 176 slices, 1 mm isotropic, TR = 2.27 ms. Additional sequences  were used to exclude incidental brain lesions.2.4.The structural T1-images were segmented into grey matter, white matter, and cerebro-spinal fluid (CSF) using SPM12  space  and smoothed by full width-half-max 3 mm filter, like what is done for usual voxel-based morphometry analyses. The segmentation results were inspected visually prior to further processing, and none of the cases had to be excluded because of obvious failures. Mesial temporal atrophy (MTA) was assessed according to the established score , ranging2.5.The definition of the final sample was made on the basis of the automatic assessment of NPH scores as described in 2.6.BrainAGE was estimated according to the approach described previously by 2.7.An automated assessment of the AD-RAI was performed using a voxel-based SVM. The basic principle is described in 2.8.Automatic MR volumetry was performed with the fully automated multi-atlas segmentation tool cNeuro 3.Demographic and clinical data of the present series are summarized in In univariate regression models and among brain aging and AD-signature areas, NPH status was associated with decreased volumes in the hippocampus, PHG, and entorhinal cortex bilaterally , 3. The Among control areas , there were no significant associations between the volumes and NPH status (data not shown). In contrast, we found a strong negative association between cerebral white matter volumes bilaterally and increased NPH values (35.6% of variance in the left and 37.8% in the right hemisphere) that persisted after adjustment for AD-RAI or Brain Age values . In a co4.Whether or not NPH structural changes depend strictly or not on brain aging and AD-related neurodegeneration remains a matter of intense debate. Our data in asymptomatic cases with the first MRI signs of increased risk for NPH status support a mixed scenario. They reveal that radiologically defined NPH positivity in asymptomatic elders was associated with decreased left hippocampal and cerebral and cerebellar white matter volumes that did not depend on the progression of brain aging and AD-related MRI volumetric changes supporting the idea of an idiopathic component of the disease. In other areas such as the right hippocampus, parahippocampal gyrus, and entorhinal cortex, the impact of NPH on brain volumetry seems to be strictly mediated by age-related neurodegeneration.Early volumetric MRI studies reported increased gray matter and white matter loss in AD compared to idiopathic NPH cases in the hippocampus and perihippocampal fissures . Later oThe present study focuses on asymptomatic, radiologically defined cases at risk for NPH and takes into account automatic MRI parameters assessing the risk of AD (AD-RAI) but also the brain age gap estimation (BrainAge). By integrating these parameters in multivariable models, we were able to define whether there is a structural signature of NPH at-risk status independently of the impact of neurodegenerative changes and the brain aging process. Our data show that radiological NPH positivity is associated with a significant decrease in the volume of the left hippocampus after controlling for AD-RAI and BrainAge values. Importantly, this parameter alone explained 27.6% of the radiological NPH variance indicating that the observed association is not marginal. Our findings are consistent with previous observations , pointinAnother set of data showed that some of the volumetric changes observed in cases at risk for NPH reflect partly the acceleration of the brain aging process. This was the case of the parahippocampal gyrus and entorhinal cortex but also right hippocampus volumes that are related to NPH scores in univariate models, yet their association did not persist when adjusting for BrainAge values. Importantly, this latter parameter alone explained more than 30% of the radiological NPH variance in models including the right hippocampus, parahippocampal gyrus, and entorhinal cortex, and its addition explained an additional 9–19% in the models including the left hippocampus and cerebral and cerebellar white matter. This finding implies that the acceleration of the brain aging process is a key player in cases at risk for NPH. In contrast, the AD-RAI values that reflect the global AD risk based on an automatic MRI volumetric assessment were not related to radiological NPH positivity at least in these asymptomatic cases. Taken together, these observations support the idea that in cases at risk for NPH, as defined by SVM assessment of NPH-related MRI markers, brain aging-related and brain aging and AD-independent volumetric changes coexist. The latter concerns volume loss in a restricted portion of the hippocampus and white matter that could be considered a possible MRI signature of idiopathic forms of NPH. It is plausible to speculate that in clinical forms of NPH in old age, and depending on the severity of concomitant AD pathology and acceleration of brain aging, the relative weight of each process changes, leading to neurodegeneration, brain aging, or idiopathic-predominant but also mixed forms of the disease. This complexity may explain the contradictory reports on the prognosis of clinically overt NPH in elderly cohorts.Among the strengths of the present study, one should note the focus on asymptomatic cases that makes it possible to control for the confounding effect of severe AD pathology, the use of multivariable models that allows for defining the added value of each of the automatic MRI parameters, and consideration of both AD-signature and control areas in the statistical analysis. Several limitations should be considered. The small sample size limits the number of predictors that can be simultaneously included in regression models. In particular, we did not explore diffusion tensor imaging variables, which are known to be involved in the evolution of NPH, and did not control for the distinct impact of concomitant amyloid pathology. In a recent study, the latter has been used to define a hydrocephalic variant of AD . MoreoveThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Commission cantonale d’éthique de la recherche (CCER). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.SH and PG conceived the study. CR and M-LM: recruitment. M-LM, CR, and PG: neuropsychology supervising. M-LM and FH: data preparation. FH, PG, and M-LM analyzed the data. PG, FH, SH, and M-LM: manuscript writing. All authors contributed to the article and approved the submitted version.


Lactobacilli have a fundamental role in the food industry as starters and probiotics, therefore, requiring special attention concerning food safety. In this work, 14 strains selected accordingly to their genetic fingerprint and physiologic characteristics are presented as representatives of a collection of 200 strains. Lactobacilli are ubiquitous lactic acid bacteria and are of particular interest, given their significant presence in fermented and non-fermented foods and in the human commensal microbiota. Thanks to their long history of human use and consumption and their use as probiotics, they have been generally recognized as safe . HoweverLacticaseibacillus rhamnosus, Lacticaseibacillus paracasei, Lacticaseibacillus casei, and Lacticaseibacillus zeae recently reclassified , the whole-genome sequencing constitutes an adequate tool to taxonomically characterize and carry out a risk assessment by verifying the presence of health concern factors in microorganisms intentionally used in the food chain . Therefoassified , 6. Thesassified –9. The cm method , and genm method .Library samples were loaded into a Flow Cell V3 600 cycles  according to the technical support guide. Draft genome sequencing was performed through the genomic platform consisting of a MiSeq , following the protocol of the supplier . Fastq files of the 250-bp paired-end reads obtained from targeted genome sequencing of the isolated strains were used as input for genome assemblies.L. casei 12A NZ_CP006690.1, L. paracasei ATCC334 NC_008526.1, and L. rhamnosus GG NC_013198.1) using Mauve v2.4.0 (The reads were analyzed and assembled with the WGA-LP pipeline  using the v2.4.0 . The fine v2.4.0 , SamToole v2.4.0 , and Quae v2.4.0 .