Tyrosine-protein kinase SYK, encoded by the SYK gene, is a non-receptor type protein kinase which mediates immune signal transduction through immunoreceptors. Tyrosine-protein kinase SYK expression has been associated with the development of various inflammatory diseases, cancer and neurodegenerative conditions. The reproducibility of tyrosine-protein kinase SYK research would help elucidate the mechanism in which it causes neuroinflammation as well as its potential as a novel target to treat Alzheimer\'s disease. This would be facilitated with the availability of high-quality tyrosine-protein kinase SYK. In this study, we characterized thirteen tyrosine-protein kinase SYK commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal diseases. Accordingly, glutamate excitotoxicity has been used as a model to study neuronal diseases. Immune proteins, such as major histocompatibility complex (MHC) class I molecules and their receptors, play important roles in many neuronal diseases, while T-cell receptors (TCR) are the primary receptors of MHCI. We previously showed that a critical component of TCR, CD3ζ, is expressed by mouse retinal ganglion cells (RGCs). The mutation of CD3ζ or MHCI molecules compromises the development of RGC structure and function. In this study, we investigated whether CD3ζ-mediated molecular signaling regulates RGC death in glutamate excitotoxicity. We show that mutation of CD3ζ significantly increased RGC survival in NMDA-induced excitotoxicity. In addition, we found that several downstream molecules of TCR, including Src (proto-oncogene tyrosine-protein kinase) family kinases (SFKs) and spleen tyrosine kinase (Syk), are expressed by RGCs. Selective inhibition of an SFK member, Hck, or Syk members, Syk or Zap70, significantly increased RGC survival in NMDA-induced excitotoxicity. These results provide direct evidence to reveal the underlying molecular mechanisms that control RGC death under disease conditions.

Developing a reliable method to predict thrombocytopenia is imperative in drug discovery. Here, we establish an assay using a microphysiological system (MPS) to recapitulate the in-vivo mechanisms of platelet aggregation and adhesion. This assay highlights the role of shear stress on platelet aggregation and their interactions with vascular endothelial cells. Platelet aggregation induced by soluble collagen was detected under agitated, but not static, conditions using a plate shaker and gravity-driven flow using MPS. Notably, aggregates adhered on vascular endothelial cells under gravity-driven flow in the MPS, and this incident increased in a concentration-dependent manner. Upon comparing the soluble collagen-induced aggregation activity in platelet-rich plasma (PRP) and whole blood, remarkable platelet aggregate formation was observed at concentrations of 30 µg/mL and 3 µg/mL in PRP and whole blood, respectively. Moreover, ODN2395, an oligonucleotide, induced platelet aggregation and adhesion to vascular endothelial cells. SYK inhibition, which mediated thrombogenic activity via glycoprotein VI on platelets, ameliorated platelet aggregation in the system, demonstrating that the mechanism of platelet aggregation was induced by soluble collagen and oligonucleotide. Our evaluation system partially recapitulated the aggregation mechanisms in blood vessels and can contribute to the discovery of safe drugs to mitigate the risk of thrombocytopenia.

Diabetes accelerates vascular senescence, which is the basis for atherosclerosis and stiffness. The activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and oxidative stress are closely associated with progressive senescence in vascular smooth muscle cells (VSMCs). The vascular protective effect of FGF21 has gradually gained increasing attention, but its role in diabetes-induced vascular senescence needs further investigation. In this study, diabetic mice and primary VSMCs are transfected with an FGF21 activation plasmid and treated with a peroxisome proliferator-activated receptor γ (PPARγ) agonist (rosiglitazone), an NLRP3 inhibitor (MCC950), and a spleen tyrosine kinase (SYK)-specific inhibitor, R406, to detect senescence-associated markers. We find that FGF21 overexpression significantly restores the level of catalase (CAT), vascular relaxation, inhibits the intensity of ROSgreen fluorescence and p21 immunofluorescence, and reduces the area of SA-β-gal staining and collagen deposition in the aortas of diabetic mice. FGF21 overexpression restores CAT, inhibits the expression of p21, and limits the area of SA-β-gal staining in VSMCs under high glucose conditions. Mechanistically, FGF21 inhibits SYK phosphorylation, the production of the NLRP3 dimer, the expression of NLRP3, and the colocalization of NLRP3 with PYCARD (ASC), as well as NLRP3 with caspase-1, to reverse the cleavage of PPARγ, preserve CAT levels, suppress ROSgreen density, and reduce the expression of p21 in VSMCs under high glucose conditions. Our results suggest that FGF21 alleviates vascular senescence by regulating the SYK-NLRP3 inflammasome-PPARγ-catalase pathway in diabetic mice.

UNASSIGNED: Psoriasis is a chronic inflammatory skin disease that has no cure. While the specific cause of psoriasis is unknown, interactions between immune cells and inflammatory cytokines are believed to be important in its pathogenesis. Thymic stromal lymphopoietin (TSLP) is a cytokine produced by epithelial cells that profoundly affects dendritic cells (DCs) and is involved in allergy and inflammatory diseases. In some studies, its expression is higher in the skin of psoriasis patients, whereas it is increased in treated psoriasis patients when compared with untreated patients in others.
UNASSIGNED: To investigate the role of TSLP in the pathogenesis of psoriasis.
UNASSIGNED: In vitro and in vivo study.
UNASSIGNED: To investigate the effect of TSLP on psoriasis in vivo, a mouse psoriasis model and shRNA targeting TSLP to reduce its expression were used. Mouse primary bone marrow dendritic cells (BMDCs) were cultured in vitro and used to investigate the signaling pathways activated by TSLP. Results: We found that reducing TSLP expression in psoriasis skin alleviated disease severity. TSLP activated the Janus kinase (JAK)/SYK pathway in psoriatic skin. In vitro studies with BMDCs demonstrated that TSLP increased DC maturation through the JAK/SYK pathway and activated DCs-secreted cytokines that stimulated CD4+ T cells to develop into T helper 17 (Th17) cells by activating STAT3 signaling. The JAK/SYK pathway inhibitor reduced the effect of TSLP on activating BMDCs and promoting Th17 differentiation by CD4+ T cells.
UNASSIGNED: These findings indicated that TSLP exerted its immune-modulating effect in psoriasis through the JAK/SYK pathway.

OBJECTIVE: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of β-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear.
METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with β-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting.
RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to β-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1β, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides β-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB.
CONCLUSIONS: Oral epithelial cells express Dectin-1 and recognize β-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

Enhanced DNA repair is an important mechanism of inherent and acquired resistance to DNA targeted therapies, including poly ADP ribose polymerase (PARP) inhibition. Spleen associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase acknowledged for its regulatory roles in immune cell function, cell adhesion, and vascular development. This study presents evidence indicating that Syk expression in high-grade serous ovarian cancer and triple-negative breast cancers promotes DNA double-strand break resection, homologous recombination (HR), and subsequent therapeutic resistance. Our investigations reveal that Syk is activated by ATM following DNA damage and is recruited to DNA double-strand breaks by NBS1. Once localized to the break site, Syk phosphorylates CtIP, a pivotal mediator of resection and HR, at Thr-847 to promote repair activity, particularly in Syk-expressing cancer cells. Inhibition of Syk or its genetic deletion impedes CtIP Thr-847 phosphorylation and overcomes the resistant phenotype. Collectively, our findings suggest a model wherein Syk fosters therapeutic resistance by promoting DNA resection and HR through a hitherto uncharacterized ATM-Syk-CtIP pathway. Moreover, Syk emerges as a promising tumor-specific target to sensitize Syk-expressing tumors to PARP inhibitors, radiation and other DNA-targeted therapies.

BACKGROUND: SIRPB1 expression is upregulated in various tumor types, including gliomas, and is known to contribute to tumor progression; nevertheless, its function in the immune milieu of gliomas is still mainly unknown.
METHODS: This study, we analyzed 1152 normal samples from the GTEx database and 670 glioma samples from the TCGA database to investigate the relationship between the expression of SIRPB1 and clinicopathological features. Moreover, SIRPB1 gene knockout THP-1 cell lines were constructed using CRISPR/Cas9 and were induced into a co-culture of macrophages and glioma cells in vitro to learn more about the role of SIRPB1 in the glioma immune milieu. Lastly, we established a prognostic model to predict the effect of SIRPB1 on prognosis.
RESULTS: Significantly higher levels of SIRPB1 expression were found in gliomas, which had an adverse effect on the immune milieu and correlated poorly with patient survival. SIRPB1 activation with certain antibodies results in SYK phosphorylation and the subsequent activation of calcium, MAPK, and NF-κB signaling pathways. This phenomenon is primarily observed in myeloid-derived cells as opposed to glioma cells. In vitro co-culture demonstrated that macrophages with SIRPB1 knockout showed decreased IL1RA, CCL2, and IL-8, which were recovered upon ectopic expression of SIRPB1 but reduced again following treatment with SYK inhibitor GS9973. Critically, a lower overall survival rate was linked to increased SIRPB1 expression. Making use of SIRPB1 expression along with additional clinicopathological variables, we established a nomogram that showed a high degree of prediction accuracy.
CONCLUSIONS: Our study demonstrates that glioma cells can be activated by macrophages via SIRPB1, subsequently reprogramming the TME, suggesting that SIRPB1 could serve as a promising therapeutic target for gliomas.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer\'s disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Sovleplenib (HMPL-523) is a selective spleen tyrosine kinase (Syk) inhibitor with anti-tumor activity in preclinical models of B-cell malignancy. We conducted a dose-escalation and dose-expansion phase I study of sovleplenib in patients with relapsed/ refractory mature B-cell tumors. Dose escalation followed a 3+3 design; patients received oral sovleplenib (200-800 mg once daily [q.d.] or 200 mg twice daily [b.i.d.], 28-day cycles). During dose expansion, patients were enrolled into four cohorts per lymphoma classification and treated at the recommended phase II dose (RP2D) (clinicaltrials gov. Identifier: NCT02857998). Overall, 134 Chinese patients were enrolled (dose escalation, N=27; dose expansion, N=107). Five patients experienced dose-limiting toxicities: one each of amylase increased (200 mg q.d.), febrile neutropenia (800 mg q.d.), renal failure (800 mg q.d.), hyperuricemia and blood creatine phosphokinase increased (200 mg b.i.d.) and blood bilirubin increased and pneumonia (200 mg b.i.d.). RP2D was determined as 600 mg (>65 kg) or 400 mg (≤65 kg) q.d.. The primary efficacy end point of independent review committee-assessed objective response rate in indolent B-cell lymphoma was 50.8% (95% confidence interval: 37.5- 64.1) in 59 evaluable patients at RP2D (follicular lymphoma: 60.5%, marginal zone lymphoma: 28.6%, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, 0%). The most common (≥10% patients) grade ≥3 treatment-related adverse events in the dose-expansion phase were decreased neutrophil count (29.9%), pneumonia (12.1%) and decreased white blood cell count (11.2%). Pharmacokinetic exposures increased dose-proportionally with ascending dose levels from 200-800 mg, without observed saturation. Sovleplenib showed anti-tumor activity in relapsed/refractory B-cell lymphoma with acceptable safety. Further studies are warranted.