[Novel immunomodulatory therapies in myasthenia gravis]. / Myasthénie grave : nouvelles thérapies immunosuppressives.

Myasthenia gravis (MG) is an autoimmune disease characterized by fluctuating weakness of skeletal muscles. Despite current treatments, a significant percentage of patients remain symptomatic. This review explores new immunosuppressive therapies and ongoing clinical trials in MG, including depletion of B lymphocytes with agents such as rituximab and inebilizumab, as well as the use of eculizumab, efgartigimod, satralizumab, tocilizumab, and CAR-T (Chimeric Antigen Receptor-T) cell therapy. These advancements aim to improve disease control and patients' quality of life.

La myasthénie grave (MG) est une maladie auto-immune caractérisée par une faiblesse fluctuante des muscles squelettiques. Malgré les traitements classiques, un pourcentage significatif de patients reste symptomatique. Cet article explore les nouvelles thérapies immunosuppressives et les essais cliniques en cours pour la MG, notamment la déplétion des lymphocytes B avec des agents tels que le rituximab et l'inébilizumab, ainsi que l'utilisation de l'éculizumab, de l'efgartigimod, du satralizumab, du tocilizumab et de la thérapie par cellules CAR-T (Chimeric Antigen Receptor-T). Ces avancées visent à améliorer le contrôle de la maladie et la qualité de vie des patients.

Ocrelizumab B cell depletion has no effect on HERV RNA expression in PBMC in MS patients.

BACKGROUND: Epstein barr virus (EBV) infection of B cells is now understood to be one of the triggering events for the development of Multiple Sclerosis (MS), a progressive immune-mediated disease of the central nervous system. EBV infection is also linked to expression of human endogenous retroviruses (HERVs) of the HERV-W group, a further risk factor for the development of MS. Ocrelizumab is a high-potency disease-modifying treatment (DMT) for MS, which depletes B cells by targeting CD20. OBJECTIVES: We studied the effects of ocrelizumab on gene expression in peripheral blood mononuclear cells (PBMC) from paired samples from 20 patients taken prior to and 6 months after beginning ocrelizumab therapy. We hypothesised that EBV and HERV-W loads would be lower in post-treatment samples. METHODS: Samples were collected in Paxgene tubes, subject to RNA extraction and Illumina paired end short read mRNA sequencing with mapping of sequence reads to the human genome using Salmon and differential gene expression compared with DeSeq2. Mapping was also performed separately to the HERV-D database of HERV sequences and the EBV reference sequence. RESULTS: Patient samples were more strongly clustered by individual rather than disease type (relapsing/remitting or primary progressive), treatment (pre and post), age, or sex. Fourteen genes, all clearly linked to B cell function were significantly down regulated in the post treatment samples. Interestingly only one pre-treatment sample had detectable EBV RNA and there were no significant differences in HERV expression (of any group) between pre- and post-treatment samples. CONCLUSIONS: While EBV and HERV expression are clearly linked to triggering MS pathogenesis, it does not appear that high level expression of these viruses is a part of the ongoing disease process or that changes in virus load are associated with ocrelizumab treatment.

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism.

Antibody responses, involving B cells, CD4 + T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.

A novel potent class I HDAC inhibitor reverses the STAT4/p66Shc apoptotic defect in B cells from chronic lymphocytic leukemia patients.

Chronic Lymphocytic Leukemia (CLL) patients have a defective expression of the proapoptotic protein p66Shc and of its transcriptional factor STAT4, which evoke molecular abnormalities, impairing apoptosis and worsening disease prognosis and severity. p66Shc expression is epigenetically controlled and transcriptionally modulated by STAT4; epigenetic modifiers are deregulated in CLL cells and specific histone deacetylases (HDACs) like HDAC1, are overexpressed. Reactivation of STAT4/p66Shc expression may represent an attractive and challenging strategy to reverse CLL apoptosis defects. New selective class I HDAC inhibitors (HDACis, 6a-g) were developed with increased potency over existing agents and preferentially interfering with the CLL-relevant isoform HDAC1, to unveil the role of class I HDACs in the upregulation of STAT4 expression, which upregulates p66Shc expression and hence normalizes CLL cell apoptosis. 6c (chlopynostat) was identified as a potent HDAC1i with a superior profile over entinostat. 6c induces marked apoptosis of CLL cells compared with SAHA, which was associated with an upregulation of STAT4/p66Shc protein expression. The role of HDAC1, but not HDAC3, in the epigenetic upregulation of STAT4/p66Shc was demonstrated for the first time in CLL cells and was validated in siRNA-induced HDAC1/HDAC3 knock-down EBV-B cells. To sum up, HDAC1 inhibition is necessary to reactivate STAT4/p66Shc expression in patients with CLL. 6c is one of the most potent HDAC1is known to date and represents a novel pharmacological tool for reversing the impairment of the STAT4/p66Shc apoptotic machinery.

Safety and biologic activity of a canine anti-CD20 monoclonal antibody in dogs with diffuse large B-cell lymphoma.

BACKGROUND: To explore the safety and utility of combining low dose single-agent doxorubicin with a canine specific anti-CD20 monoclonal antibody (1E4-cIgGB) in client owned dogs with untreated B-cell lymphoma. ANIMALS: Forty-two client-owned dogs with untreated B-cell lymphoma. METHODS: A prospective, single arm, open label clinical trial of dogs with B-cell lymphoma were enrolled to receive 1E4-cIgGB and doxorubicin in addition to 1 of 3 immunomodulatory regimens. B-cell depletion was monitored by flow cytometry performed on peripheral blood samples at each visit. RESULTS: Dogs demonstrated a statistically significant depletion in CD21+ B-cells 7 days following the first antibody infusion (median fraction of baseline at 7 days = 0.04, P < .01) that persisted throughout treatment (median fraction of baseline at 21 days = 0.01, P < .01) whereas CD5+ T-cells remained unchanged (median fraction of baseline at 7 days = 1.05, P = .88; median fraction of baselie at 7 days = 0.79, P = .42; Figure 1; Supplemental Table 3). Recovery of B-cells was delayed, with at Day 196, only 6/17 dogs (35%) remaining on the study had CD21+ counts >0.5 of baseline, indicating sustained B cell depletion at 4+ months after the final treatment. 1E4-cIgGB was well tolerated with only 1 dog exhibiting a hypersensitivity event within minutes of the last antibody infusion. CONCLUSIONS: The canine 1E4-cIgGB anti-CD20 monoclonal antibody is apparently safe when administered with doxorubicin and effectively depletes B-cells in dogs with DLBCL.

Inhibitors of Bruton's tyrosine kinase as emerging therapeutic strategy in autoimmune diseases.

Bruton's tyrosine kinase (BTK) is a cytoplasmic, non-receptor signal transducer, initially identified as an essential signaling molecule for B cells, with genetic mutations resulting in a disorder characterized by disturbed B cell and antibody development. Subsequent research revealed the critical role of BTK in the functionality of monocytes, macrophages and neutrophils. Various immune cells, among which B cells and neutrophils, rely on BTK activity for diverse signaling pathways downstream of multiple receptors, which makes this kinase an ideal target to treat hematological malignancies and autoimmune diseases. First-generation BTK inhibitors are already on the market to treat hematological disorders. It has been demonstrated that B cells and myeloid cells play a significant role in the pathogenesis of different autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren's syndrome. Consequently, second-generation BTK inhibitors are currently being developed to treat these disorders. Despite the acknowledged involvement of BTK in various cell types, the focus on B cells often overshadows its impact on innate immune cells. Among these cell types, neutrophils are often underestimated in the pathogenesis of autoimmune diseases. In this narrative review, the function of BTK in different immune cell subsets is discussed, after which an overview is provided of different upcoming BTK inhibitors tested for treatment of autoimmune diseases. Special attention is paid to BTK inhibition and its effect on neutrophil biology.

Long-Term Clinical Outcomes and B Cell Immune Reconstitution Following Allo-HCT With Prophylactic, Post-Transplant Rituximab.

Chronic graft-versus-host disease (cGVHD) remains a significant source of morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Post-transplant, prophylactic rituximab has successfully decreased cGHVD rates in clinical trials, but the durability of this strategy is uncertain. The long-terms effect of post-HCT B cell depletion on immune reconstitution, B cell function, and infectious complications are also unknown. In this study, we provide 10 yr follow-up and correlative analyses on patients given post-HCT, prophylactic rituximab. The objective of the study is to examine the durability of cGVHD protection as well as the long-term effect of rituximab prophylaxis on protective immune reconstitution, B cell function, and alloantibody formation. We analyzed 35 patients given prophylactic rituximab on phase II clinical trial. Clinical outcomes included cGVHD development, relapse and survival outcomes, and infectious outcomes. Correlative analyses included B cell subset analysis, development of antibodies to infectious antigens, and, for male patients receiving female donor grafts, development of antibodies to HY antigens. To further investigate the effect of rituximab on immune reconstitution and function, we also analyzed 43 similarly transplanted patients who did not receive post- or peri-HCT rituximab as a comparator group. For patients who received rituximab, the 8-yr cumulative incidence of cGHVD and freedom from immunosuppression were 20.0% and 76.2%, respectively. Importantly, no late incidences of cGVHD developed beyond 14 mo post-HCT. Relative to patients who did not receive rituximab, post-HCT rituximab was associated with increased B cell aplasia at 1 yr post-HCT (42.9% versus 11% of patients, P = .037); by 3 yr post-HCT, this aplasia resolved. Patients who received rituximab also had a significantly lower proportion of IgD+/CD38+ transitional B cells at 3 yr post-HCT (78.8% versus 89.9%, P = .039); at 10 yr post-HCT, this percentage remained markedly decreased at 50.7%. Rituximab prophylaxis altered B cell function. In male patients receiving female donor grafts, fewer patients developed HY antibodies at 3 yr post-HCT (20% versus 78%, P = .04). At 10 yr post-HCT, HY antibody production remained decreased at 33%. Rituximab prophylaxis was also associated with significantly lower antibody response to tetanus and EBV infectious antigens as well as lower IgG levels. Despite these changes, post-HCT was not associated with increased infections, although patients who received rituximab required intravenous immunoglobulin (IVIG) supplementation more frequently than those who did not (62.9% versus 32.6% of patients, P = .01). Prior data on the efficacy and feasibility of rituximab prophylaxis are durable, with persistent reduction in cGVHD. Rituximab prophylaxis also results in lasting B cell immunologic changes, with altered B cell subset composition and decreased alloantibody formation. Associated infectious risks were not increased, perhaps mitigated by high IVIG use.

Tertiary Lymphoid Structure-Associated B Cells Enhance CXCL13+CD103+CD8+ Tissue-Resident Memory T-Cell Response to Programmed Cell Death Protein 1 Blockade in Cancer Immunotherapy.

BACKGROUND & AIMS: Although the presence of tertiary lymphoid structures (TLS) correlates with positive responses to immunotherapy in many solid malignancies, the mechanism by which TLS enhances antitumor immunity is not well understood. The present study aimed to investigate the underlying cross talk circuits between B cells and tissue-resident memory T (Trm) cells within the TLS and to understand their role in the context of immunotherapy. METHODS: Immunostaining and H&E staining of TLS and chemokine (C-X-C motif) ligand 13 (CXCL13)+ cluster of differentiation (CD)103+CD8+ Trm cells were performed on tumor sections from patients with gastric cancer (GC). The mechanism of communication between B cells and CXCL13+CD103+CD8+ Trm cells was determined in vitro and in vivo. The effect of CXCL13+CD103+CD8+ Trm cells in suppressing tumor growth was evaluated through anti-programmed cell death protein (PD)-1 therapy. RESULTS: The presence of TLS and CXCL13+CD103+CD8+ Trm cells in tumor tissues favored a superior response to anti-PD-1 therapy in patients with GC. Additionally, our research identified that activated B cells enhanced CXCL13 and granzyme B secretion by CD103+CD8+ Trm cells. Mechanistically, B cells facilitated the glycolysis of CD103+CD8+ Trm cells through the lymphotoxin-α/tumor necrosis factor receptor 2 (TNFR2) axis, and the mechanistic target of rapamycin signaling pathway played a critical role in CD103+CD8+ Trm cells glycolysis during this process. Moreover, the presence of TLS and CXCL13+CD103+CD8+ Trm cells correlated with potent responsiveness to anti-PD-1 therapy in a TNFR2-dependent manner. CONCLUSIONS: This study further reveals a crucial role for cellular communication between TLS-associated B cell and CXCL13+CD103+CD8+ Trm cells in antitumor immunity, providing valuable insights into the potential use of the lymphotoxin-α/TNFR2 axis within CXCL13+CD103+CD8+ Trm cells for advancing immunotherapy strategies in GC.

Promising therapies for the treatment of myasthenia gravis.

INTRODUCTION: Myasthenia gravis (MG) is an autoimmune condition targeting the neuromuscular junction, which manifests with neuromuscular symptoms of varying severity and significant morbidity. The mainstay of treatment in MG is mitigation of the immune cascade with steroids and non-steroidal immunosuppressive therapies. The therapeutic strategies in MG are transitioning from broad and indiscriminate immunosuppression to novel agents targeting key steps in MG pathogenesis, including T cell activation, B cell proliferation, complement activation, maintenance of pathogenic antibody production, and proinflammatory cytokine production. AREAS COVERED: In this review, an overview of the pathogenesis of MG and traditional MG therapies is presented, followed by a discussion of the novel MG drugs that have been evaluated in phase 3 clinical trials with an emphasis on those which have received regulatory approval. EXPERT OPINION: Novel MG therapeutics belonging to the classes of complement inhibitors, neonatal Fc receptor (FcRn) inhibitors and B cell depletors, as well as the other emerging MG drugs in the pipeline constitute promising treatment strategies with potentially better efficacy and safety compared to the conventional MG treatments. However, further long-term research is needed in order to optimize the implementation of these new treatment options for the appropriate patient populations.

Momordicine-I Suppresses Head and Neck Cancer Growth by Reprogrammimg Immunosuppressive Effect of the Tumor-Infiltrating Macrophages and B Lymphocytes.

Head and neck cancer (HNC) is prevalent worldwide, and treatment options are limited. Momordicine-I (M-I), a natural component from bitter melon, shows antitumor activity against these cancers, but its mechanism of action, especially in the tumor microenvironment (TME), remains unclear. In this study, we establish that M-I reduces HNC tumor growth in two different immunocompetent mouse models using MOC2 and SCC VII cells. We demonstrate that the anticancer activity results from modulating several molecules in the monocyte/macrophage clusters in CD45+ populations in MOC2 tumors by single-cell RNA sequencing. Tumor-associated macrophages (TAM) often pose a barrier to antitumor effects, but following M-I treatment, we observe a significant reduction in the expression of Sfln4, a myeloid cell differentiation factor, and Cxcl3, a neutrophil chemoattractant, in the monocyte/macrophage populations. We further find that the macrophages must be in close contact with the tumor cells to inhibit Sfln4 and Cxcl3, suggesting that these TAMs are impacted by M-I treatment. Coculturing macrophages with tumor cells shows inhibition of Agr1 expression following M-I treatment, which is indicative of switching from M2 to M1 phenotype. Furthermore, the total B-cell population in M-I-treated tumors is significantly lower, whereas spleen cells also show similar results when cocultured with MOC2 cells. M-I treatment also inhibits PD1, PD-L1, and FoxP3 expression in tumors. Collectively, these results uncover the potential mechanism of M-I by modulating immune cells, and this new insight can help to develop M-I as a promising candidate to treat HNCs, either alone or as adjuvant therapy.

A randomized, non-comparative phase 2 study of neoadjuvant immune-checkpoint blockade in retroperitoneal dedifferentiated liposarcoma and extremity/truncal undifferentiated pleomorphic sarcoma.

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS.

High-Dose ERT, Rituximab, and Early HSCT in an Infant with Wolman's Disease.

Wolman's disease, a severe form of lysosomal acid lipase deficiency, leads to pathologic lipid accumulation in the liver and gut that, without treatment, is fatal in infancy. Although continued enzyme-replacement therapy (ERT) in combination with dietary fat restriction prolongs life, its therapeutic effect may wane over time. Allogeneic hematopoietic stem-cell transplantation (HSCT) offers a more definitive solution but carries a high risk of death. Here we describe an infant with Wolman's disease who received high-dose ERT, together with dietary fat restriction and rituximab-based B-cell depletion, as a bridge to early HSCT. At 32 months, the infant was independent of ERT and disease-free, with 100% donor chimerism in the peripheral blood.

Effect of Plasmapheresis on the Efficacy of Rituximab in Antibody-Mediated Rejection Patients.

BACKGROUND: Rituximab and plasmapheresis (PP) suppress and eliminate antibody production in patients experiencing antibody-mediated rejection (AMR). Herein, we discuss a case where rituximab was less effective after PP for treating AMR. CASE: A 55-year-old male patient underwent kidney transplantation. His renal function remained normal for 1 year. Subsequently, renal function declined, and (donor-specific antibodies showed positive results. A biopsy of the transplanted kidney revealed AMR. On the day of the biopsy, the medical staff administered 200 mg of rituximab, followed by IV immunoglobulin (IVIg) and PP the next day. The time interval between PP + IVIg treatment and rituximab was 12 h. As a result, the B-cell markers CD19 and CD20 did not decrease sufficiently, and the patient's creatinine and glomerular filtration rate muscles did not recover adequately. CONCLUSION: We report a case in which PP was administered shortly after rituximab injection, resulting in insufficient B-cell inhibition due to the removal of rituximab.

CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment.

Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here, we probe the decline of B cells in human CTLA-4 knock-in mice by using anti-human CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.

EZH2 inhibition stimulates repetitive element expression and viral mimicry in resting splenic B cells.

Mammalian cells repress expression of repetitive genomic sequences by forming heterochromatin. However, the consequences of ectopic repeat expression remain unclear. Here we demonstrate that inhibitors of EZH2, the catalytic subunit of the Polycomb repressive complex 2 (PRC2), stimulate repeat misexpression and cell death in resting splenic B cells. B cells are uniquely sensitive to these agents because they exhibit high levels of histone H3 lysine 27 trimethylation (H3K27me3) and correspondingly low DNA methylation at repeat elements. We generated a pattern recognition receptor loss-of-function mouse model, called RIC, with mutations in Rigi (encoding for RIG-I), Ifih1 (MDA5), and Cgas. In both wildtype and RIC mutant B cells, EZH2 inhibition caused loss of H3K27me3 at repetitive elements and upregulated their expression. However, NF-κB-dependent expression of inflammatory chemokines and subsequent cell death was suppressed by the RIC mutations. We further show that inhibition of EZH2 in cancer cells requires the same pattern recognition receptors to activate an interferon response. Together, the results reveal chemokine expression induced by EZH2 inhibitors in B cells as a novel inflammatory response to genomic repeat expression. Given the overlap of genes induced by EZH2 inhibitors and Epstein-Barr virus infection, this response can be described as a form of viral mimicry.

Thallium exposure induces changes in B and T cell generation in mice.

Thallium (Tl) is a high-priority toxic metal that poses a severe threat to human health. The toxicity characteristics induced by Tl have been partially discussed. However, the immunotoxic effects of Tl exposure have remained largely unexplored. Our findings demonstrated that 50 ppm of Tl exposure for one week induced severe weight loss in mice, which was accompanied by appetite suppression. Moreover, although Tl exposure did not induce significant pathological damage to skeletal muscle and bone, Tl inhibited the expression of B cell development-related genes in the bone marrow. Additionally, Tl exposure increased B cell apoptosis and reduced its generation in the bone marrow. Analysis of B cells in the blood indicated that the percentage of B-2 cells decreased significantly, whereas B-2 cell proportions in the spleen did not. The percentage of CD4+ T cells in the thymus increased significantly, and the proportion of CD8+ T cells did not. Furthermore, although the proportion of the total CD4+ and CD8+ T cells was not significantly altered in the blood and spleen, Tl exposure promoted the migration of naïve CD4+ T cells and recent thymic emigrants (RTEs) from the thymus to the spleen. These results suggest that Tl exposure can affect B and T cell generation and migration, which provides new evidence for Tl-induced immunotoxicity.

Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Baicalein alleviates fibrosis and inflammation in systemic sclerosis by regulating B-cell abnormalities.

BACKGROUND: Systemic sclerosis (SSc; also known as "scleroderma") is an autoimmune disorder characterized by extensive fibrosis, vascular changes, and immunologic dysregulation. Baicalein (phenolic flavonoid derived from Scutellaria baicalensis Georgi) has been used to treat the pathological processes of various fibrotic and inflammatory diseases. In this study, we investigated the effect of baicalein on the major pathologic characteristics of SSc: fibrosis, B-cell abnormalities, and inflammation. METHODS: The effect of baicalein on collagen accumulation and expression of fibrogenic markers in human dermal fibroblasts were analyzed. SSc mice were produced by injecting bleomycin and treated with baicalein (25, 50, or 100 mg/kg). The antifibrotic features of baicalein and its mechanisms were investigated by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay, western blotting and flow cytometry. RESULTS: Baicalein (5-120 µM) significantly inhibited the accumulation of the extracellular matrix and fibroblast activation in transforming growth factor (TGF)-ß1- and platelet derived growth factor (PDGF)-induced human dermal fibroblasts, as evidenced by abrogated deposition of total collagen, decreased secretion of soluble collagen, reduced collagen contraction capability and downregulation of various fibrogenesis molecules. In a bleomycin-induced model of dermal fibrosis in mice, baicalein (25-100 mg/kg) restored dermal architecture, ameliorated inflammatory infiltrates, and attenuated dermal thickness and collagen accumulation in a dose-dependent manner. According to flow cytometry, baicalein reduced the proportion of B cells (B220+ lymphocytes) and increased the proportion of memory B cells (B220+CD27+ lymphocytes) in the spleens of bleomycin-induced mice. Baicalein treatment potently attenuated serum levels of cytokines (interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-17A, tumor necrosis factor-α), chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1 beta) and autoantibodies (anti-scleroderma 70 (Scl-70), anti-polymyositis-scleroderma (PM-Scl), anti-centromeres, anti-double stranded DNA (dsDNA). In addition, baicalein treatment can significantly inhibit the activation of TGF-ß1 signaling in dermal fibroblasts and bleomycin-induce mice of SSc, evidenced by reducing the expression of TGF-ß1 and IL-11, as well as inhibiting both small mother against decapentaplegic homolog 3 (SMAD3) and extracellular signal-related kinase (ERK) activation. CONCLUSIONS: These findings suggest that baicalein has therapeutic potential against SSc, exerting modulating B-cell abnormalities, anti-inflammatory effects, and antifibrosis.

Role of the Bruton tyrosine kinase pathway in multiple sclerosis.

Multiple sclerosis (MS) is a chronic, immune-mediated, neurodegenerative condition that results in progressive accumulation of disability over the course of the disease. MS presents heterogeneously, and, as the disease progresses, patients develop a range of physical and neurologic problems that include reduced mobility, cognitive impairment, weakness, fatigue, pain, and defects in speech or vision. Economically, MS is costly, including both direct costs stemming from clinical care and medications and the indirect costs of productivity losses. These costs pose a substantial burden to patients, families, caregivers, employers, and society. There are 21 approved disease-modifying therapies for MS across several drug classes. The importance of early MS treatment has been confirmed, and progress has been made in the treatment of relapsing-remitting MS, although this progress has not been replicated for progressive presentations of the disease. Ongoing research continues to elucidate the exact mechanisms of disease in MS as well as potential new treatment strategies that may better address current gaps, such as disability progression in secondary progressive MS without activity. One of the novel pathways under investigation is the inhibition of Bruton tyrosine kinase, a cytoplasmic tyrosine kinase, which is expressed in B cells and other potentially targetable hematopoietic lineage cells. This review examines emerging hypotheses that targeting both B cells and myeloid cells within the periphery and central nervous system could yield clinical effects in key areas of MS pathophysiology that are currently unaddressed.

Modeling the B-cell receptor signaling on single cell level reveals a stable network circuit topology between nonmalignant B cells and chronic lymphocytic leukemia cells and between untreated cells and cells treated with kinase inhibitors.

B-cell receptor (BCR) signaling is central for the pathomechanism of chronic lymphocytic leukemia (CLL), and inhibitors of BCR signaling have substantially improved treatment options. To model malignant and nonmalignant BCR signaling, we quantified five components of BCR signaling (ZAP70/SYK, BTK, PLCγ2, AKT, ERK1/2) in single cells from primary human leukemic cells and from nonmalignant tissue. We measured signaling activity in a time-resolved manner after stimulation with BCR crosslinking by anti-IgM and/or anti-CD19 and with or without inhibition of phosphatases with H2 O2 . The phosphorylation of BCR signaling components was increased in malignant cells compared to nonmalignant cells and in IGHV unmutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2 O2 led to higher phosphorylation levels of BCR components in CLL cells with mutated IGHV compared to unmutated IGHV. We modeled the connectivity of the cascade components by correlating signal intensities across single cells. The network topology remained stable between malignant and nonmalignant cells. To additionally test for the impact of therapeutic compounds on the network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib), LYN (dasatinib) and SYK (entospletinib). Idelalisib treatment resulted in similar effects in malignant and nonmalignant cells, whereas ibrutinib was mostly active on CLL cells. Idelalisib and ibrutinib had complementary effects on the BCR signaling cascade whose activity was further reduced upon dasatinib and entospletinib treatment. The characterization of the molecular circuitry of leukemic BCR signaling will allow a more refined targeting of this Achilles heel.