A genome-wide meta-analysis of palmoplantar pustulosis implicates Th2 responses and cigarette smoking in disease pathogenesis.

BACKGROUND: Palmoplantar pustulosis (PPP) is an inflammatory skin disorder that mostly affects smokers and manifests with painful pustular eruptions on the palms and soles. While the disease can present with concurrent plaque psoriasis, TNF and IL-17/IL-23 inhibitors show limited efficacy. There is therefore a pressing need to uncover PPP disease drivers and therapeutic targets. OBJECTIVES: To identify genetic determinants of PPP and investigate whether cigarette smoking contributes to disease pathogenesis. METHODS: We performed a genome-wide association meta-analysis of three North-European cohorts (n=1,456 PPP cases and 402,050 controls). We then used the scGWAS program to investigate the cell-type specificity of the association signals. We also undertook genetic correlation analyses to examine the similarities between PPP and other immune-mediated diseases. Finally, we applied Mendelian randomization to analyze the causal relationship between cigarette smoking and PPP. RESULTS: We found that PPP is not associated with the main genetic determinants of plaque psoriasis. Conversely, we identified genome-wide significant associations with the FCGR3A/FCGR3B and CCHCR1 loci. We also observed 13 suggestive (P<5X10-6) susceptibility regions, including the IL4/IL13 interval. Accordingly, we demonstrated a significant genetic correlation between PPP and Th2-mediated diseases like atopic dermatitis and ulcerative colitis. We also found that genes mapping to PPP-associated intervals were preferentially expressed in dendritic cells and often implicated in T-cell activation pathways. Finally, we undertook a Mendelian randomization analysis, which supported a causal role of cigarette smoking in PPP. CONCLUSIONS: The first genome-wide association study of PPP points to a pathogenic role for deregulated Th2 responses and cigarette smoking.

Single-cell analysis of psoriasis resolution demonstrates an inflammatory fibroblast state targeted by IL-23 blockade.

Biologic therapies targeting the IL-23/IL-17 axis have transformed the treatment of psoriasis. However, the early mechanisms of action of these drugs remain poorly understood. Here, we perform longitudinal single-cell RNA-sequencing in affected individuals receiving IL-23 inhibitor therapy. By profiling skin at baseline, day 3 and day 14 of treatment, we demonstrate that IL-23 blockade causes marked gene expression shifts, with fibroblast and myeloid populations displaying the most extensive changes at day 3. We also identify a transient WNT5A+/IL24+ fibroblast state, which is only detectable in lesional skin. In-silico and in-vitro studies indicate that signals stemming from these WNT5A+/IL24+ fibroblasts upregulate multiple inflammatory genes in keratinocytes. Importantly, the abundance of WNT5A+/IL24+ fibroblasts is significantly reduced after treatment. This observation is validated in-silico, by deconvolution of multiple transcriptomic datasets, and experimentally, by RNA in-situ hybridization. These findings demonstrate that the evolution of inflammatory fibroblast states is a key feature of resolving psoriasis skin.

The role of the interleukin-36 axis in generalized pustular psoriasis: a review of the mechanism of action of spesolimab.

Generalized pustular psoriasis (GPP) is a rare, chronic, inflammatory skin disorder characterized by recurrent flares associated with skin erythema, desquamation, and widespread superficial sterile pustules, which may be severe ("lakes of pus"). Systemic symptoms are often present, including malaise, fever, and skin pain. In GPP, innate immune responses are driven by abnormal activation of the interleukin (IL)-36-chemokine-neutrophil axis and excessive neutrophil infiltration. This review highlights the IL-36 pathway in the context of the IL-1 superfamily and describes how unopposed IL-36 signaling can lead to the development of GPP. Targeted inhibition of the IL-36 receptor (IL-36R) is an attractive therapeutic strategy in the treatment of GPP, including flare prevention and sustained disease control. Spesolimab is a first-in-class, humanized, monoclonal antibody that binds specifically to the IL-36R and antagonizes IL-36 signaling. Spesolimab was approved by the US Food and Drug Administration in September 2022 to treat GPP flares in adults and was subsequently approved for GPP flare treatment in other countries across the world. Anti-IL-36R therapy, such as spesolimab, can mitigate flares and address flare prevention in GPP, presumably through rebalancing IL-36 signaling and modulating the pro-inflammatory response of the downstream effectors.

Macrophage and neutrophil heterogeneity at single-cell spatial resolution in human inflammatory bowel disease.

Ulcerative colitis and Crohn's disease are chronic inflammatory intestinal diseases with perplexing heterogeneity in disease manifestation and response to treatment. While the molecular basis for this heterogeneity remains uncharacterized, single-cell technologies allow us to explore the transcriptional states within tissues at an unprecedented resolution which could further understanding of these complex diseases. Here, we apply single-cell RNA-sequencing to human inflamed intestine and show that the largest differences among patients are present within the myeloid compartment including macrophages and neutrophils. Using spatial transcriptomics in human tissue at single-cell resolution (CosMx Spatial Molecular Imaging) we spatially localize each of the macrophage and neutrophil subsets identified by single-cell RNA-sequencing and unravel further macrophage diversity based on their tissue localization. Finally, single-cell RNA-sequencing combined with single-cell spatial analysis reveals a strong communication network involving macrophages and inflammatory fibroblasts. Our data sheds light on the cellular complexity of these diseases and points towards the myeloid and stromal compartments as important cellular subsets for understanding patient-to-patient heterogeneity.

Clinical and Biomarker Responses to BI 655064, an Antagonistic Anti-CD40 Antibody, in Patients With Active Lupus Nephritis: A Randomized, Double-Blind, Placebo-Controlled, Phase II Trial.

OBJECTIVE: To characterize its dose-response relationship, BI 655064 (an anti-CD40 monoclonal antibody) was tested as an add-on to mycophenolate and glucocorticoids in patients with active lupus nephritis (LN). METHODS: A total of 121 patients were randomized (2:1:1:2) to receive placebo or BI 655064 120, 180, or 240 mg and received a weekly loading dose for 3 weeks followed by dosing every 2 weeks for the 120 and 180 mg groups, and 120 mg weekly for the 240 mg group. The primary endpoint was complete renal response (CRR) at week 52. Secondary endpoints included CRR at week 26. RESULTS: A dose-response relationship with CRR at week 52 was not shown (BI 655064 120 mg, 38.3%; 180 mg, 45.0%; 240 mg, 44.6%; placebo, 48.3%). At week 26, 28.6% (120 mg), 50.0% (180 mg), 35.0% (240 mg), and 37.5% (placebo) achieved CRR. The unexpected high placebo response prompted a post hoc analysis evaluating confirmed CRR (cCRR, at weeks 46 and 52). cCRR was achieved in 22.5% (120 mg), 44.3% (180 mg), 38.2% (240 mg), and 29.1% (placebo) of patients. Most patients reported ≥1 adverse event (BI 655064, 85.7-95.0%; placebo, 97.5%), most frequently infections and infestations (BI 655064 61.9-75.0%; placebo 60%). Compared with other groups, higher rates of serious (20% vs. 7.5-10%) and severe infections (10% vs. 4.8-5.0%) were reported with 240 mg BI 655064. CONCLUSION: The trial failed to demonstrate a dose-response relationship for the primary CRR endpoint. Post hoc analyses suggest a potential benefit of BI 655064 180 mg in patients with active LN.

Single-cell analysis implicates TH17-to-TH2 cell plasticity in the pathogenesis of palmoplantar pustulosis.

BACKGROUND: Palmoplantar pustulosis (PPP) is a severe inflammatory skin disorder characterized by eruptions of painful, neutrophil-filled pustules on the palms and soles. Although PPP has a profound effect on quality of life, it remains poorly understood and notoriously difficult to treat. OBJECTIVE: We sought to investigate the immune pathways that underlie the pathogenesis of PPP. METHODS: We applied bulk and single-cell RNA sequencing (RNA-Seq) methods to the analysis of skin biopsy samples and peripheral blood mononuclear cells. We validated our results by flow cytometry and immune fluorescence microscopy RESULTS: Bulk RNA-Seq of patient skin detected an unexpected signature of T-cell activation, with a significant overexpression of several TH2 genes typically upregulated in atopic dermatitis. To further explore these findings, we carried out single-cell RNA-Seq in peripheral blood mononuclear cells of healthy and affected individuals. Memory CD4+ T cells of PPP patients were skewed toward a TH17 phenotype, a phenomenon that was particularly significant among cutaneous lymphocyte-associated antigen-positive skin-homing cells. We also identified a subset of memory CD4+ T cells that expressed both TH17 (KLRB1/CD161) and TH2 (GATA3) markers, with pseudotime analysis suggesting that the population was the result of TH17 to TH2 plasticity. Interestingly, the GATA3+/CD161+ cells were overrepresented among the peripheral blood mononuclear cells of affected individuals, both in the single-cell RNA-Seq data set and in independent flow cytometry experiments. Dual-positive cells were also detected in patient skin by immune fluorescence microscopy. CONCLUSIONS: PPP is associated with complex T-cell activation patterns and may explain why biologic drugs that target individual T helper cell populations have shown limited therapeutic efficacy.

Pustular psoriasis: Molecular pathways and effects of spesolimab in generalized pustular psoriasis.

BACKGROUND: The IL-36 pathway plays a key role in the pathogenesis of generalized pustular psoriasis (GPP). In a proof-of-concept clinical trial, treatment with spesolimab, an anti-IL-36 receptor antibody, resulted in rapid skin and pustular clearance in patients presenting with GPP flares. OBJECTIVE: We sought to compare the molecular profiles of lesional and nonlesional skin from patients with GPP or palmoplantar pustulosis (PPP) with skin from healthy volunteers, and to investigate the molecular changes after spesolimab treatment in the skin and blood of patients with GPP flares. METHODS: Pre- and post-treatment skin and blood samples were collected from patients with GPP who participated in a single-arm, phase I study (n = 7). Skin biopsies from patients with PPP (n = 8) and healthy volunteers (n = 16) were obtained for comparison at baseline. Biomarkers were assessed by RNA-sequencing, histopathology, and immunohistochemistry. RESULTS: In GPP and PPP lesions, 1287 transcripts were commonly upregulated or downregulated. Selected transcripts from the IL-36 signaling pathway were upregulated in untreated GPP and PPP lesions. In patients with GPP, IL-36 pathway-related signatures, TH1/TH17 and innate inflammation signaling, neutrophilic mediators, and keratinocyte-driven inflammation pathways were downregulated by spesolimab as early as week 1. Spesolimab also decreased related serum biomarkers and cell populations in the skin lesions from patients with GPP, including CD3+ T, CD11c+, and IL-36γ+ cells and lipocalin-2-expressing cells. CONCLUSIONS: In patients with GPP, spesolimab showed rapid modulation of commonly dysregulated molecular pathways in GPP and PPP, which may be associated with improved clinical outcomes.

Safety, pharmacokinetics and pharmacodynamics of BI 705564, a highly selective, covalent inhibitor of Bruton's tyrosine kinase, in Phase I clinical trials in healthy volunteers.

AIMS: To evaluate the safety, pharmacokinetics and pharmacodynamics of single- and multiple-rising doses (MRDs) of BI 705564 and establish proof of mechanism. METHODS: BI 705564 was studied in 2 placebo-controlled, Phase I clinical trials testing single-rising doses (1-160 mg) and MRDs (1-80 mg) of BI 705564 over 14 days in healthy male volunteers. Blood samples were analysed for BI 705564 plasma concentration, Bruton's tyrosine kinase (BTK) target occupancy (TO) and CD69 expression in B cells stimulated ex vivo. A substudy was conducted in allergic, otherwise healthy, MRD participants. Safety was assessed in both studies. RESULTS: All doses of BI 705564 were well tolerated. Geometric mean BI 705564 plasma terminal half-life ranged from 10.1 to 16.9 hours across tested doses, with no relevant accumulation after multiple dosing. Doses ≥20 mg resulted in ≥85% average TO that was maintained for ≥48 hours after single-dose administration. Functional effects of BTK signalling were demonstrated by dose-dependent inhibition of CD69 expression. In allergic participants, BI 705564 treatment showed a trend in wheal size reduction in a skin prick test and complete inhibition of basophil activation. Mild bleeding-related adverse events were observed with BI 705564; bleeding time increased in 1/12 participants (8.3%) who received placebo vs 26/48 (54.2%) treated with BI 705564. CONCLUSION: BI 705564 showed efficient target engagement through durable TO and inhibition of ex vivo B-cell activation, and proof of mechanism through effects on allergic skin responses. Mild bleeding-related adverse events were probably related to inhibition of platelet aggregation by BTK inhibition.

Transcriptional insights into pathogenesis of cutaneous systemic sclerosis using pathway driven meta-analysis assisted by machine learning methods.

Pathophysiology of systemic sclerosis (SSc, Scleroderma), an autoimmune rheumatic disease, comprises of mechanisms that drive vasculopathy, inflammation and fibrosis. Understanding of the disease and associated clinical heterogeneity has advanced considerably in the past decade, highlighting the necessity of more specific targeted therapy. While many of the recent trials in SSc failed to meet the primary end points that predominantly relied on changes in modified Rodnan skin scores (MRSS), sub-group analysis, especially those focused on the basal skin transcriptomic data have provided insights into patient subsets that respond to therapies. These findings suggest that deeper understanding of the molecular changes in pathways is very important to define disease drivers in various patient subgroups. In view of these challenges, we performed meta-analysis on 9 public available SSc microarray studies using a novel pathway pivoted approach combining consensus clustering and machine learning assisted feature selection. Selected pathway modules were further explored through cluster specific topological network analysis in search of novel therapeutic concepts. In addition, we went beyond previously described SSc class divisions of 3 clusters (e.g. inflammation, fibro-proliferative, normal-like) and expanded into a much finer stratification in order to profile SSc patients more accurately. Our analysis unveiled an important 80 pathway signatures that differentiated SSc patients into 8 unique subtypes. The 5 pathway modules derived from such signature successfully defined the 8 SSc subsets and were validated by in-silico cellular deconvolution analysis. Myeloid cells and fibroblasts involvement in different clusters were confirmed and linked to corresponding pathway activities. Collectively, our findings revealed more complex disease subtypes in SSc; Key gene mediators such as IL6, FGFR1, TLR7, PLCG2, IRK2 identified by network analysis underscored the scientific rationale for exploring additional targets in treatment of SSc.

Dysregulation of club cell biology in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.

Phoenix from the flames: Rediscovering the role of the CD40-CD40L pathway in systemic lupus erythematosus and lupus nephritis.

Lupus nephritis (LN) is a significant complication of systemic lupus erythematosus (SLE), increasing its morbidity and mortality. Although the current standard of care helps suppress disease activity, it is associated with toxicity and ultimately does not cure SLE. At present, there are no therapies specifically indicated for the treatment of LN and there is an unmet need in this disease where treatment remains a challenge. The CD40-CD40L pathway is central to SLE pathogenesis and the generation of autoantibodies and their deposition in the kidneys, resulting in renal injury in patients with LN. CD40 is expressed on immune cells (including B cells, monocytes and dendritic cells) and also non-haematopoietic cells. Interactions between CD40L on T cells and CD40 on B cells in the renal interstitium are critical for the local expansion of naive B cells and autoantibody-producing B cells in LN. CD40L-mediated activation of myeloid cells and resident kidney cells, including endothelial cells, proximal tubular epithelial cells, podocytes and mesangial cells, further amplifies the inflammatory milieu in the interstitium and the glomeruli. Several studies have highlighted the upregulated expression of CD40 in LN kidney biopsies, and preclinical data have demonstrated the importance of the CD40-CD40L pathway in murine SLE and LN. Blocking this pathway is expected to ameliorate inflammation driven by infiltrating immune cells and resident kidney cells. Initial experimental therapeutic interventions targeting the CD40-CD40L pathway, based on CD40L antibodies, were associated with an increased incidence of thrombosis. However, this safety issue has not been observed with second-generation CD40/CD40L antibodies that have been engineered to prevent platelet activation. With these advancements, together with recent preclinical and clinical findings, it is anticipated that selective blockade of the CD40-CD40L pathway may address the unmet treatment needs in SLE, LN and other autoimmune diseases.

Loss-of-Function Myeloperoxidase Mutations Are Associated with Increased Neutrophil Counts and Pustular Skin Disease.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031-2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031-2A>C;c.2031-2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031-2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031-2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10-6 and p = 3.6 × 10-5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10-10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.

Cell-specific expression of lung disease risk-related genes in the human small airway epithelium.

BACKGROUND: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. METHODS: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. RESULTS: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. CONCLUSIONS: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Fecal MicroRNAs Show Promise as Noninvasive Crohn's Disease Biomarkers.

BACKGROUND: Short non-coding microRNAs (miRNAs) are involved in various cellular processes during disease progression of Crohn's disease (CD) and remarkably stable in feces, which make them attractive biomarker candidates for reflecting intestinal inflammatory processes. Here we investigated the potential of fecal miRNAs as noninvasive and translational CD biomarkers. METHODS: MiRNAs were screened in feces of 52 patients with CD and 15 healthy controls using RNA sequencing and the results were confirmed by PCR. The relationship between fecal miRNA levels and the clinical CD activity index (CDAI) or CD endoscopic index of severity (CDEIS) was explored, respectively. Additionally, fecal miRNAs were investigated in dextran sodium sulfate, adoptive T-cell transfer, and Helicobacter typhlonius/stress-induced murine colitis models using the NanoString platform. RESULTS: Nine miRNAs (miR-15a-5p, miR-16-5p, miR-128-3p, miR-142-5p, miR-24-3p, miR-27a-3p, miR-223-3p, miR-223-5p, and miR-3074-5p) were significantly (adj. P < 0.05, >3-fold) increased whereas 8 miRNAs (miR-10a-5p, miR-10b-5p, miR-141-3p, miR-192-5p, miR-200a-3p, miR-375, miR-378a-3p, and let-7g-5p) were significantly decreased in CD. MiR-192-5p, miR-375, and miR-141-3p correlated (P < 0.05) with both CDAI and CDEIS whereas miR-15a-5p correlated only with CDEIS. Deregulated expression of miR-223-3p, miR-16-5p, miR-15a-5p, miR-24-3p, and miR-200a-3p was also observed in murine models. The identified altered fecal miRNA levels reflect pathophysiological mechanisms in CD, such as Th1 and Th17 inflammation, autophagy, and fibrotic processes. CONCLUSIONS: Our translational study assessed global fecal miRNA changes of patients with CD and relevant preclinical models. These fecal miRNAs show promise as translational and clinically useful noninvasive biomarkers for mechanistic investigation of intestinal pathophysiology, including monitoring of disease progression.

Global skin gene expression analysis of early diffuse cutaneous systemic sclerosis shows a prominent innate and adaptive inflammatory profile.

OBJECTIVES: Determine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease. METHODS: Skin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic Sclerosis (PRESS) cohort (mean disease duration 1.3 years) and 33 matched healthy controls was examined by next-generation RNA sequencing. Data were analysed for cell type-specific signatures and compared with similarly obtained data from 55 previously biopsied patients in Genetics versus Environment in Scleroderma Outcomes Study cohort with longer disease duration (mean 7.4 years) and their matched controls. Correlations with histological features and clinical course were also evaluated. RESULTS: SSc patients in PRESS had a high prevalence of M2 (96%) and M1 (94%) macrophage and CD8 T cell (65%), CD4 T cell (60%) and B cell (69%) signatures. Immunohistochemical staining of immune cell markers correlated with the gene expression-based immune cell signatures. The prevalence of immune cell signatures in early diffuse SSc patients was higher than in patients with longer disease duration. In the multivariable model, adaptive immune cell signatures were significantly associated with shorter disease duration, while fibroblast and macrophage cell type signatures were associated with higher modified Rodnan Skin Score (mRSS). Immune cell signatures also correlated with skin thickness progression rate prior to biopsy, but did not predict subsequent mRSS progression. CONCLUSIONS: Skin in early diffuse SSc has prominent innate and adaptive immune cell signatures. As a prominently affected end organ, these signatures reflect the preceding rate of disease progression. These findings could have implications in understanding SSc pathogenesis and clinical trial design.

Characterization of an immortalized human small airway basal stem/progenitor cell line with airway region-specific differentiation capacity.

BACKGROUND: The pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways ≥ 6th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line. METHODS: Small airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF. RESULTS: The hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for > 200 doubling levels and > 70 passages, continuing to maintain basal cell features (TP63+, KRT5+). When cultured on ALI, hSABCi-NS1.1 cells consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1+), mucous (MUC5AC+, MUC5B+), neuroendocrine (CHGA+), ionocyte (FOXI1+) and surfactant protein positive cells (SFTPA+, SFTPB+, SFTPD+), observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that "cilium" and "immunity" were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2-1, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF. CONCLUSIONS: The immortalized hSABCi-NS1.1 cell line has diverse differentiation capacities and retains SAE features, which will be useful for understanding the biology of SAE, the pathogenesis of SAE-related diseases, and testing new pharmacologic agents.

Effects of BI 655064, an antagonistic anti-CD40 antibody, on clinical and biomarker variables in patients with active rheumatoid arthritis: a randomised, double-blind, placebo-controlled, phase IIa study.

OBJECTIVE: To evaluate the safety, efficacy and therapeutic mechanism of BI 655064, an antagonistic anti-CD40 monoclonal antibody, in patients with rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX-IR). METHODS: In total, 67 patients were randomised to receive weekly subcutaneous doses of 120 mg BI 655064 (n=44) or placebo (n=23) for 12 weeks. The primary endpoint was the proportion of patients who achieved 20% improvement in American College of Rheumatology criteria (ACR20) at week 12. Safety was assessed in patients who received at least one dose of study drug. RESULTS: At week 12, the primary endpoint was not met, with 68.2% of patients treated with BI 655064 achieving an ACR20 vs 45.5% with placebo (p=0.064); using Bayesian analysis, the posterior probability of seeing a difference greater than 35% was 42.9%. BI 655064 was associated with greater changes in CD40-CD40L pathway-related markers, including reductions in inflammatory and bone resorption markers (interleukin-6, matrix metalloproteinase-3, receptor activator of nuclear factor-κB ligand), concentration of autoantibodies (immunoglobulin [Ig]G rheumatoid factor [RF], IgM RF, IgA RF) and CD95+ activated B-cell subsets. No serious adverse events (AEs) related to BI 655064 treatment or thromboembolic events occurred; reported AEs were mainly of mild intensity. CONCLUSION: Although blockade of the CD40-CD40L pathway with BI 655064 in MTX-IR patients with RA resulted in marked changes in clinical and biological parameters, including reductions in activated B-cells, autoantibody production and inflammatory and bone resorption markers, with a favourable safety profile, clinical efficacy was not demonstrated in this small phase IIa study. TRIAL REGISTRATION NUMBER: NCT01751776.