Extensive physiologic characterization of the transporter has begun to illuminate its role in endosomal ion homeostasis. Nevertheless, we have yet to understand how loss of ClC-5 function in the kidney proximal tubule impairs membrane traffic of megalin and cubilin receptors to cause the low molecular weight proteinuria characteristic of DD. This review identifies open questions that remain to be answered, evaluates the current literature addressing these questions, and suggests new testable models that may link loss of ClC-5 function to tubular proteinuria in DD.Dent disease (DD) is a rare kidney disorder caused by mutations in the Cl Protein recovery occurs through endocytosis mediated by the multi-ligand receptors megalin and cubilin.,CLCN5 have been described, the majority of which result in missense or frameshift mutations with many resulting in truncation of the protein.,,Dent disease (DD) is a rare, X-linked disorder primarily resulting from the loss of function of the voltage-gated chloride/proton exchanger ClC-5, which presents with low molecular weight (LMW) proteinuria and frequently progresses to chronic kidney disease and end-stage renal disease.Since mutations in ClC-5 were first identified as the cause of DD, we have developed a greater understanding of the physiologic role of ClC-5 in endosomal ion homeostasis. Despite this, how loss of function of ClC-5 results in decreased megalin and cubilin expression to cause LMW proteinuria remains elusive. Specifically, there has been little consideration given to how alterations in endosomal pH or chloride content translate mechanistically to loss of megalin and cubilin receptors. This is particularly a puzzling question because impaired acidification along the endocytic pathway typically leads to protein stabilization rather than loss. This review focuses on the interface between the cell biological and physiological approaches needed to determine the function of ClC-5 in the proximal tubule and to understand how trafficking goes awry in its absence. We posit critical issues that remain to be addressed, describe the data to date and the limitations of these studies, and suggest testable hypotheses to resolve the many open questions in the field.−] in the PT. However, we still do not fully understand whether it is the changes in endosomal [Cl−] and/or [H+] in PT cells lacking functional ClC-5 that result in aberrant trafficking of megalin and cubilin. Moreover, it remains unclear how endosome maturation and cargo sorting are impaired by defective ion homeostasis in DD to result in reduced levels of megalin and cubilin.ClC-5 activity is clearly required to maintain proper endosomal pH and  rather than pH may be the primary function of ClC-5. In an early test of this hypothesis, Novarino et al. generated a knock-in mouse model with a novel mutation in ClC-5 that uncouples Cl−/H+ transport (E211A), rendering the mutant a Cl− channel.in vitro acidification assays are performed using nonphysiologic conditions and because endosomal pH in vivo is not easily measured, the uncoupled mutant may in fact be partially defective at regulating endosomal acidification in the PT. A more recent study examined a similar disease-causing mutation (E211G), which like E211A functions as a Cl− channel rather than as a H+/Cl− exchanger.Progressive luminal acidification of endosomes is well known to be critical for proper sorting and function along the endolysosomal pathway, and + and Cl− transport in early endosomes is a critical function of ClC-5, and that [Cl−] as well as [H+] concentration may be an important factor in endosome maturation. In normal endosome maturation, the decrease in pH within endosomes is necessary for ligand dissociation, sorting, and degradation. Chloride concentration increases as endosomes mature,−], a reduction in lysosomes and/or an increase in number and size of endosomes would be predicted. To date, no major structural differences have been reported in the endolysosomal pathway of Dent patients or mouse models.,trans-Golgi network, and dissociate upon reaching more acidic late endosomes for their ultimate delivery to lysosomes.These recent studies suggest that the coupling of H,The two cytosolic CBS (cystathionine β-synthase) domains at the C-terminus of ClC-5 form a nucleotide-binding pocket that binds to, but does not hydrolyze, ATP and ADP. The binding of ATP to the two CBS domains enhances ClC-5 activity− or H+ is the primary mediator of ClC-5 endosomal function, it is clear that many disease-causing mutants do alter endosomal acidification in vivo. Because impaired acidification of endocytic pathway compartments is well known to impair lysosomal delivery and inhibit protein degradation, this raises a conundrum—Why are megalin and cubilin protein levels reduced in DD PTs? An ancillary question is why disease-causing uncoupling mutations that may not impact pH apparently have similar effects on steady-state receptor levels. In addition, knowing pH and [Cl−] of endosomes in vivo may provide better correlation of genotype to phenotype. Resolving these questions will require a better understanding of the uniquely specialized endocytic pathway of the PT and how it is regulated.Regardless of whether ClReduced uptake of filtered proteins and expression of endocytic receptors are hallmarks of DD. The drop in expression of megalin and cubilin protein levels in DD mouse models occurs without corresponding changes in mRNA,,Both imaging and biochemical data support the loss of megalin and cubilin receptor expression in DD animal and cell models. The difference between megalin and cubilin expression in ClC-5 KO versus WT cells is most readily obvious in PTs of heterozygous female mice.in vivo. Ultrastructural studies of PTs in megalin KO mice and in kidney biopsies from Donnai–Barrow syndrome patients with megalin mutations reveal a striking reduction in the density of coated pits, early endosomes, and lysosomes.The relatively modest (∼30%) reduction in megalin levels observed by blotting belies the dramatic reduction observed by immunostaining. Resolving the discrepancy is important, because megalin expression has an essential function in maintaining the integrity of the apical endocytic pathway ,In addition to its global function in maintaining the PT apical endocytic pathway, recent studies suggest that megalin has distinct roles in regulating endocytic function in different PT nephron segments.,,,Although most studies have monitored changes in megalin expression in DD, albumin and many other filtered ligands bind to cubilin rather than megalin.In addition to a reduction in the total number of receptors, the distribution of megalin and cubilin appears to be altered in ClC-5 KO mice. Christensen et al. found reduced expression of megalin and cubilin in brush border relative to endosomal membranes.In order to pinpoint how loss of ClC-5 affects endocytic receptor levels, we need to know the endocytosis, recycling, and degradation rates of megalin and cubilin in WT and ClC-5 KO cells. These rates, combined with quantitative assessment of megalin and cubilin expression levels and their distribution in PT cells, including the fraction at the surface, within AEEs, and within AVs will provide insight into whether the endosome maturation and/or degradation rates are affected. Using these aggregate data to develop mathematical models of megalin and cubilin trafficking will enable identification of which rates are altered in DD. The incorporation of individual rate constants with data from steady-state distribution and protein level measurements into a kinetic model makes this a powerful approach to identify small differences in the kinetics or sorting efficiency within a single trafficking step that result in a large effect in receptor expression over many cycles. Ideally, a detailed model would further enable distinction between slow and fast recycling pathways to determine if one or both are affected in DD.,,,The trafficking defect in cells lacking ClC-5 is presumably not limited to megalin and cubilin, and indeed, altered trafficking of other PT apical membrane proteins including NHE3 and NaPi-2 has been reported.+] and other ions impact myriad downstream pathways, and the molecular mechanism by which loss of ClC-5 activity translates into reduced megalin and cubilin expression remains elusive. Additionally, specific DD-causing mutations may differentially affect downstream steps, contributing to phenotypic heterogeneity in cellular responses and disease severity.,Alterations in endosomal  may be altered in the absence of a change in pH.,Alternatively (or in addition), ClC-5 may influence endosomal cholesterol levels through the ARNO/Arf6 pathway . Arf6 is+] and [Cl−] levels. Whether improper acidification is key to dysfunction in DD remains unclear. Continued optimization of fluorescent probes to quantify organelle ion composition will facilitate a better understanding of the role of ClC-5 in its native tissues. Additionally, the downstream consequences of impaired ion homeostasis that lead to LMW proteinuria are unresolved. The loss of ClC-5 leads to reduced uptake of filtered proteins that likely reflects the reduced expression of megalin and cubilin observed in DD models. How ion homeostasis is linked to this outcome is the next major question to tackle. Deciphering these mechanisms will require an experimental shift toward cell biological and computational approaches. Specifically, a better appreciation of the trafficking pathway and kinetics of megalin and cubilin in normal and DD PTs will provide much needed insight into the role of ClC-5 in regulating endosome maturation and trafficking. The availability of improved PT cell models that better approximate endocytic function of the PT in vivo will facilitate this effort together with advanced gene-editing technologies to introduce pathogenic mutations in ClC-5.After considerable study, we are beginning to understand how ClC-5 functions within endosomes to contribute to [H


The skin is constantly exposed to various types of chemical stresses that challenge the immune cells, leading to the activation of T cell-mediated hypersensitivity reactions including atopic dermatitis. Previous studies have demonstrated that a variety of natural compounds are effective against development of atopic dermatitis by modulating immune responses. Cardamonin is a natural compound abundantly found in cardamom spices and many other medicinal plant species. In the present study, we attempted to examine whether cardamonin could inhibit oxazolone-induced atopic dermatitis in vivo. Our results show that topical application of cardamonin onto the ear of mice suppressed oxazolone-induced inflammation in the ear and hyperplasia in the spleen. Cardamonin also inhibited oxazolone-induced destruction of connective tissues and subsequent infiltration of mast cells into the skin. In addition, we found that the production of Th2 cytokines is negatively regulated by NRF2, and the induction of NRF2 by cardamonin contributed to suppressing oxazolone-induced Th2 cytokine production and oxidative damages in vivo. Together, our results demonstrate that cardamonin is a promising natural compound, which might be effective for treatment of atopic dermatitis. Atopic dermatitis, also known as atopic eczema, is an inflammatory skin disorder that is characterized by intense pruritus, scratching, and cutaneous hypersensitivity to allergens . It is wcis-DNA element existing in the promoter of phase II cytoprotective enzymes J rearrangement. TCR is bound to the membrane and recognize antigens only when presented in the context of MHC I or MHC II . T cellsAs mentioned earlier, NRF2 is responsible for transcriptional activation of phase II cytoprotective genes. On the other hand, there is also evidence that NRF2 can suppress the production of pro-inflammatory cytokines in certain circumstances . HoweverBecause NRF2 suppresses oxazolone-induced production of Th2 cytokines , we specWe have demonstrated that cardamonin suppresses oxazolone-induced atopic dermatitis in vivo . SuppresCardamonin stimulates ARE-dependent gene expression A in HaCaIn conclusion, our study provides evidence that cardamonin inhibits atopic dermatitis in vivo and that this effect could be attributed to the induction of NRF2 and the inhibition of Th2 cytokine production.


Noninvasive brain stimulation (NIBS) such a transcranial magnetic stimulation, intermittent theta burst stimulation, transcranial direct current stimulation and electroconvulsive therapy have emerged as an efficacious and well-tolerated therapy for treatment-resistant psychiatric disorders. While novel NIBS techniques are an exciting addition to the current repertoire of neuropsychiatric therapies, their success is somewhat limited by the wide range of treatment responses seen among treated patients.In this study, the authors will review the studies on relevant genetic polymorphisms and discuss the role of RNA genotyping in personalizing NIBS.Genome studies have revealed several genetic polymorphisms that may contribute for the heterogeneity of treatment response to NIBS where the presence of certain single nucleotide polymorphisms (SNPs) are associated with responders versus nonresponders.Historically, mental illnesses have been arguably some of the most challenging disorders to study and to treat because of the degree of biological variability across affected individuals, the role of genetic and epigenetic modifications, the diversity of clinical symptomatology and presentations and the interplay with environmental factors. In lieu of these challenges, there has been a push for personalized medicine in psychiatry that aims to optimize treatment response based on one’s unique characteristics. These revolutionary neuromodulation techniques permit healthcare providers to alter cortical excitability without physically penetrating into brain tissue, such that sessions can be delivered in minutes on an outpatient basis, and patients may return to their daily activities on the same day. Additionally, these brain stimulation methods are postulated to induce changes on local neural activity that outlast the duration of stimulation and may be synergistically combined with pharmacotherapies to deliver therapeutic effects more quickly and with greater efficacy and durability than either modality alone  magnetic fields that pass through the skull and induce focal electric currents that depolarize axons in target areas of brain tissue, modulating local neural activity with downstream effects in neural networks throughout the cortex . The effCurrently, TMS is approved by the Food and Drug Administration (FDA) for treatment of treatment-resistant major depression  gene encodes BDNF protein, which plays an important role in neuronal growth and differentiation, synaptic transmission, neuroprotection and neuroplasticity. BNDF binds tropomyosin receptor kinase B (TrkB) and activates signaling cascades, including the Ras/MAPK-ERK, IRS-1/P13K/AKT and PLC/DAG/IP3 pathways, which activate downstream survival and growth genes . BDNF prBDNF is believed to be an important regulator rehabilitation-induced recovery following stroke by enhancing synaptic plasticity, increasing angiogenesis and neurogenesis and stimulating brain repair  in ValVal patients versus ValMet patients (3.2The dopamine D2 receptor (DRD2) gene encodes a G-protein-coupled receptor that is highly expressed in the striatum and nucleus accumbens that mediates a variety of cognitive functions, including learning and memory, attention, reward behavior and pain response . The mosBecause treatment with NIBS involves long-term modulation of cortical excitability, DRD2 variants are especially relevant in patients receiving NIBS because the D2 receptor has been shown to have dose-dependent effects on neuroplasticity. In healthy adults, D2 receptor blockade with sulpiride abolished tDCS-induced changes of excitability in both anodal and cathodal tDCS after-effects 5 min following stimulation  enzyme has been hypothesized to play a role in DRD2 variants because of its function in metabolizing and degrading dopamine. The COMT missense variant Val158Met modulates COMT activity and subsequently decreased dopamine levels in the DLPFC, where the Val/Val carriers demonstrate the most rapid dopamine metabolism, Val/Met carriers demonstrate intermediate dopamine metabolism and Met/Met carriers demonstrate the slowest metabolism , has been widely studied in the pathogenesis of major depressive disorder in which the monoamine hypothesis of depression states that serotonin deficiency is linked with depression. Repetitive TMS to the DLPFC is currently an adjuvant treatment for treatment-resistant depression and theoretically enhances neuronal activity of serotonergic and/or dopaminergic systems. Animal studies have shown increased levels of 5-HT in the hippocampus and amygdala  score reduction than s/s homozygotes, although the influence of the 5-HTT genotype was the same between active and sham TMS  gene encodes for nonselective cation channels that regulate glutamate release in response to a variety of stimuli such as endocannabinoids, eicosanoids, vanilloid compounds, voltage and heat . TRPV1 h3.5Granulin (GRN) encodes progranulin, a secreted neuroregulatory growth factor believed to play a central role in brain development and neurodegeneration (3.6


The therapeutic alliance between patients and physical therapists has been shown to influence clinical outcomes in patients with chronic low back pain when consulting in-person. However, no studies have examined whether the therapeutic alliance developed between patients with knee osteoarthritis and physical therapists during telephonic consultations influences clinical outcomes.This study aims to investigate whether the therapeutic alliance between patients with knee osteoarthritis and physical therapists measured after the second consultation is associated with outcomes following telephone-delivered exercise and advice.Secondary analysis of 87 patients in the intervention arm of a randomized controlled trial allocated to receive 5 to 10 telephone consultations with one of 8 physical therapists over a period of 6 months, involving education and prescription of a strengthening and physical activity program. Separate regression models investigated the association between patient and therapist ratings of therapeutic alliance (measured after the second consultation using the Working Alliance Inventory Short Form) and outcomes  at 6 and 12 months, with relevant covariates included.There was some evidence of a weak association between patient ratings of the alliance and some outcomes at 6 months . In addition, there was some evidence of a weak association between patient ratings of the alliance and some outcomes at 12 months . The data suggest that associations between therapist ratings of therapeutic alliance and outcomes were not strong, except for improved quality of life at 12 months .Higher patient ratings, but not higher therapist ratings, of the therapeutic alliance were weakly associated with improvements in some clinical outcomes and with worsening in one outcome. Although the findings suggest that patients who perceive a stronger alliance with their therapist may achieve better clinical outcomes, the observed relationships were generally weak and unlikely to be clinically significant. The limitations include the fact that measures of therapeutic alliance have not been validated for use in musculoskeletal physical therapy settings. There was a risk of type 1 error; however, findings were interpreted on the basis of clinical significance rather than statistical significance alone.Australian New Zealand Clinical Trials Registry ACTRN12616000054415; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=369204 Knee osteoarthritis (OA) is highly prevalent and leading cause of functional limitation in older adults ,2. GivenAn important aspect of health care is the strength of the relationship developed between the patient and the health professional. This relationship, known as the therapeutic alliance, is conceptualized as a sense of collaboration, warmth, and support between a patient and clinician , and it Most existing studies evaluating relationships between therapeutic alliance and outcomes of physical therapy practice have focused on in-person consultations between patients and therapists. Thus, it is not clear if findings from such studies can be generalized to telephone-delivered models of physical therapy care, where patients and physical therapists have no physical or visual contact. Our research provides some evidence from qualitative studies that physical therapists and patients with OA perceive a strong alliance when consulting via video  and teleThis exploratory study used data collected from physical therapists and patients in the intervention arm of a randomized controlled trial  12616000054415), which evaluated the effectiveness of incorporating physical therapist-delivered exercise advice and support into an existing musculoskeletal telephonic service delivered by nurses ,29. The The intervention arm of the RCT included 87 randomized patients with knee OA. Inclusion criteria were meeting the National Institute for Health and Care Excellence OA clinical criteria  . PatientA total of 8 physical therapists were recruited in Victoria, Australia, to deliver the intervention for the trial. Selection criteria included a physical therapy qualification, at least two years of musculoskeletal professional experience, and current Australian registration to practice. Before the commencement of the trial, the physical therapists underwent a 2.5-day training program in the delivery of person-centered care and behavior change  ,31. ThisDetails of the RCT have been published , includiPatients were provided with a study folder containing information about OA and management, exercise instructions and access to a study website containing video demonstrations of each exercise. Patients were provided with three exercise resistance bands for home exercises.Outcome measures  in the RCT that were included in this secondary analysis were as follows:Overall average knee pain in the past week .Physical function , as well as change in physical activity (descriptors much less to much more). Scores were dichotomized into 1  and 0 . Global changes at 6 and 12 months  via 7-point scales . Scores were dichotomized into 1  and 0 .Satisfaction with care collected at 6 and 12 months via a 7-point scale , only collected at 6 months.Physical therapist-rated patient adherence to home exercise program collected at 6-months via an 11-point scale  rated at 6 and 12 months.Self-rated adherence to (a) prescribed exercises and (b) physical activity plan via an 11-point scale  that way, to always feeling (or thinking) that way. The WAI has 3 subscales: (1) task ; (2) bond , and (3) goal , which are summed together to give a total score ranging from 12 to 84  [Therapeutic alliance was measured using the Working Alliance Inventory-Short Form (WAI) ,35, a colliance) ,35.As recommended , both paMeans and SDs of the patient and physical therapist characteristics and therapeutic alliance ratings were calculated. Separate regression models were used to investigate whether the therapeutic alliance was associated with each outcome. For each continuous outcome, linear regression models for the 6 and 12-month outcomes (change from baseline) were fit, with random effects for each patient to account for the two measurements. The baseline outcome measurement, where available, was included in the model, as were terms for patient  and physical therapist characteristics  that could potentially influence both the therapeutic alliance measure and outcomes at 6 and 12 months. The effect of therapeutic alliance on outcomes at 6 and 12 months was estimated by including terms for the outcome measurement time point, therapeutic alliance score, and an interaction between the two. Global change scores were dichotomized and analyzed using mixed-effects logistic regression models. Separate models were fit for the patient and physical therapist ratings of the alliance. As data for physical therapist ratings of adherence were only collected at 6 months, a standard linear regression model was fit. Analysis was performed using Stata .Most of the 87 patients  were femHalf of the 8 physical therapists  were malAssociations between patient ratings of therapeutic alliance and continuous and binary outcomes at 6 and 12 months are displayed in Data suggest that patient-rated therapeutic alliance was associated with some outcomes at 12 months. A one-unit increase in the therapeutic alliance score was associated with (1) a 0.15 (95% CI 0.03 to 0.27) unit improvement in self-efficacy; (2) increased odds of a global improvement in pain ; (3) increased odds of a global improvement in physical function , and (4) increased odds of a global improvement overall .Associations between physical therapist ratings of the therapeutic alliance and continuous and binary outcomes at 6 and 12 months are displayed in The aim of this study was to investigate whether the therapeutic alliance between patients and physical therapists during telephonic consultations was associated with outcomes following exercise and advice for people with knee OA. The findings suggest that patient-rated therapeutic alliance was weakly associated with some outcomes at 6 and 12 months, including improvements in pain, self-efficacy, global function, and overall global improvement, in addition to a worsening in fear of movement. The data indicated that associations between physical therapist-rated therapeutic alliance and outcomes were not meaningful. The observed relationships were generally weak and thus unlikely to be clinically significant.This is the first study to investigate the relationship between therapeutic alliance and clinical outcomes following telephone-delivered physical therapy care in adults with OA. Existing reviews focusing on traditional, in-person consultations among those with musculoskeletal conditions have found that a stronger therapeutic alliance between the patient and their physical therapist is associated with improved outcomes, including better adherence to physical-therapist–prescribed exercise and physical activity , improveOur findings are broadly similar to those of previous research exploring therapeutic alliance in tele-rehabilitation consultations with clinicians outside of the physical therapy profession. One study of 22 adolescents (mean age 15 years) with idiopathic arthritis who received care via 12 telephonic consultations from trained nonprofessional health coaches over 12 weeks found that therapeutic alliance was correlated with improved treatment outcomes, including decreased pain . HoweverAlthough we observed associations between therapeutic alliance and outcomes, the coefficients were very small and confidence intervals contained values that were close to zero. Thus, the clinical significance of our observed relationships is unclear. A single unit increase in therapeutic alliance score  corresponded to a very small, 0.10-unit improvement in overall average knee pain  at 6 months. This magnitude of change is similar to that observed by Ferreira et al , who invOur patient and physical therapist ratings of the therapeutic alliance were high , and theOur study is relevant to clinicians and researchers. The findings suggest that the strength of the therapeutic alliance with the physical therapist as perceived by the patient is associated with some clinical outcomes after telephonic consultations focused on exercise management. Thus, physical therapists should be mindful about the therapeutic alliance they build with their patients. To enhance the therapeutic alliance, it has been recommended that clinicians focus on fostering person-centered interactions with patients, including offering emotional support and facilitating patient involvement in decision-making -51. It iFuture research should consider evaluating relationships between therapeutic alliance and clinical outcomes in real-world clinical practice, as both alliance and clinical outcomes may be more varied than observed within the context of a clinical trial. Importantly, we found that physical therapist ratings of therapeutic alliance were generally not related to clinical outcomes, suggesting that their own perceptions of the alliance may not be as important as those of the patient. Our study was the first to investigate the relationship between therapeutic alliance and clinical outcomes following telephone-delivered physical therapy care in adults with OA, and thus further research is required to compare therapeutic alliance during tele-rehabilitation and traditional in-person consultations, and how it moderates treatment outcomes.Our study has some limitations. As with any study, there is a risk of type 1 error. However, in accordance with recommendations from the American Statistical Association , we did In conclusion, higher patient ratings but not higher physical therapist ratings of the therapeutic alliance were weakly associated with improvements in some clinical outcomes. Although these findings suggest that patients who perceive a stronger alliance with their physical therapist may achieve some better clinical outcomes, the observed relationships were generally weak and unlikely to be clinically significant. Limitations include the fact that measures of therapeutic alliance have not been validated for use in musculoskeletal physical therapy settings. There was a risk of type 1 error; however, findings were interpreted based on clinical significance rather than statistical significance alone.


Retroperitoneum is an uncommon site for Schwannoma tumors and among adrenal incidentaloma the Schwannoma is rare. This condition lacks of a specific clinical and radiological features, but correct diagnosis before pathological examination should be very important for clinical management and surgical decision. We describe a case of voluminous retroperitoneal incidental mass, that was proved to be an adrenal Schwannoma. Schwannomas arise from Schwann cells, so they can appear in any portion of neural tissue where cells of Schwann are present but they prefer neck and head regions or the flexor surfaces of the upper and lower extremities.19,20 makes difficult to diagnose adrenal Schwannomas as other malignant tumors of adrenal glands or retroperitoneal organs.4,8,21A correct pre-operative diagnosis of adrenal Schwannoma is essential to avoiding unnecessary extensive surgery and/or regional lymph nodes dissection; in routine clinical practice, the lack of specific clinical and radiological featuresT1 weighted images and heterogeneously high signal intensity on T2 weighted images, with progressive contrast enhancement after contrast medium injection and without drop of signal in T1 weighted opposition phase sequence. The tumors showed mild and progressive peripheral enhancement involving especially the wall and the internal septa with irregular pattern due to cystic intralesional components  was performed to characterize the mass: a voluminous solid and heterogeneous mass with fluid components was detected in the left adrenal gland. The mass measured 9 × 6 × 7 cm and showed low signal intensity on mponents . The adrmponents . Unenhanmponents . The rigmponents . After amponents . Gross e13 Firstly Schwannomas were described by Verocay in 1908 and Antonini in 1920 subclassified these tumors into two distinct histologic patterns.22 Typical Schwannoma is a solitary mass, with ovoid or spherical morphology and well-defined margins; when Schwannoma is very large (>8–10 cm) it is frequently affected by degenerative changes with cystic areas, calcifications, interstitial fibrosis and hyalinization. The degenerative pattern is predominant in the “ancient” Schwannoma, a variant with a very good clinical outcome.23 It should be known that 5–18% of Schwannomas are associated with von Recklinghausen’s disease; in this pathological context Schwannomas are generally malignant with a trend of manifestation in multiple areas.24 The medulla of adrenal gland receives double innervation by two different groups of myelinated nerve fibers, respectively derived from the sympathetic trunk (or vagus nerve) and from the phrenic nerve. Adrenal Shwannomas are thought to origin from one of those nerves and to arise from medulla because of the uninterrupted continuance between the tumor and the adrenal medulla in absence of septum around the tumor. They grow up from adrenomedullary site compressing the adrenal cortex.16 Schwannomas present two main microscopic pattern at histological analysis, that are respectively characterized by high cellularity component (areas of Antoni A) and pure myxoid component (areas of Antoni B). Each component may result prevalent in the lesion determining a specular radiological specific findings on CT or MRI scans. Morever, as tumors with neuroectodermal origin, almost all Schwannomas exhibit intense immunohistochemical staining for S-100 protein.13,25 A limited number of Shwannomas may be not easily distinguishable from neurofibromas because of their similar histologic aspects and positive staining for S-100 protein.14 Moreover, in the assessment of differential diagnosis between these two entities, Fine et al26 have demonstrated that a positive stain for calretinin  helps for discrimination and allows a diagnosis of Shwannoma. Behavior of these tumors may be biologically benign or not. Malignant Schwannomas are often ex-novo lesions, with locally invasive pattern of growth and tendency to develop distant metastases.25 On the contrary, benign lesions do not present invasive trend and during expansion they simply displace surrounding structures and organs; however, there is not proven correlation between size and biological malignant potential.27 One percent of all retroperitoneal tumors are Schwannomas and only 1–3% of all Schwannomas are located in profound structure such as retroperitoneum and posterior mediastinum, representing uncommon siting of this tumor.28,29 Retroperitoneum is a flexible space and Schwannomas detection is often accidental because large size can be reached before they become symptomatic, showing for long time only incipient and vague sign of adjacent organ compression. In advanced stages, space-occupying mass signs manifest with lumbar heaviness or pain, neuralgia and paresthesia in the distribution of the affected nerves, ureteral obstruction; in malignant forms with infiltration of surrounding structures there are generally hematuria and bone pain.27 The insufficiency of specific or typical imaging findings on ultrasound, CT or MRI makes the pre-operative diagnosis and the differential diagnosis with other retroperitoneal malignant entity very difficult.30 However, the role of radiological imaging is essential in management and treatment planning through examination of tumor size, location, possible invasion of other structures.13,31 In accordance with literature, at MRI the Shwannomas show low signal intensity on T1 weighted images and heterogeneous high signal intensity on T2 weighted images with cystic areas and smooth profile. However, these imaging characteristics are not specific. Our tumor showed regular margins, heterogeneously high signal intensity on T2 weighted images, described as necrotic components, and small areas of degeneration on histological examination were present. Most of the head and neck schwannomas are characterized by typical appearance of free diffusion in the centre and restricted diffusion in the periphery of the mass.32 In our case, there was not this evidence. Infact, myxoid matrix, abundant in Schwannomas , is composed by elevated water content, due to poor cellularity, high mucin and low collagen content, and this likely explain their high diffusivity.33 Previous studies described the apparent diffusion coefficient (ADC) values of some adrenal lesions concluding that ADC values could not be used to differentiate between benign and malignant lesions;34 in the differential diagnosis between paragangliomas and schwannomas the ADC quantitative assessment could not provide significantly for the differentiation of both tumors.35 Classic CT findings of retroperitoneal Schwannomas are rounded and well-marginated masses with various enhancements. Adrenal Schwannomas are hypovascular tumors with mild heterogeneous and delayed progressive enhancement pattern involving equally the tissue, the septa and the walls of the lesion; however, the presence of degenerative changes contributes to make more heterogeneous the enhancement.31 The intravenous contrast medium injection should facilitate the differentiation with the adrenocortical carcinoma, characterized by rapid enhancement in hepatic arterial phase, and with pheochromocytoma, which generally is an hypervascular lesion with significant early heterogeneous enhancement.31 Calcifications and cystic components are frequently identified in these tumors.11,12,31 In our case, unenhanced CT performed during FDG CT-PET examination confirmed the presence of well-circumscribed round mass without evidence of calcifications. Cystic-like areas were visible and also detected trough small region of interest placement. The mass showed also an increased FDG uptake. Non-functioning solid tumors represent the main differential diagnoses of adrenal Schwannoma, which is not easily distinguishable from adrenal adenoma, adrenal myelolipoma, groups of neuroblastomas , adrenocortical carcinoma and adrenal metastasis. Adrenal adenomas and adrenal myelolipomas generally have a fat content that makes possible to differentiate them from other tumors. Neuroblastomas are positive during metaiodobenzylguanidine (MIBG) scintigraphy in seventy percent of cases and, among the neuroblastomas, ganglioneuromas are similar to retroperitoneal Schwannomas on imaging studies. However, although MIBG-negative ganglioneuromas are difficult to differentiate from Schwannomas36 , Ichikawa et al described delayed heterogeneous enhancement of ganglioneuroma on contrast- enhanced CT and whorled appearance with curvilinear bands of low signal intensity on T2 weighted images on MRI.36 Radiologist should keep in mind these findings that could be helpful in the differential diagnosis between MIBG-negative ganglioneuromas and other tumors. In our experience, the preoperative diagnosis was erroneous and misinterpreted the nature of the lesion, suggesting the possibility to be facing an adrenal ganglioneuroma or pheochromocytoma. Our experience confirms the difficulties in obtaining a correct diagnosis before pathological examination.Schwannoma is a neurogenic tumor, generally occuring between the third and the sixth decades of life, with no sex or location predilection. In fact, it may arise everywhere in organs or nerve trunks and the with sole exception of cranial nerves I and II, lacking Schwann cells.In summary, primary adrenal Schwannomas are rare and difficult to diagnose and frequently pre-operative workup is non-diagnostic, only postulating a non-secreting adrenal mass.For this reason, adrenal Schwannomas should be always included in the differential diagnosis of solid nonfunctioning adrenal tumors.T1 weighted images and heterogeneous high signal intensity on T2 weighted images.MRI is a valuable imaging technique for the diagnosis of adrenal Schwannoma, which appears as a well-circumscribed unilateral mass with cystic degenerative changes, characterized by low signal intensity on As confirmed by literature and by our experience, radical excision must be considered the most appropriate treatment, resulting in satisfactory prognosis.Given the rarity of this tumor and lack of definitive nonhistologic diagnostic modalities, adrenal Schwannoma remains a diagnosis of exclusion, but it should occupy an important place in the differential diagnosis set of incidentally discovered adrenal masses, as illustrated by this case.


Growing evidence suggests that the immune component of the tumor microenvironment (TME) may be highly involved in the progression of high-grade serous ovarian cancer (HGSOC), as an immunosuppressive TME is associated with worse patient outcomes. Due to the poor prognosis of HGSOC, new therapeutic strategies targeting the TME may provide a potential path forward for preventing disease progression to improve patient survival. One such postulated approach is the repurposing of the type 2 diabetes medication, metformin, which has shown promise in reducing HGSOC tumor progression in retrospective epidemiological analyses and through numerous preclinical studies. Despite its potential utility in treating HGSOC, and that the immune TME is considered as a key factor in the disease’s progression, little data has definitively shown the ability of metformin to target this component of the TME. In this brief review, we provide a summary of the current understanding of the effects of metformin on leukocyte function in ovarian cancer and, coupled with data from other related disease states, posit the potential mechanisms by which the drug may enhance the anti-tumorigenic effects of immune cells to improve HGSOC patient survival. Among gynecologic carcinomas, high-grade serous ovarian cancer (HGSOC) is the highest cause of cancer-related mortality, bearing a poor survival rate of roughly 50% over five years due to the extremely metastatic nature of this disease ,2,3. EarDuring metastasis, HGSOC tumor cells undergo the processes of adhesion, proliferation, migration, and invasion into the peritoneal cavity and preferentially latch onto the adipose tissue depot known as the omentum . Many stAs the survival rates of patients with HGSOC have not dramatically improved over the last few decades , it is cMetformin, a biguanide antidiabetic medication, is one such drug that has gained overwhelming attention in the treatment of inflammatory diseases, as well as a number of cancers. Early retrospective epidemiological case-control studies have indicated a possible association for metformin to enhance progression-free survival in patients with diabetes when compared to controls, which include patients with diabetes receiving other medications, as well as patients without diabetes ,14. ConsIn ovarian cancer, in vivo xenograft studies have shown that clinically relevant doses of metformin given in a preventative regimen, including pretreatment prior to and maintenance during engraftment, have decreased the size of the primary tumor and inhibited the number of metastatic implants , suggest+ TILs (CTC) has often been associated with improved overall survival [+ regulatory T cells (Treg) in the TME correlates with poorer prognosis [+ TILs and CD4+/CD25+ Treg cells in the epithelium and stroma of several histologic subtypes of ovarian cancer (OvCa). Most T lymphocyte subtypes failed to demonstrate an increase in survival rate, except for the intraepithelial CD8+ TILs. Importantly, they found that a high intraepithelial CD8+/CD4+ TIL ratio was associated with an improvement in the survival rate, whereas CD4+ TILs alone did not. These findings demonstrated the influence of CD8+ TILs on the prognosis for OvCa. Similarly, intraepithelial CD4+/CD25+ Tregs alone failed to show an increase in survival rate, while a high CD8+/Treg (CTC/Treg) ratio was associated with improved survival of patients with ovarian cancer [The diverse activity of tumor-infiltrating lymphocytes (TILs) may play a significant role in the progression of many types of cancers, as an increased presence of cytotoxic CD8survival , whereasrognosis . In generognosis , allowin+ TIL function is of great importance in the prognosis and the survival rate in patients with HGSOC. To date, the ability to pharmacologically modulate cytotoxic T cell behavior in the metastatic TME of HGSOC has not yet been evaluated using preclinical models. However, several studies demonstrate the potential impact of metformin on maintaining CD8+ TIL function and possibly enriching a high CD8+/CD4+ ratio to promote an immunoreactive environment in other cancers. In a study by Eikawa et al. [+ TILs in the TME. These findings were exemplified through the increase of three cytokines that the PD1−/Tim3+ phenotypic CD8+ TILs produce, consisting of interleukin-2 (IL-2), tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ). They also illustrated how metformin induced the differentiation from central memory T cells (Tmem) to effector memory T cells, demonstrating a stronger memory immune response to recurring tumor development. The authors attributed these findings in part to 5′-AMP-activated protein kinase (AMPK)-dependent inhibition of the mammalian target of rapamycin (mTOR) [+/CD25− T cells into Tregs [+ T cells. When treated with metformin, an increase in CD8+ TILs was observed, suggesting that metformin promoted either increased recruitment or differentiation toward an antitumor T cell phenotype. Moreover, in vivo findings showed that metformin effectively delayed tumor growth by causing a shift from oxidative phosphorylation (OXPHOS) to glycolysis, which then decreased the Treg expression of interleukin-10 (IL-10) and cytotoxic T lymphocyte antigen-4 (CTLA-4) [+ T cell phenotypes in vivo was associated with its activation of mTOR, as the mTOR inhibitor rapamycin ablated the ability of metformin to suppress Treg abundance. This mechanism is in contrast with the observed prevention of CD8+ TIL exhaustion resulting from the inhibition of mTOR by AMPK in the previous study by Eikawa et al. While convincing, it is contradictory to its canonical negative regulation of mTOR that was observed in most reports across disease states  . More reto Tregs . Subsequ(CTLA-4) . Intriguiewed in ). Taken iewed in , as welliewed in . However+ T cell phenotype [+ T cells into perivascular adipose depots in vivo in a murine model of hypertension [While the molecular mechanisms underlying metformin’s potential enhancement of T cell function in the TME are still poorly understood, it is clear that its primary intracellular target upstream of AMPK, liver kinase B1 (LKB1) , directlhenotype . Similarhenotype . These drtension ; whetherThe potential influence of AMPK-dependent metabolic reprogramming on T cell phenotypes has recently been reviewed , and as Although direct evidence identifying the effects of metformin on T cell infiltration and immunoreactive phenotypes in HGSOC has yet to be conducted, indirect evidence suggests that metformin may inhibit an immunosuppressive microenvironment in OvCa through the inactivation of MDSCs. MDSCs are proposed to target a number of immune cells, including T lymphocytes, which can result in decreased responsivity to antigens or other factors (reviewed in ). A rece2, an HIF1α activator, warrants caution with the interpretation of the results mentioned above due to the non-specificity of both drugs in their intracellular activity . How. How2, ay  on metformin. Consistent with their in vitro and in vivo data, metformin treatment significantly decreased CD39 and CD73 expression in MDSCs but increased CD8+ T cell function, as measured using granzyme B production. Intriguingly, these effects were observed in patients with diabetes before and after metformin treatment using a pairwise comparison  [To complement their preclinical findings, the level of expression of CD39 and CD73 in MDSCs, as well as CD8eatment) . PresumaTaken together, this study by Li et al. supports a possible mechanism for metformin that prevents T cell exhaustion through inhibition by other immune cells, such as MDSCs, to maintain an immunoreactive TME. While promising, the in vitro data reached significance only at supraphysiologic levels of metformin at 2mM, which is markedly higher than the micromolar range (<100 µM) experimental equivalent to the clinically used doses of the drug . It is uCancer cells produce an inflammatory response, which results in increased levels of neutrophils and often decreased levels of functional lymphocytes. Neutrophils induce several cytokines, including vascular endothelial growth factor (VEGF), which aids in angiogenesis and tumor growth . LymphocThere is no clear indication that metformin may impact the NLR in patients with HGSOC. However, data in other inflammatory disease states suggest that the drug could potentially reduce NLR in several contexts. When looking at metformin in diabetes, a disease that has been observed to have an elevated NLR , it was An excess neutrophil count could predictably lead to the increased formation of NETs, which are the secretion of a network of fibers, including chromatin and proteins, that generally have microbicidal activity (reviewed in ). NET foWhile there is no direct evidence in HGSOC, these studies suggest that metformin’s anti-inflammatory effects may include a reduced neutrophil count, a subsequent decreased NLR, and the inhibition of NETosis  . The putAs mentioned above , one of Metformin has been shown to induce AMPK activation and broadly alter the metabolic profile of a number of cell types in OvCa in vitro, including HGSOC tumor cells ,85, fibrThis dualistic nature of AMPK in regulating glycolysis may thus depend on the cell type and tissue, as its intracellular activity is likely dependent on the proteomic composition  and the intrinsic activity of its downstream kinases may determine the outcome of its activation. In light of these conflicting data regarding AMPK and the glycolytic shift, in order to elucidate how metformin may modulate immune cell metabolic reprogramming and consequent function, it is thus critical to either (1) perform a systematic analysis of the necessity of AMPK for metformin’s action using dominant-negative and other genetic constructs in vitro and in vivo , with thLastly, it is important to consider that glycolysis mediated by GLUT1 expression and AMPK activation has been observed to markedly enhance OvCa tumor cell proliferation . More reOne of the difficulties in appreciating the potential capacity for metformin to restore immunoreactivity and minimize tumor development or progression is the inherently complicated relationship between the immune system, inflammation, and cancer. While inflammatory responses to tumor growth display a number of mechanisms that are known to stimulate tumor cell self-renewal, proliferation, and motility , the abiToo many patients succumb to the aggressive behavior of OvCa tumor cells. Recent advances in improving personalized medicine in HGSOC patients via the high-throughput screening of biopsies combining genomics, proteomics, and metabolomics is a notable potential approach in future clinical decision-making to improve survival, as using these strategies may allow for targeted therapy in patients to exploit therapeutic liabilities of tumors to PARP inhibitors  or anti-angiogenic agents  (reviewed in ). As met+ T lymphocytes, inhibit MDSC immunosuppressive function, decrease neutrophil recruitment and the NLR, and possibly repress TAM infiltration into the TME all suggest that the drug may enhance the immune sensitivity in HGSOC and thereby prevent metastasis. Further research is still crucial for examining additional mechanisms that metformin is involved with in HGSOC, as well as validating current results found from previous studies, before metformin can be confidently utilized as a new therapy in patients with HGSOC. As seen comprehensively in the aforementioned studies, the addition of metformin in the immunosuppressed TME of patients from multiple cancers has shown to improve their immune cell function and sensitivity, which may relate to its ability to improve the outcome in highly aggressive diseases, such as HGSOC. While it has yet to be shown directly, the overlap in the role of immune cells across cancers implies that metformin may also be useful in restoring the immune-mediated inhibition of tumor development and progression in ovarian cancer. The capacity for metformin to promote cytotoxic CD8+/CD133+ cancer stem-like cells (CSCs) and increase CSC sensitivity to carboplatin ex vivo. Unfortunately, to date, only the study by Li et al. (It is important to note that much of the clinical data suggesting the benefit of metformin regarding patient outcomes are still predominantly in retrospective analyses, which makes it difficult to determine how the drug may be best utilized in therapeutic strategies. However, recent prospective clinical data do in fact support an improvement in the overall survival of patients with ovarian cancer treated with metformin , which ii et al.  above haCurrently, the preclinical data generally support the idea that metformin may prohibit an immunosuppressed TME and, if this is the case, could prevent the success of HGSOC cells in terms of metastasizing within the peritoneum. These data would then suggest that metformin may be most effective as a chemopreventive measure; however, with the nonspecific symptoms that are present in the early stages of disease development, it introduces another obstacle with regard to appropriately timing the initiation of metformin as a potential chemoprevention therapy. Moreover, the current prospective trials evaluating metformin in HGSOC and other ovarian carcinomas are intervention strategies in patients with stage II disease or above , which tOverall, these possibilities warrant extensive investigation of how metformin may impact the local and systemic immune responses to de novo tumorigenesis and the subsequent metastasis to distant tissues. Only by using clinically relevant doses in physiologically representative systems, such as in vitro organotypic or organoid model systems, as well as complementary in vivo approaches utilizing advanced cell-specific genetic techniques, will it become clear whether the drug may in fact suppress tumor development or progression. It may be specifically beneficial to focus on experimental strategies that reflect the epidemiological findings in which benefits were observed retrospectively in individuals on the medication to manage diabetes, and possibly for extended periods that may have preceded significant events during neoplastic growth and dissemination. While technically challenging, this could be accomplished through the chronic pretreatment of in vivo animal models prior to engraftment, or in vitro by utilizing continuous peristaltic systems for reliably consistent drug delivery coupled with recently developed organoid cultures that incorporate immune cells, as well as other stromal cells ,104. Fur


Autotaxin (ATX) is an exoenzyme which, due to its unique lysophospholipase D activity, is responsible for the synthesis of lysophosphatidic acid (LPA). ATX activity is responsible for the concentration of LPA in the blood. ATX expression is increased in various types of cancers, including breast cancer, where it promotes metastasis. The expression of ATX is also remarkably increased under inflammatory conditions, particularly in the osteoarticular compartment, where it controls bone erosion. Biological actions of ATX are mediated by LPA. However, the phosphate head group of LPA is highly sensitive to degradation by the action of lipid phosphate phosphatases, resulting in LPA inactivation. This suggests that for efficient action, LPA requires protection, which is potentially achieved through docking to a carrier protein. Interestingly, recent reports suggest that ATX might act as a docking molecule for LPA and also support the concept that binding of ATX to the cell surface through its interaction with adhesive molecules  could facilitate a rapid route of delivering active LPA to its cell surface receptors. This new mechanism offers a new vision of how ATX/LPA works in cancer metastasis and inflammatory bone diseases, paving the way for new therapeutic developments. ENPP2, the ATX gene, is regulated by cytokines, growth factors and hormones (The name autotaxin (ATX), proposed by Stracke and colleagues in 1992, arose during the characterization of a new potent autocrine motility-stimulating protein produced by human A2058 melanoma cells ,4. ATX lhormones .ATX is secreted in the extracellular space. Mechanisms responsible for ATX secretion were characterized in adipocytes showing the requirement of post-translational maturation of the N-terminal signal peptide of ATX and N-glycosylation of amino acids N53 and N410 . In physENPP2) correlates with a poor outcome in several types of cancer  [10A [2].  cancers ,28,29),  cancers . Our gro cancers ,32. Brin cancers ,34.ENPP2 has indeed been identified as a candidate gene causing drug resistance in the long-term treatment of ovarian cancer, and stable ectopic expression of ATX in OVCAR-3 ovarian cancer cells delays apoptosis induced by carboplatin [ENPP2 mRNA expression was found to be significantly downregulated in lung cancer samples, both immunohistochemistry analysis of lung tissue biopsies and serum ATX activity levels revealed that lung cancer in humans is associated with increased levels of ATX protein and its activity [ATX lysoPLD/LPA axis upregulation is not only related to tumor growth, but also contributes to other critical aspects of cancer biology including inflammation, angiogenesis, invasion and metastasis ,36,37. Uboplatin . Severalboplatin . It has boplatin . Shao anboplatin . Althougactivity .Several studies are underway to assess the therapeutic potential of ATX lysoPLD inhibitors . Since LLPA is a very simple lipid subjected to strong degradation in the extracellular medium by a family of three enzymes called the lipid phosphate phosphatases (LPPs). At the plasma membrane, the catalytic site of the LPPs is oriented to the extracellular environment, enabling them to access and hydrolyze extracellular LPA . BindingSMB domains present in the ATX primary sequence are small cysteine-rich domains known to mediate protein–protein interactions, an example being the vitronectin SMB domain that binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) . Thus, sCrystallographic studies of ATX have been completed, revealing a complex organization: the PDE core domain interacts with both the SMB domains on one side and the NUC domain on the other ,64. WhilENPP2 gene. This insertion directs ATXα to the plasma membrane, thereby ensuring a localized LPA production and signaling [Integrins are not the only adhesive molecules involved in maintaining ATX at the cell surface. Moolenaar’s team found that ATXα, the less abundant isoform, binds to cell surface heparan sulfate (HS) proteoglycans through a unique insertion of a 52 polybasic amino acid sequence generated following alternative splicing of the ignaling . Our groignaling . In addiignaling . Even thignaling . Taken tRheumatoid arthritis is a human autoimmune disease characterized in the clinic by highly inflammatory joints that cause swelling and destruction of both cartilage and bone . MoleculhTNF+/−), mimicking the human pathogenesis. Conditional knockout of the ATX gene, enpp2, in mesenchymal cells in Col6a-Cre+ mice, mainly affecting synovial fibroblasts, attenuated the development of arthritis [+/−hTNF mice [−/−hTNF mice displayed a significant reduction of local bone erosion. Thus, pharmacological inhibition of ATX using BMP22 causes distinctively different effects on inflammation versus bone resorption as it does not protect hTNF+/− transgenic mice from inflammation or swelling but protects them from inflammation-induced bone loss by reducing osteoclastic bone resorption.LPA is detected at high levels in the synovial fluid of RA patients due to the elevated expression of ATX, mediated at least in part through TNFα action on synovial fibroblasts ,74,75. A+/− mice . The sim1 [lpar1 knockout mice have been shown to be protected against collagen-induced arthritis (CIA) by reducing bone destruction and infiltration of pro-inflammatory T helper (Th)17 lymphocytes into the joints [1 is the most ubiquitous LPA receptor. As a consequence, global lpar1 knockout animals exhibit multiple defects, particularly in their neural and metabolic systems [We previously demonstrated that LPA controls the bone resorption activity of osteoclasts by a direct activation of its cell surface receptor LPA1 ,76. Globe joints . LPA1 is systems ,78,79. Fenpp2 floxed mice with Ctsk-Cre mice, we generated ∆CtskATX animals with specific ablation of ATX expression in mature osteoclasts. As expected, ∆CtskATX mature osteoclasts were defective in mineral matrix degradation in vitro but this was potently rescued by adding exogenous ATX, suggesting that the environment can supply ATX if necessary under specific pathophysiological conditions. In this context, variable sources of ATX and/or LPA are potentially available in the bone microenvironment with the presence of fibroblasts [CtskATX mice were remarkably protected against bone degradation induced by two independent powerful inflammatory challenges such as lipopolysaccharide (LPS) treatment and transfer of the serum from mice expressing the transgenic T cell receptor (TCR) and the MHC class II allele Ag7 (K/BxN), while, as expected, specific inhibition of ATX expression in mature osteoclasts does not affect the onset and progression of the inflammatory process or the accumulation of ATX in the synovitis of ∆CtskATX mice [Through in vitro studies, we recently demonstrated that ATX is upregulated during the course of osteoclastogenesis, and as such, ATX belongs to late osteoclastic markers . In addiroblasts , chondroroblasts , osteoblroblasts , adipocyroblasts  and endoroblasts . Howevertsk mice . These r1 [Bone metastases frequently form at advanced stages in a large range of solid tumors such as breast and prostate cancers, which are the most osteophyle cancers, but bone metastases are also secondary sites for tumor cells that have escaped from primary lung, thyroid and renal cancers . In this1 . HoweverMature osteoclasts express multiple adhesive molecules that have been described to interact with ATX . One of these, the integrin αvβ3, is a marker of osteoclast differentiation . In addiIn conclusion, all these results suggest that binding of ATX to the cell surface through its interaction with different adhesion molecules could achieve two major objectives. The first objective is to allow the production of LPA in the immediate vicinity of its membrane receptors. LPA is highly sensitive to degradation by phospholipases that are extensively represented at cell surfaces and in biological liquids. Therefore, LPA requires protection that can be provided by docking to a carrier protein. ATX associated to the cell membrane loaded with LPA might offer the fastest way to deliver active LPA to its cell surface receptors.The second objective is to increase the production of LPA through the amplification of ATX/lysoPLD activity. This unexpected finding was confirmed from a series of recent independent studies opening new opportunities for the future design of ATX inhibitors. Targeting the ATX-bound lysoPLD instead of the ATX-soluble lysoPLD may provide more efficient therapeutic drugs. In addition, instead of targeting the enzymatic activity of ATX, the development of drugs directed against the binding domains of ATX or to ATX adhesion molecules could be considered. These new therapeutic strategies could have several advantages. They could compensate for the mode of action of current ATX inhibitors that all induce a massive decrease in LPA concentration in the bloodstream, which during long-term treatments can potentially provoke unknown side effects with a major concern for the central nervous system and the reproductive system. Collectively, the works described in this review show a fairly wide diversity in cell surface “receptor-like” interactions for ATX. Because of the large variety of cells in the organism that express the specific pattern of adhesive molecules and as a consequence specific types of “receptor-like” molecules for ATX, it is conceivable to consider the development of new blockers of ATX action that could restrict the therapies to specific sub-types of cells and tissues.


The current and future therapeutic strategies for this category of patients are based on well-established and complex preclinical data to which the authors paid insufficient attention.An interesting and particularly welcome article by Taieb et al.2 This effect is attributed predominantly to paradoxical ERK activation .3 Figure 1 makes no mention of this tissue-specific molecular aspect, which is important not only for mechanistic reasons, but is also of therapeutic interest since BRAFi, which evades paradoxical MAPK pathway activation, is currently in clinical development.4 Moreover, a definition of a paradox index has been set for BRAFi  as a means of quantifying a therapeutic window of high clinical efficacy (at least in BRAF-mutant melanoma cells) with minimal paradoxical ERK activation.5One of the major advances in BRAF-mutated CRC is the setting of BRAFi–MEKi combinations. Of importance, although not discussed by the authors, this association of a BRAFi with a MEKi was dictated by the fact that BRAFi can induce neoplasia, most often cutaneous squamous cell carcinoma.6 This explains why, in the case of colorectal cancer cells and not melanoma cells , the addition of EGFRi is a necessary complement to the BRAFi–MEKi association. When advocating EGFRi in CCR, and more broadly, the use of monoclonal antibodies, the respective order of application of cetuximab versus bevacizumab is still a matter of debate. Cetuximab is an IgG1 and this characteristic confers to the drug the capacity to develop the clinically important mechanism of antibody-dependent cellular cytotoxicity (ADCC). ADCC was recently highlighted by others and us for its significant contribution to the global action mechanism of cetuximab.The authors did mention another level of the underlying molecular complexity, i.e. the interruption by MEKi of negative feedback of ERK signalling at the initiation steps of MAPKinase signalling.7 In brief, based on preclinical and clinical observations, cetuximab-mediated ADCC, through natural killer-cell release of INFα, results in priming of cytotoxic T cells.7 Recent experimental and clinical data have revealed that bevacizumab, through its interaction with VEGF, may in fact restore normal endothelial cell diapedesis. This results in favourable tissular diffusion of cytotoxic T lymphocytes instead of regulatory T cells, the diffusion of which is facilitated by the deleterious impact of VEGF on endothelial cells.8 Thus, under bevacizumab treatment, adequate tumoural tissue redistribution of beneficial antitumour CD8 T cells is achieved in place of detrimental regulatory T cells. This background may support recent clinical results regarding the optimal order for cetuximab (priming CD8 T cells) versus bevacizumab  in first-line metastatic CRC9 and strengthens combination strategies also including immunotherapy by checkpoint inhibitors in unstable microsatellite BRAF-mutated CRC patients. The fact that BRAF + MEK inhibition positively affects the tumour microenvironment and immune modulation is thus a strong argument in favour of adding immunotherapy in the context of the cetuximab–BRAFi–MEKi/bevacizumab combination with attention being paid to an optimal sequencing. The included figure recapitulates the molecular mechanisms considered above and seeks to clarify the particularly complex background-sustaining treatment options for metastatic BRAF-mutated CRC.Of importance is the immune modulation generated by ADCC.


Superspreading events have distinguished the COVID-19 pandemic from the early outbreak of the disease. Our studies of exhaled aerosol suggest that a critical factor in these and other transmission events is the propensity of certain individuals to exhale large numbers of small respiratory droplets. Our findings indicate that the capacity of airway lining mucus to resist breakup on breathing varies significantly between individuals, with a trend to increasing with the advance of COVID-19 infection and body mass index multiplied by age . Understanding the source and variance of respiratory droplet generation, and controlling it via the stabilization of airway lining mucus surfaces, may lead to effective approaches to reducing COVID-19 infection and transmission. COVID-19 transmits by droplets generated from surfaces of airway mucus during processes of respiration within hosts infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. We studied respiratory droplet generation and exhalation in human and nonhuman primate subjects with and without COVID-19 infection to explore whether SARS-CoV-2 infection, and other changes in physiological state, translate into observable evolution of numbers and sizes of exhaled respiratory droplets in healthy and diseased subjects. In our observational cohort study of the exhaled breath particles of 194 healthy human subjects, and in our experimental infection study of eight nonhuman primates infected, by aerosol, with SARS-CoV-2, we found that exhaled aerosol particles vary between subjects by three orders of magnitude, with exhaled respiratory droplet number increasing with degree of COVID-19 infection and elevated BMI-years. We observed that 18% of human subjects (35) accounted for 80% of the exhaled bioaerosol of the group (194), reflecting a superspreader distribution of bioaerosol analogous to a classical 20:80 superspreader of infection distribution. These findings suggest that quantitative assessment and control of exhaled aerosol may be critical to slowing the airborne spread of COVID-19 in the absence of an effective and widely disseminated vaccine. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmits through the air by a combination of the large droplets exhaled when people cough or sneeze and the very small droplets people generate in their airways when they naturally breathe –4. How eGeneration of respiratory droplets in exhaled breath can occur by the force of the fast airflows in the upper airways that arise when we breathe, talk, cough, and sneeze. At peak inspiratory flows during normal breathing, air speeds in the trachea and main bronchi can reach turbulent velocities . The rusTo ascertain whether COVID-19 infection and other phenotypical differences associated with severity of infection risk might alter airborne droplet generation from airway lining fluid during acts of breathing, we conducted two studies in human and nonhuman primates (NHPs). In our first study, we evaluated the exhaled breath of 194 human subjects at two sites to determine exhaled breath particle variations in the human population. In our second study, we measured the exhaled breath from two species of NHPs following experimental infection via inhalation of SARS-CoV-2. We then assessed exhaled breath particle evolution over the time course of exhaled aerosol particles as a function of nasal viral titer. We report on these studies here.We evaluated the exhaled aerosol of 194 human volunteers at two sites in North Carolina (74 subjects) and Michigan (120 subjects). We conducted observational cohort studies of essential workers at No Evil Foods in Asheville, NC, and of students, staff, and faculty at Grand Rapids Community College in Grand Rapids, MI, over a total period of 4 d. The results from these measurements are shown in We categorized subjects by those exhaling greater or less than 156 particles per liter of air. We chose this demarcation since the individuals above this threshold aerosol number exhaled 80% of the total particle production from the 194 human volunteers while being less than 20% of the total members of the group—analogous to the conventional definition of superspreading of airborne infectious disease . Within P < 0.015). The BMI-year results are shown in We evaluated relationships between exhaled aerosol particle number and sex, age, and body mass index (BMI). No correlation was found with sex, while significant correlations were observed between exhaled aerosol, age, and BMI—and particularly BMI-years. We characterized each of the 146 individuals for whom we obtained age and BMI information by their age multiplied by their BMI, or by their BMI-years. We noted that half of the group  with lowest BMI-years (less than 650 BMI-years) exhaled significantly less aerosol than the half of the group  with highest BMI-years (above 650 BMI-years)  to assess interspecies differences in comparison to the human model. We experimentally infected the NHPs (n = 8) with SARS-CoV-2 by small-particle aerosol (∼2 µm) and closely monitored them thereafter. Results of mucosal sampling  showed productive infection in both species, with viral RNA detected as early as +1 d postinfection, viral titers reaching a crescendo in most animals by day +7 postinfection, and clear declination by day +14 postinfection, and undetectable concentrations by study terminus (+28 d postinfection). Corresponding clinical signs of COVID-19 in both species of animals over the course of postinfection observation were considered self-limiting and generally mild. Animals experienced no significant weight loss, a transient, unremarkable fever, and rare lung sounds upon auscultation during clinical examination.We studied exhaled aerosol in an NHP model to explore the dependence of exhaled aerosol on COVID-19 infection. We used two NHP species in the study  and two of four of the African green monkeys .Although increase of total particles was observed in both species, aerosol particle production increase relative to preinfection totals was more profound in the rhesus macaques A than inThe particle distributions of the exhaled breath particles, averaged among species-segregated cohort, changed as COVID-19 disease progressed . The shiMycobacterium tuberculosis (Mtb). The rhesus macaque represents a highly refined model of clinical TB, with development of active pulmonary disease that shares remarkable similarities with clinical TB in humans (n = 4) were experimentally infected by small-particle aerosol exposure (∼2 μm) with Mtb (Erdman strain) at a target inhaled dose of 400 colony-forming units (CFU). Animals were monitored for development of disease thereafter, including biological outcome parameters correlative with development of disease . Aerosol parameters measured within the face masks, which also was used in our COVID-19 primate model, included cumulative  particle counts and distribution-specific data collection. Results showed animals experimentally aerosol-infected with Mtb strain experienced development of active pulmonary TB over the next several weeks postinfection. Aerosol monitoring of the infected primates via face mask during postexposure observation showed a peak in total particles  at week 4 postinfection  lung infection. Particularly, that exhaled breath particles in the NHP COVID-19 infection model rise to a crescendo and decrease in size with growth in viral load  and 4, aAging , diet 11, and luIn a companion article , exhaledThe strong correlation observed here between advanced BMI-years and greater propensity to generate respiratory droplets  may be sWhile those with low BMI-years, including children, appear to be at smallest risk of airway lining mucus breakup and respiratory droplet generation, our NHP results suggest that all individuals, including those with low BMI-years, can be at risk for generating large numbers of respiratory droplets, particularly following lung infection, and therefore argue for the vigilant hygienic protection of the young as well as the old when it comes to the gathering of people within indoor environments where respiratory droplets can linger and accumulate.Our finding that the proportion of small respiratory droplets  increasThe scientific response to the COVID-19 pandemic has largely focused on the development of curative drugs and preventive vaccines. In the wait for a cure or an effective widely adopted vaccine, it may be advisable for the scientific community to additionally focus on management of COVID-19 through the restoration of airway lining mucus barrier function, and, notably, in the reduction of the propensity of airway lining mucus to disintegrate under the force of natural breathing, which it is otherwise disinclined to do in the airways of the young and uninfected.Exhaled aerosol numbers appear to be not only an indicator of disease progression, but a marker of disease risk in noninfected individuals. Monitoring (as a diagnostic) might also be an important strategy to consider in the control of transmission and infection of COVID-19 and other respiratory infectious diseases, including TB and influenza.SI Appendix. For the North Carolina study, an independent review board  determined formal Institutional Review Board (IRB) review to be unnecessary when considering the observational nature of the study and the corresponding minimal impact on human subject research. For the Grand Rapids Community College study, the college’s IRB committee approved the protocol.We conducted observational cohort human volunteer studies in North Carolina and Michigan designed to evaluate exhaled aerosol particle size and number during normal breathing in noninfected humans. In the conducting of the studies and the reporting of our results, we followed Strengthening the Reporting of Observational Studies in Epidemiology statement reporting guidelines. Eligible participants were healthy adults 19 y to 66 y of age, either essential workers at No Evil Foods in Asheville, NC, or students, faculty, staff, and other human volunteers at Grand Rapids Community College in Michigan. Participants were not screened for SARS CoV-2 infection by serology or PCR before enrollment. The trial was conducted on the premises of No Evil Foods and at Grand Rapids Community College. An illustrative (North Carolina) protocol is available in Participants spent up to 30 min per session while away from work to have their exhaled aerosol particles measured. Exhaled particles were measured by a particle detector (Climet 450-t) designed to count airborne particles in the size range of 0.3 μm to 5 μm. The particle detector was connected to a standard nebulizer tubing and mouthpiece that filters incoming air through a high-efficiency particulate air (HEPA) filter. Each standard nebulizer tubing and mouthpiece was removed from sealed packaging before each subject prior to the subject’s first exhaled particle detection. On subsequent counting maneuvers, the same mouthpiece, tubing, and HEPA filter were replaced into the particle counter system by the participant to insure effective hygiene. Subjects performed normal tidal breathing through a mouthpiece while plugging their noses over 1 to 2 min—beginning with two deep breaths to empty their lungs of environmental particles. Over this time frame, particle counts per liter diminished to a lower baseline number, reflecting particles emitted from breakup of airway lining fluid surfaces in the subject’s airways. Once the lower plateau of particle counts was reached, subjects continued to breathe normally. Three to eight particle counts  were then averaged to determine the mean exhaled particle count and SD. Participants sat opposite to the study administrator with a Plexiglas barrier separating the participant and the administrator.Mtb (Erdman) by small-particle aerosol previously characterized in our laboratory inhalation system (3 tissue culture infectious dose 50 (SI Appendix) and (for the TB studies) an inhaled dose of 466 ± 237 CFU. Animals were observed for 28 d or 60 d postinfection  including twice daily monitoring by veterinary staff. In our COVID-19 studies, mucosal and other biosamples were collected at 7 d before infection, at days 1, 3, 7, 14, and at necropsy (day 28) after infection. In our TB studies, mucosal and other biosamples were collected at 7 d before infection, at days 1, 7, 14, 21, 28, 35, 42, and at necropsy. During biosampling events and physical examination while anesthetized and in dorsal recumbency, and experiencing normal respiration, animals were individually sampled for exhaled breath aerosols. This sampling was performed using a modified pediatric face mask fitted with a HEPA-filtered inspiration port, and a corresponding sampler for exhalation. A particle counter (Thermo Systems Inc. AeroTrak handheld particle counter Model 9306-V2) was used to sample exhaled breath particles for five 1-min intervals at every sampling time point. Exhaled breath particle data were collected in a cumulative fashion.A total of eight male (>7 y of age), purpose-bred rhesus macaques and wild-caught African green monkeys were used in our NHP study of COVID-19; four rhesus macaque monkeys were used in the TB study. NHPs are extremely limited in allocation for the purposes of biomedical research studies, and represent a scarce scientific resource. Therefore, acquisition and use may, at times, trump the balancing of particular desirable characteristics  in the design of particular experimental cohorts. The use of “all males” in this study was not intentional inasmuch as it was imposed by the process of acquisition and allocation of animals for this study. The African green monkeys species used in a portion of the studies were acquired from a source that does not purpose-breed animals and rather acquires from natural habitat. Accordingly, demographics on these animals are limited  and are only estimated from dental record. Animals were exposed to SARS-CoV-2  and n system . AnimalsSI Appendix, Methods).Nasal swabs were collected in 200 µL of DNA/RNA Shield and extracted for viral RNA using the Quick-RNA Viral kit. Samples were then quantified using RT-qPCR (Guide for the Care and Use of Laboratory Animals (The Institutional Animal Care and Use Committee of Tulane University reviewed and approved all the procedures for this experiment. The Tulane National Primate Research Center is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All animals are cared for in accordance with  Animals . The TulSupplementary File


Since 2005, The Michael J. Fox Foundation for Parkinson’s Research (MJFF) has invested significant funding and non-funding effort to accelerate research and drug development activity around the Parkinson disease (PD)-associated protein LRRK2. MJFF has spearheaded multiple public/private pre-competitive collaborations that have contributed to our understanding of LRRK2 function; de-risked potential safety questions around the therapeutic use of LRRK2 kinase inhibitors; and generated critical research tools, biosamples, and data for the field. Several LRRK2-targeted therapies are now in human testing due to the hard work of so many in the PD community. In this perspective, we present a holistic description and model of how our Foundation’s support targeted important barriers to LRRK2 research and helped move the field into clinical trials. LRRK2) gene represent one of the most common genetic causes of Parkinson disease (PD), explaining an estimated 1–2% of cases overall [LRRK2 locus are also associated with increased risk for idiopathic PD (iPD) [Discovered in 2004 ,2, mutat overall . In somePD (iPD) ,6. The mPD (iPD) . These fGiven its strong genetic link to PD and compelling therapeutic rationale, The Michael J. Fox Foundation for Parkinson’s Research (MJFF) has been a consistent supporter and funder of studies to facilitate translation of LRRK2 research into treatments for people with PD. Our efforts have focused on a multi-faceted strategy with the goal of informing optimal clinical trials of LRRK2-targeted therapies. Through multiple initiatives and programs, MJFF has sought to (1) define the role of LRRK2 in PD; (2) establish and improve measures of the LRRK2 pathway; (3) foster translation of LRRK2 into therapies; and (4) promote development and distribution of tools and resources to facilitate LRRK2 and PD research. To maximize the impact of these strategies and speed delivery of promising treatments to patients, we instilled a philosophy of collaboration and sharing throughout the LRRK2 field by creating and coordinating a broad “Consortium” approach to LRRK2 research and drug development .The nomination of genes, proteins, and pathways potentially involved in PD pathogenesis drives research to characterize these targets and pathways and the cascade of events underlying disease onset, progression and disability. Researchers often need to first clarify the impact of genetic mutations and variations on protein structure and function. This is then expanded by work to define normal and pathological roles of a new target within cells and in more complex biological systems. Ultimately, a mechanistic and pathogenic model linking a protein, its proximal biology, and distal downstream effects on disease becomes clearer, providing validation and support for therapeutic development.It is through this strategic lens that MJFF established its approach to expanding understanding of the role of LRRK2 in PD. Working closely with a global community of researchers, drug makers, clinicians, and people with and without PD, MJFF has shaped funding strategies to address key questions in the field. We frequently bring key opinion leaders together at scientific meetings and workshops to identify critical gaps and challenges and subsequently find and support expert groups to address these challenges. Below, we describe several key areas where this model has proven particularly successful in furthering our understanding of LRRK2.First, MJFF has been a leader in supporting work to understand the structure of the LRRK2 protein ,9,10. LRSecond, using a similar collaborative approach, MJFF funded a global team of scientists and drug makers who had access to critical tools and model systems to identify and validate major downstream substrates of LRRK2. This study resulted in the identification of a set of Ras-related in brain (Rab) GTPases as endogenous LRRK2 substrates , a findiFinally, MJFF support aims to define the upstream modifiers of LRRK2 activation and downstream mechanisms of LRRK2-mediated neurodegeneration using novel methods and model systems. These studies are yielding interesting data on other proteins and pathways that could be targeted to decrease the pathogenic effects of LRRK2 ,21. MoreObjective biomarkers of LRRK2 protein expression and activity can speed current therapeutic development and incentivize additional industry groups to start new drug programs against this target. Through multiple approaches, MJFF has supported work with the goal of providing a suite of biochemical assays for use in identifying those in the PD population who might benefit from LRRK2 targeted therapies.www.michaeljfox.org/mjff-scientific-publications-clinician-articles). The Consortium is now discussing methods to detect LRRK2 and its activation in the cerebrospinal fluid (CSF) and in immune cells such as monocytes and neutrophils given the high expression of LRRK2 in these cells [Given many early challenges in methods for isolating and assaying the LRRK2 protein, MJFF launched a precompetitive consortium with industry groups to optimize the detection of LRRK2 and its activation in human biosamples. The group convenes frequently to discuss various assay development efforts for detecting LRRK2 levels and activation. In addition, MJFF provided the group standardized clinical samples to enable head-to-head comparison of various high-throughput detection platforms. These efforts identified changes in LRRK2 activation in G2019S subjects by employing a highly sensitive pS935 LRRK2 assay  and demose cells . These eMJFF-supported investigators recently identified bis(monoacylglycerol)phosphate (BMP) as an important indicator of LRRK2 activity . ResultsLRRK2 . Although these variants alter LRRK2 kinase activity in cellular studies [MJFF is also supporting research using clinical samples to determine the biochemical impact of other disease-linked or associated variants in  studies , MJFF ge studies .In the earliest days of research on LRRK2, it was evident that the medicinal chemistry understanding to develop therapies targeting the protein’s kinase activity was far ahead of the biological understanding of its role in cells. This is evidenced by numerous publications and patents describing chemical LRRK2 protein inhibitors . DespiteWhile the first leading compounds neared the clinic, a critical question arose as to whether LRRK2 inhibitors might lead to side effects or toxicity that would halt progress. Observations in animal models lacking the LRRK2 gene had already suggested that loss of LRRK2 is associated with morphological changes in cells in the kidney and lungs ,33,34,35To address this, MJFF established a pre-competitive LRRK2 Safety Initiative involving three pharmaceutical companies . Demonstrating a clear willingness to tackle a common challenge together, each company agreed to contribute LRRK2 kinase inhibitors of different structural classes in order to address the question of LRRK2 inhibitor safety. Results of the collaborative study showed that the changes seen in lung were indeed due to an apparent direct action on LRRK2. However, these changes were completely reversible after a two-week washout of drug and the group found no lung functional consequences despite the high drug exposures achieved . In a sehttps://investors.biogen.com/news-releases/news-release-details/biogen-and-denali-collaborate-lrrk2-program-parkinsons-disease).At the time of this writing, two companies are testing LRRK2 treatments in human trials. Denali Therapeutics was the first company to enter the clinic with a LRRK2 kinase inhibitor in 2017. DNL201, a small molecule LRRK2 inhibitor, completed Phase 1 studies (NCT03710707) and was deemed safe and well-tolerated. Importantly, the company reported no clinically meaningful changes or dose-related trends in the functional pulmonary readouts they tested. Denali Therapeutics is also testing in phase 1b trials another oral LRRK2 inhibitor, DNL151 (NCT04056689). Based on results of these trials, Denali Therapeutics has recently announced that they will be moving forward with DNL151 due to its pharmacokinetic properties and in an exciting new development has partnered with Biogen to develop and commercialize its LRRK2 kinase inhibitor . This work complements the efforts of other organizations who have also launched programs to enhance capacity for finding and educating people with genetic forms of PD (www.parkinson.org/PDGENEration). We are also working to identify people who carry other mutations and variants in LRRK2 in Spain, China, Singapore, and Malaysia. Biosamples from these individuals will be important for further confirming the potential benefits of LRRK2 inhibitors and establishing novel biomarkers. Finally, MJFF is piloting studies within the US for engaging PD patients of Asian descent in genetics research. These efforts are not only addressing recruitment challenges for LRRK2 trials but are also educating and empowering a more diverse and inclusive patient community to participate and contribute to research.With three LRRK2-targeted therapies in clinical testing and additional therapies likely to move into trials soon, a big challenge will be identifying and recruiting sufficient numbers of G2019S www.michaeljfox.org/research-tools-catalog).In addition to funding, MJFF has long been a leader in providing the community with critical resources such as preclinical tools and models as well as clinical data from cohorts of people with PD and access to human biosamples. MJFF works with contract research organizations and a global network of investigators to generate, distribute and, when needed, further characterize these tools. Through periodic input from the research community via surveys and meetings, we continuously monitor the need for and prioritize those tools that address key gaps in the PD field and speed progress. Through such efforts, the Foundation has made available and continues to generate multiple animal models, antibodies, cell lines, immunoassays, and viral vectors, to study the function of LRRK2 in various cellular and in vivo systems , we are following 156 people with PD carrying a LRRK2 mutation and 216 non-manifesting carriers. These volunteers are contributing valuable clinical data and biosamples longitudinally to guide biomarker studies for PD. Through PPMI, the Foundation has made available fibroblasts and induced pluripotent stem cells (iPSCs) from LRRK2 manifesting and non-manifesting carriers that are being used by laboratories around the world for exploring and validating LRRK2′s biological function (www.ppmi-info.org/access-data-specimens/request-pbmcs-cell-lines). With new data emerging on the role of LRRK2 in specialized cells, future efforts are directed at generating isogenic controls for these lines and differentiating these iPSCs into various cell types such as dopamine neurons, astrocytes, and microglia to compare the cell-type specificity of observed LRRK2 effects. Data generated through PPMI and other cohorts are readily available through various platforms for access by researchers that in turn encourages generation of new hypotheses and data standardization and replication (www.michaeljfox.org/data-sets).As part of our PPMI study . The Foundation works closely with academic laboratories and industry groups to guide sample selection and enforces data return and dissemination of results through MJFF-led calls. These efforts have proved to be extremely beneficial for drug developers as it provides them with the opportunity to leverage our biosample collections for biomarker studies and has benefited the community as they can apply lessons learned from these studies in their laboratories to validate these findings.MJFF has also strived to provide standardized and high-quality biosamples from human Importantly, in addition to being a resource provider, MJFF has encouraged and incentivized progress through effective models of collaboration between key academic and industry stakeholders. MJFF is a strong advocate for open science policies, requiring data and tools generated through our funding to be made available and when possible deposited in publicly accessible repositories. The Foundation frequently convenes stakeholders to evaluate progress and challenges in the field and to revisit and reshape our strategies as needed. We are also aligning our efforts with many other organizations and funders to promote greater transparency in research results through promotion of open-access publication and archiving models. These approaches have been instrumental to success in advancing the LRRK2 field and have provided a model that MJFF now uses across many internal strategic priorities.In the last 15 years, the LRRK2 field has overcome challenges in understanding of a complex, disease-associated protein. As a leading strategic funder and facilitator of PD research and drug development, MJFF has used a highly collaborative model to support work around key challenges and barriers to progress. The outcomes of these efforts are evident in the growing number of LRRK2 therapeutic programs moving closer to or currently in clinical trials. MJFF remains dedicated to fueling and fostering the drug discovery pipeline for PD with the goal of bringing new therapies into the hands of patients.