Safety, pharmacokinetics, and efficacy of ruxolitinib cream in children and adolescents with atopic dermatitis.

BACKGROUND: Therapies for children with atopic dermatitis (AD) have safety and tolerability concerns that may limit long-term use. Ruxolitinib cream, a Janus kinase (JAK) inhibitor, is effective and well tolerated in adolescents and adults with AD. OBJECTIVE: To analyze the safety and tolerability of ruxolitinib cream in pediatric patients. Pharmacokinetics and efficacy were also evaluated in this phase 1 study (NCT03257644). METHODS: Patients aged 2 to 17 years with AD (affected body surface area 8%-20%; Investigator's Global Assessment score ≥2) were enrolled stepwise in 6 age-descending, strength-increasing cohorts to apply 0.5%, 0.75%, or 1.5% ruxolitinib cream twice daily for 28 days. Safety, pharmacokinetics, and efficacy were analyzed at baseline, week 2 (day 10), and week 4 (day 29). RESULTS: Among 71 patients, 44 (62.0%) had a baseline Investigator's Global Assessment score of 3; median (range) body surface area affected at baseline was 12.2% (1.7%-20.4%). Ruxolitinib cream was well tolerated, with 4 patients (5.6%) experiencing treatment-related adverse events (all grades 1/2). No clinically meaningful changes in mean chemistry or hematology values were observed, and no consistent pattern of change in bone biomarkers was detected. Mean plasma ruxolitinib levels within each cohort (range, 23.1-97.9 nM) were well below the half-maximal inhibitory concentration for thrombopoietin phosphorylation of STAT3 (281 nM). All cohorts experienced improvements in exploratory efficacy end points. CONCLUSION: Ruxolitinib cream was well tolerated in pediatric patients with AD, with no effect on blood counts or bone biomarkers. Mean plasma concentration was low. Efficacy was consistent with data from previous studies in adolescents and adults. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03257644.

Long-term safety and disease control with ruxolitinib cream in atopic dermatitis: Results from two phase 3 studies.

BACKGROUND: Ruxolitinib cream demonstrated safety and efficacy over 8 weeks in 2 double-blind phase 3 atopic dermatitis studies (NCT03745638/NCT03745651). OBJECTIVE: To evaluate long-term safety (LTS) and disease control with ruxolitinib cream. METHODS: Patients initially randomized to twice-daily 0.75%/1.5% ruxolitinib cream maintained their regimen during the 44-week LTS period (as-needed treatment). Patients on vehicle were rerandomized (1:1) at week 8 to either ruxolitinib cream strength. Safety and disease control (Investigator's Global Assessment score 0/1 and affected body surface area) were assessed. RESULTS: Over 52 weeks, adverse events were reported in 67.4%/62.6%/53.5%/57.6% of patients in 0.75%/1.5% ruxolitinib cream/vehicle to 0.75% ruxolitinib cream/vehicle to 1.5% ruxolitinib cream groups (n = 426/446/101/99). Most common adverse events were upper respiratory tract infection (10.3%/11.4%/5.9%/7.1%) and nasopharyngitis (8.9%/9.9%/7.9%/14.1%). Most adverse events were considered unrelated to treatment. Application site reactions were infrequent (3.8%/1.8%/1.0%/1.0%). Disease control was achieved throughout the LTS; 74.1% to 77.8% of patients had Investigator's Global Assessment 0/1 at week 52, and mean affected body surface area was low (1.4%-1.8%). LIMITATIONS: LTS had no control treatment. CONCLUSION: During 44 weeks of as-needed treatment, ruxolitinib cream demonstrated effective disease control and tolerability; low ruxolitinib plasma concentrations alongside safety findings reflecting known risk factors suggest physiologically meaningful systemic Janus kinase inhibition is highly unlikely.

Rapid pruritus reduction with ruxolitinib cream treatment in patients with atopic dermatitis.

BACKGROUND: Ruxolitinib cream is a topical formulation of ruxolitinib, a Janus kinase (JAK) 1/JAK2 inhibitor. OBJECTIVES: To report timing and magnitude of effect of ruxolitinib cream on itch in patients with atopic dermatitis (AD), a highly pruritic inflammatory skin disease. METHODS: Two phase 3 trials (TRuE-AD1 [NCT03745638]/TRuE-AD2 [NCT03745651]) enrolled patients aged ≥12 years with AD for ≥2 years, Investigator's Global Assessment score of 2 or 3, and 3%-20% affected body surface area. Patients (total N = 1249; median age, 32 years) were randomised (2:2:1) to twice daily 0.75% ruxolitinib cream, 1.5% ruxolitinib cream or vehicle cream for 8 weeks of double-blinded treatment. Worst itch was measured using the numerical rating scale (NRS). RESULTS: Significantly more patients who applied ruxolitinib cream (either strength) achieved a ≥2-point itch reduction (NRS2) within approximately 12 h versus vehicle (0.75%/1.5% ruxolitinib cream, 16.3%/13.1%; vehicle, 6.9%; both P < 0.05), with further improvements through Week 8 (58.3%/65.1% vs 29.4%; both P < 0.0001). A ≥4-point itch reduction (NRS4) was achieved by significantly more patients who applied 0.75%/1.5% ruxolitinib cream versus vehicle by Day 2 (8.9%/11.2% vs 2.1%; P < 0.005); higher rates were observed at Week 8 (41.5%/51.5% vs 15.8%; P < 0.0001). Median time for the 0.75%/1.5% ruxolitinib cream groups to achieve NRS4 from baseline was 15.0/13.0 days; this endpoint was not reached by the vehicle group. CONCLUSIONS: Ruxolitinib cream demonstrated rapid improvement in itch in patients with mild to moderate AD that was sustained for 8 weeks. Significantly more patients applying ruxolitinib cream achieved itch NRS2 within approximately 12 h and itch NRS4 by Day 2 versus vehicle.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

Endocytosis and recycling of immune complexes by follicular dendritic cells enhances B cell antigen binding and activation.

Stromal-derived follicular dendritic cells (FDCs) are a major reservoir for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate. A long-standing question is how FDCs retain antigen in its native form for extended periods and how they display it to specific B cells. Here we found that FDCs acquired complement-coated immune complexes (ICs) from noncognate B cells via complement receptors 1 and 2 (CD35 and CD21, respectively) and rapidly internalized them by an actin-dependent pathway. ICs were retained intact within a nondegradative cycling compartment and were displayed periodically on the cell surface where they were accessible to antigen-specific B cells. This would explain how antigens are protected from damage and retained over long periods of time, while remaining accessible for B cells.

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Efficacy and safety of ruxolitinib cream for the treatment of atopic dermatitis: Results from 2 phase 3, randomized, double-blind studies.

BACKGROUND: Ruxolitinib (RUX) cream demonstrated potent anti-inflammatory and antipruritic efficacy in a phase 2 study in adults with atopic dermatitis (AD). OBJECTIVE: To evaluate 8-week efficacy and safety in 2 phase 3 studies of RUX cream in patients with AD. METHODS: Topical Ruxolitinib Evaluation in Atopic Dermatitis Study 1 (NCT03745638) and Study 2 (NCT03745651) enrolled patients aged ≥12 years with AD for ≥2 years, an Investigator's Global Assessment score of 2/3, and 3%-20% affected body surface area. Patients were randomized 2:2:1 to twice-daily 0.75% RUX cream, 1.5% RUX cream, or vehicle cream for 8 continuous weeks. The primary endpoint was Investigator's Global Assessment treatment success at week 8 (Investigator's Global Assessment score of 0/1 and ≥2-grade improvement from baseline). RESULTS: In the Topical Ruxolitinib Evaluation in Atopic Dermatitis Study 1 and 2, 631 and 618 patients were randomized (631/577 analyzed for efficacy). Significantly more patients achieved Investigator's Global Assessment treatment success with 0.75% RUX cream (50.0%/39.0%) and 1.5% RUX cream (53.8%/51.3%) versus vehicle (15.1%/7.6%; P < .0001) at week 8. Significant itch reductions versus vehicle were reported within 12 hours of first application of 1.5% RUX (P < .05). Application site reactions were infrequent (<1%) and lower with RUX versus vehicle; none were clinically significant. LIMITATIONS: Longer-term safety data are not yet available. CONCLUSIONS: RUX cream showed anti-inflammatory and prompt antipruritic effects with superior efficacy versus vehicle and was well tolerated.

Treatment of atopic dermatitis with ruxolitinib cream (JAK1/JAK2 inhibitor) or triamcinolone cream.

BACKGROUND: Atopic dermatitis (AD) is a highly pruritic chronic inflammatory skin disorder. Ruxolitinib, a selective inhibitor of Janus kinase 1 and Janus kinase 2, potently suppresses cytokine signaling involved in AD pathogenesis. OBJECTIVE: We sought to evaluate the efficacy and safety of ruxolitinib (RUX) cream in adults with AD. METHODS: In this phase 2 study (NCT03011892), 307 adult patients with AD, an Investigator's Global Assessment score of 2 or 3 (mild or moderate), and 3% to 20% affected body surface area were equally randomized for 8 weeks of double-blind treatment to RUX (1.5% twice daily [BID], 1.5% once daily [QD], 0.5% QD, 0.15% QD), vehicle, or triamcinolone cream (0.1% BID for 4 weeks, then vehicle for 4 weeks). Subsequently, patients could apply 1.5% RUX BID for 4 additional weeks of open-label treatment. The primary end point was the comparison between 1.5% RUX cream BID and vehicle in mean percentage change from baseline in Eczema Area and Severity Index at week 4. RESULTS: All RUX regimens demonstrated therapeutic benefit at week 4; 1.5% BID provided the greatest improvement in Eczema Area and Severity Index (71.6% vs 15.5%; P < .0001) and Investigator's Global Assessment responses (38.0% vs 7.7%; P < .001) versus vehicle. Rapid reductions in the itch numerical rating scale score occurred within 36 hours (1.5% BID vs vehicle, ‒1.8 vs ‒0.2; P < .0001) and were sustained through 12 weeks. Patients who transitioned to 1.5% RUX BID improved in all measures. RUX was not associated with clinically significant application-site reactions. CONCLUSIONS: RUX cream provided rapid and sustained improvements in AD symptoms and was well tolerated.

Effects of ruxolitinib cream on pruritus and quality of life in atopic dermatitis: Results from a phase 2, randomized, dose-ranging, vehicle- and active-controlled study.

BACKGROUND: Atopic dermatitis (AD), a chronic, highly pruritic skin disorder, impairs quality of life (QoL). Janus kinase inhibitors suppress inflammatory and pruritus-associated cytokine signaling in AD. OBJECTIVE: To report the effects of ruxolitinib (RUX) cream on itch and QoL in AD. METHODS: A total of 307 adult patients with an Investigator's Global Assessment (score of 2 or 3) and 3% to 20% affected body surface area were randomly assigned for 8 weeks to receive double-blind treatment with RUX (1.5% twice daily, 1.5% once daily, 0.5% once daily, or 0.15% once daily), vehicle twice daily, or triamcinolone cream (0.1% twice daily for 4 weeks then vehicle for 4 weeks). Itch was measured by using the numerical rating scale, and patient QoL was assessed with Skindex-16. RESULTS: Improvements in itch numerical rating scale and Skindex-16 were observed with RUX cream. Overall, 42.5% of patients who applied 1.5% RUX twice daily experienced minimal clinically important difference in itch within 36 hours of treatment (vehicle, 13.6%; P < .01); near-maximal improvement was observed by week 4. Itch reduction was associated with improved QoL burden (Pearson correlation, 0.67; P < .001). Significant improvements in Skindex-16 overall scores were noted at week 2. LIMITATIONS: Facial AD lesions were not treated. CONCLUSION: RUX cream provides a clinically meaningful reduction in itch and QoL burden.

Microglial cell hyper-ramification and neuronal dendritic spine loss in the hippocampus and medial prefrontal cortex in a mouse model of PTSD.

Few animal models exist that successfully reproduce several core associative and non-associative behaviours relevant to post-traumatic stress disorder (PTSD), such as long-lasting fear reactions, hyperarousal, and subtle attentional and cognitive dysfunction. As such, these models may lack the face validity required to adequately model pathophysiological features of PTSD such as CNS grey matter loss and neuroinflammation. Here we aimed to investigate in a mouse model of PTSD whether contextual fear conditioning associated with a relatively high intensity footshock exposure induces loss of neuronal dendritic spines in various corticolimbic brain regions, as their regression may help explain grey matter reductions in PTSD patients. Further, we aimed to observe whether these changes were accompanied by alterations in microglial cell number and morphology, and increased expression of complement factors implicated in the mediation of microglial cell-mediated engulfment of dendritic spines. Adult male C57Bl6J mice were exposed to a single electric footshock and subsequently underwent phenotyping of various PTSD-relevant behaviours in the short (day 2-4) and longer-term (day 29-31). 32 days post-exposure the brains of these animals were subjected to Golgi staining of dendritic spines, microglial cell Iba-1 immunohistochemistry and immunofluorescent staining of the complement factors C1q and C4. Shock exposure promoted a lasting contextual fear response, decreased locomotor activity, exaggerated acoustic startle responses indicative of hyperarousal, and a short-term facilitation of sensorimotor gating function. The shock triggered loss of dendritic spines on pyramidal neurons was accompanied by increased microglial cell number and complexity in the medial prefrontal cortex and dorsal hippocampus, but not in the amygdala. Shock also increased expression of C1q in the pyramidal layer of the CA1 region of the hippocampus but not in other brain regions. The present study further elaborates on the face and construct validity of a mouse model of PTSD and provides a good foundation to explore potential molecular interactions between microglia and dendritic spines.

Genetic deletion of P-glycoprotein alters stress responsivity and increases depression-like behavior, social withdrawal and microglial activation in the hippocampus of female mice.

P-glycoprotein (P-gp) is an ABC transporter expressed at the blood brain barrier and regulates the brain uptake of various xenobiotics and endogenous mediators including glucocorticoid hormones which are critically important to the stress response. Moreover, P-gp is expressed on microglia, the brain's immune cells, which are activated by stressors and have an emerging role in psychiatric disorders. We therefore hypothesised that germline P-gp deletion in mice might alter the behavioral and microglial response to stressors. Female P-gp knockout mice displayed an unusual, frantic anxiety response to intraperitoneal injection stress in the light-dark test. They also tended to display reduced conditioned fear responses compared to wild-type (WT) mice in a paradigm where a single electric foot-shock stressor was paired to a context. Foot-shock stress reduced social interaction and decreased microglia cell density in the amygdala which was not varied by P-gp genotype. Independently of stressor exposure, female P-gp deficient mice displayed increased depression-like behavior, idiosyncratic darting behavior, age-related social withdrawal and hyperactivity, facilitated sensorimotor gating and altered startle reactivity. In addition, P-gp deletion increased microglia cell density in the CA3 region of the hippocampus, and the microglial cells exhibited a reactive, hypo-ramified morphology. Further, female P-gp KO mice displayed increased glucocorticoid receptor (GR) expression in the hippocampus. In conclusion, this research shows that germline P-gp deletion affected various behaviors of relevance to psychiatric conditions, and that altered microglial cell activity and enhanced GR expression in the hippocampus may play a role in mediating these behaviors.

Efficacy and Safety of Naftifine HCl Cream 2% in the Treatment of Pediatric Subjects With Tinea Corporis.

BACKGROUND: Tinea corporis is fungal infection of body surfaces other than the feet, groin, scalp, or beard. Naftifine hydrochloride is a topical antifungal of the allylamine class used to treat tinea corporis, displaying fungicidal activity and clinically significant anti-bacterial and anti-inflammatory effects.
OBJECTIVE: To evaluate the efficacy and safety of two-weeks once daily application of naftifine cream 2% in the treatment of tinea corporis among pediatric subjects.
METHODS: At baseline, 231 subjects were randomly assigned 1:1 to naftifine cream 2% (n=116) and vehicle (n=115). Treatment effect consisting of mycologic determination (KOH and dermatophyte cultures) and scoring of clinical symptom severity was evaluated at baseline, week 2 (end of treatment) and week 3. Efficacy was analyzed in 181 subjects (n=88, naftifine; n=93, vehicle) with a positive baseline dermatophyte culture and KOH for whom week 3 assessments were available. Safety was evaluated by adverse events (AE) and laboratory values in 231 subjects (n=116, naftifine; n=115, vehicle).
RESULTS: Children with tinea corporis treated with naftifine cream 2% demonstrated significantly greater improvements from baseline over vehicle for mycological cure (P<0.0001) and treatment effectiveness (P=0.003) as early as 2 weeks (end of treatment). Response rates continued to increase post-treatment and were the highest 1-week after completion of the therapy (P=0.003 for complete cure; and P<0.001 for mycological cure and treatment effectiveness). Treatment related adverse events were minimal.
CONCLUSIONS: Treatment with naftifine cream 2% applied once daily for two weeks was well-tolerated and was effective in treating tinea corporis in children. Further improvement was observed 1-week after treatment completion for all key outcome measures (complete cure, mycological cure, treatment effectiveness, clinical cure, and clinical success) and clinical signs and symptoms (erythema, induration, and pruritus).

J Drugs Dermatol. 2016;15(6):743-748.

Efficacy, Safety, and Long-Term Disease Control of Ruxolitinib Cream Among Adolescents with Atopic Dermatitis: Pooled Results from Two Randomized Phase 3 Studies.

BACKGROUND: Atopic dermatitis (AD), a highly pruritic, inflammatory skin disease, affects approximately 7% of adolescents globally. A topical formulation of ruxolitinib, a Janus kinase (JAK) 1/JAK2 inhibitor, demonstrated safety and efficacy among adolescents/adults in two phase 3 studies (TRuE-AD1/TRuE-AD2). OBJECTIVE: To describe safety and efficacy of 1.5% ruxolitinib cream versus vehicle and long-term disease control of ruxolitinib cream among adolescents aged 12-17 years from pooled phase 3 study data. METHODS: Patients [≥ 12 years old with AD for ≥ 2 years, Investigator's Global Assessment score (IGA) 2/3, and 3-20% affected body surface area (BSA) at baseline] were randomized 2:2:1 to ruxolitinib cream (0.75%/1.5%) or vehicle for 8 weeks of continuous use followed by a long-term safety (LTS) period up to 52 weeks with as-needed use. Patients originally applying vehicle were rerandomized 1:1 to 0.75%/1.5% ruxolitinib cream. Efficacy measures at week 8 included IGA treatment success (IGA-TS; i.e., score of 0/1 with ≥ 2 grade improvement from baseline), ≥ 75% improvement in Eczema Area and Severity Index (EASI-75), and ≥ 4-point improvement in itch numerical rating scale (NRS4). Measures of disease control during the LTS period included IGA score of 0 (clear) or 1 (almost clear) and percentage affected BSA. Safety was assessed throughout the study. RESULTS: Of 1249 randomized patients, 245 (19.6%) were aged 12-17 years. Of these, 45 patients were randomized to vehicle and 92 patients to 1.5% ruxolitinib cream. A total of 104/137 (75.9%) patients continued on 1.5% ruxolitinib cream in the LTS period [82/92 (89.1%) continued on 1.5% ruxolitinib cream; 22/45 (48.9%) patients on vehicle were reassigned to 1.5% ruxolitinib cream], and 83/104 (79.8%) of these patients completed the LTS period. At week 8, substantially more patients who applied 1.5% ruxolitinib cream versus vehicle achieved IGA-TS (50.6% versus 14.0%), EASI-75 (60.9% versus 34.9%), and NRS4 (52.1% versus 17.4%; P = 0.009). The mean (SD) reduction in itch NRS scores was significantly greater in patients applying 1.5% ruxolitinib cream versus vehicle from day 2 [- 0.9 (1.9) versus -0.2 (1.4); P = 0.03]. During the LTS period, mean (SD) trough steady-state ruxolitinib plasma concentrations at weeks 12/52 were 27.2 (55.7)/15.5 (31.5) nM. The percentage of patients achieving IGA score of 0 or 1 was sustained or further increased with 1.5% ruxolitinib cream; mean affected BSA was generally low (< 3%; i.e., mild disease). Through 52 weeks, application site reactions occurred in 1.8% of adolescent patients applying 1.5% ruxolitinib cream at any time; no patients had serious adverse events. There were no serious infections, malignancies, major adverse cardiovascular events, or thromboembolic events. CONCLUSIONS: Meaningful anti-inflammatory and antipruritic effects were demonstrated with 1.5% ruxolitinib cream in the subset of adolescent patients with AD, comparable with those observed in the overall study population; long-term, as-needed use maintained disease control and was well tolerated. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifiers NCT03745638 (registered 19 November 2018) and NCT03745651 (registered 19 November 2018).

Complement-dependent transport of antigen into B cell follicles.

Since the original proposal by Fearon and Locksley (Fearon and Locksley. 1996. Science 272: 50-53) that the complement system linked innate and adaptive immunity, there has been a rapid expansion of studies on this topic. With the advance of intravital imaging, a number of recent papers revealed an additional novel pathway in which complement C3 and its receptors enhance humoral immunity through delivery of Ag to the B cell compartment. In this review, we discuss this pathway and highlight several novel exceptions recently found with a model influenza vaccine, such as mannose-binding lectin opsonization of influenza and uptake by macrophages, and the capture of virus by dendritic cells residing in the medullary compartment of peripheral lymph nodes.

Activation of innate and humoral immunity in the peripheral nervous system of ALS transgenic mice.

During injury to the nervous system, innate immune cells mediate phagocytosis of debris, cytokine production, and axon regeneration. In the neuro-degenerative disease amyotrophic lateral sclerosis (ALS), innate immune cells in the CNS are activated. However, the role of innate immunity in the peripheral nervous system (PNS) has not been well defined. In this study, we characterized robust activation of CD169/CD68/Iba1+ macrophages throughout the PNS in mutant SOD1(G93A) and SOD1(G37R) transgenic mouse models of ALS. Macrophage activation occurred pre-symptomatically, and expanded from focal arrays within nerve bundles to a tissue-wide distribution following symptom onset. We found a striking dichotomy for immune cells within the spinal cord and PNS. Flow cytometry and GFP bone marrow chimeras showed that spinal cord microglia were mainly tissue resident derived, dendritic-like cells, whereas in peripheral nerves, the majority of activated macrophages infiltrated from the circulation. Humoral antibodies and complement localized to PNS tissue in tandem with macrophage recruitment, and deficiency in complement C4 led to decreased macrophage activation. Therefore, cross-talk between nervous and immune systems occurs throughout the PNS during ALS disease progression. These data reveal a progressive innate and humoral immune response in peripheral nerves that is separate and distinct from spinal cord immune activation in ALS transgenic mice.

An Open-Source AI Framework for the Analysis of Single Cells in Whole-Slide Images with a Note on CD276 in Glioblastoma.

Routine examination of entire histological slides at cellular resolution poses a significant if not insurmountable challenge to human observers. However, high-resolution data such as the cellular distribution of proteins in tissues, e.g., those obtained following immunochemical staining, are highly desirable. Our present study extends the applicability of the PathoFusion framework to the cellular level. We illustrate our approach using the detection of CD276 immunoreactive cells in glioblastoma as an example. Following automatic identification by means of PathoFusion's bifocal convolutional neural network (BCNN) model, individual cells are automatically profiled and counted. Only discriminable cells selected through data filtering and thresholding were segmented for cell-level analysis. Subsequently, we converted the detection signals into the corresponding heatmaps visualizing the distribution of the detected cells in entire whole-slide images of adjacent H&E-stained sections using the Discrete Wavelet Transform (DWT). Our results demonstrate that PathoFusion is capable of autonomously detecting and counting individual immunochemically labelled cells with a high prediction performance of 0.992 AUC and 97.7% accuracy. The data can be used for whole-slide cross-modality analyses, e.g., relationships between immunochemical signals and anaplastic histological features. PathoFusion has the potential to be applied to additional problems that seek to correlate heterogeneous data streams and to serve as a clinically applicable, weakly supervised system for histological image analyses in (neuro)pathology.

Platelet recruitment to the inflamed glomerulus occurs via an alphaIIbbeta3/GPVI-dependent pathway.

Recruitment of leukocytes to glomeruli is fundamental to the pathogenesis of many forms of glomerulonephritis. In a model of glomerulonephritis induced by in situ immune complex deposition, we previously observed that, in addition to leukocytes, platelets accumulate in glomerular capillaries, where they contribute to leukocyte recruitment. However, the mechanisms of platelet recruitment and the role of platelet-expressed P-selectin in leukocyte recruitment require further investigation. We used intravital microscopy to examine the mechanisms of platelet and leukocyte recruitment to glomeruli of mice following administration of an antibody against the glomerular basement membrane (anti-GBM antibody). Platelet recruitment was initiated within five minutes of administration of anti-GBM antibody. This was unaltered by inhibition of platelet GPIbalpha but was prevented by the absence of platelet GPVI. Fibrinogen was deposited in glomerular capillaries via a partially intercellular adhesion molecule 1 (ICAM-1)-dependent mechanism, and inhibition of alpha(IIb)beta(3), fibrinogen and ICAM-1 inhibited platelet recruitment. Notably, neutrophil depletion also reduced platelet accumulation, indicating a cooperative interaction underlying recruitment of platelets and neutrophils. Finally, using bone marrow chimeras to restrict expression of P-selectin to platelets or endothelial cells, platelet but not endothelial P-selectin was required for glomerular leukocyte recruitment. Together these data indicate that platelet recruitment in this model is dependent on the combined actions of GPVI and the alpha(IIb)beta(3)/fibrinogen/ICAM-1 pathway and that platelet P-selectin is crucial for subsequent leukocyte recruitment.

Antimyeloperoxidase antibodies rapidly induce alpha-4-integrin-dependent glomerular neutrophil adhesion.

Patients with antineutrophil cytoplasmic antibodies (ANCAs) frequently develop severe vasculitis and glomerulonephritis. Although ANCAs, particularly antimyeloperoxidase (anti-MPO), have been shown to promote leukocyte adhesion in postcapillary venules, their ability to promote adhesion in the glomerular vasculature is less clear. We used intravital microscopy to examine glomerular leukocyte adhesion induced by anti-MPO. In mice pretreated with LPS, 50 microg anti-MPO induced LFA-1-dependent adhesion in glomeruli. In concert with this finding, in mice pretreated with LPS, more than 80% of circulating neutrophils bound anti-MPO within 5 minutes of intravenous administration. However, even in the absence of LPS, more than 40% of circulating neutrophils bound anti-MPO in vivo, a response not seen in MPO(-/-) mice. In addition, a higher dose of anti-MPO (200 microg) induced robust glomerular leukocyte adhesion in the absence of LPS. The latter response was beta2-integrin independent, instead requiring the alpha4-integrin, which was up-regulated on neutrophils in response to anti-MPO. These data indicate that anti-MPO antibodies bind to circulating neutrophils, and can induce glomerular leukocyte adhesion via multiple pathways. Lower doses induce adhesion only after an infection-related stimulus, whereas higher doses are capable of inducing responses in the absence of an additional inflammatory stimulus, via alternative adhesion mechanisms.

Pharmacokinetics of Ruxolitinib in Patients with Atopic Dermatitis Treated With Ruxolitinib Cream: Data from Phase II and III Studies.

BACKGROUND: Pathogenesis of atopic dermatitis (AD) involves the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. A cream formulation of ruxolitinib, a potent selective JAK1/JAK2 inhibitor, was developed for topical delivery. METHOD: Pharmacokinetic data were obtained from three double-blind, vehicle-controlled studies in patients with AD: a phase II study with ruxolitinib cream 0.15%, 0.5%, or 1.5% once daily or 1.5% twice daily (BID), and two phase III studies with 0.75% or 1.5% BID. Effects of baseline characteristics on pharmacokinetics were examined. Correlations were attempted between plasma concentrations and change in hematological parameters over time. RESULTS: Ruxolitinib plasma concentrations at steady-state (Css) increased with cream strength in a less-than-dose-proportional manner. In the phase III studies, overall mean (standard deviation [SD]) Css after ruxolitinib cream 0.75% and 1.5% BID (23.8 [35.0] and 35.7 [55.0] nM) were a fraction of the half-maximal inhibitory concentration for thrombopoietin-stimulated phosphorylated STAT3 inhibition (281 nM), a JAK/STAT signaling marker. Three covariates were identified for Css: dose, percent body surface area (%BSA) treated, and baseline Investigator's Global Assessment score. Mean (SD) bioavailability of ruxolitinib cream 1.5% BID was 6.22% (7.66%). There were no correlations between Css and any hematological changes except for a transient increase in platelets at week 2. CONCLUSIONS: Plasma ruxolitinib concentrations after treatment with topical ruxolitinib cream in patients with up to 20% BSA affected by AD are not expected to lead to systemic plasma concentrations that may be associated with adverse effects commonly associated with oral JAK inhibitors. CLINICALTRIALS.GOV: NCT03011892; NCT03745638; NCT03745651.