BACKGROUND Gastrointestinal diffuse large B-cell lymphoma (GI-DLBCL) is the most common histological subtype of extra-nodal DLBCL, but the risk factors, prognostic biomarkers, histopathological classifications, and treatment strategies have not had significant progress. Emerging evidence shows that cystatin SN (CST1) is involved in tumor progression in several cancer types, but its role in GI-DLBCL has not been revealed. MATERIAL AND METHODS We established a cohort consisting of 84 patients with GI-DLBCL who underwent surgical resection. The expression of CST1 in the cohort was investigated by immunohistochemistry, which divided the patients into subgroups with low or high expression of CST1. Moreover, the CST1 expression in GI-DLBCL tissues or adjacent GI tissues were compared with RT-qPCR. The correlation between CST1 expression and clinicopathological factors was analyzed with the chi-square test. The prognostic significance of CST1 was estimated by univariate and multivariate analysis, and statistical significance was analyzed with the log-rank test. RESULTS CST1 was aberrantly upregulated in GI-DLBCL tissues compared with in non-tumor GI tissues. High expression of CST1 indicated poor prognosis of GI-DLBCL (P=0.012), and CST1 can be regarded as an independent prognostic biomarker of GI-DLBCL (hazard ratio=3.07). In our study, serum lactate dehydrogenase (P=0.002), performance status (P=0.003), Lugano stage (P=0.002), and International Prognostic Index (P=0.001) were also prognostic factors of GI-DLBCL. CONCLUSIONS CST1 is an independent prognostic biomarker of GI-DLBCL, indicating unfavorable prognosis. Our results suggested that CST1 detection can be a promising method to stratify high-risk patients and guide individual treatment.

OBJECTIVE: Cystatin SA (CST2) belongs to the superfamily of cysteine protease inhibitors. Emerging research indicates that CST2 is often dysregulated across various cancers. Its role and molecular mechanisms in gastric cancer remain underexplored. This study aims to explore the expression and function of CST2 in gastric cancer.
METHODS: CST2 expression was analyzed and validated through Western blot. CST2 overexpression was induced by lentivirus in GC cells, and the correlation between CST2 expression levels and downstream signaling pathways was assessed. In addition, multiple assays, including cell proliferation, colony formation, wound-healing, and transwell migration/invasion, were considered to ascertain the influence of CST2 overexpression on gastric cancer. The cell cycle and apoptosis were detected by flow cytometry.
RESULTS: CST2 expression at the protein level was decreased to be reduced in both gastric cancer tissues and cell lines, and CST2 expression attenuate gastric cancer growth, an effect restricted to gastric cancer cells and absent in gastric epithelial GES-1 cells. Furthermore, CST2 was demonstrated to improve chemosensitivity to Oxaliplatin in gastric cancer cells through the PI3K/AKT signaling pathway.
CONCLUSIONS: These findings indicate that CST2 is downregulated at the protein level in gastric cancer tissues and cell lines. Additionally, CST2 was found to attenuate the growth of gastric cancer cells and to enhance sensitivity to Oxaliplatin through the PI3K/AKT signaling pathway, specific to gastric cancer cell lines. CST2 may serve as a tumor suppressor gene increasing sensitivity to Oxaliplatin in gastric cancer.

BACKGROUND: Asthma is the most common chronic disease in children with an increasing prevalence. Its development is caused by genetic and environmental factors and allergic sensitization is a known trigger. Dog allergens affect up to 30% of all children and dog dander-sensitized children show increased expression of cystatin-1 (CST1) and eotaxin-3 (CCL26) in nasal epithelium. The aim of our study was to investigate the functional mechanism of CST1 and CCL26 in the alveolar basal epithelial cell line A549.
METHODS: A549 cells were transfected with individual overexpression vectors for CST1 and CCL26 and RNA sequencing was performed to examine the transcriptomics. edgeR was used to identify differentially expressed genes (= DEG, |log2 FC | ≥ 2, FDR < 0.01). The protein expression levels of A549 cells overexpressing CST1 and CCL26 were analyzed using the Target 96 inflammation panel from OLINK (antibody-mediated proximity extension-based assay; OLINK Proteomics). Differentially expressed proteins were considered with a |log2 FC| ≥ 1, p < .05.
RESULTS: The overexpression of CST1 resulted in a total of 27 DEG (1 upregulated and 26 downregulated) and the overexpression of CCL26 in a total of 137 DEG (0 upregulated and 137 downregulated). The gene ontology enrichment analysis showed a significant downregulation of type I and III interferon signaling pathway genes as well as interferon-stimulated genes. At the protein level, overexpression of CST1 induced a significantly increased expression of CCL3, whereas CCL26 overexpression led to increased expression of HGF, and a decrease of CXCL11, CCL20, CCL3 and CXCL10.
CONCLUSIONS: Our results indicate that an overexpression of CST1 and CCL26 cause a downregulation of interferon related genes and inflammatory proteins. It might cause a higher disease susceptibility, mainly for allergic asthma, as CCL26 is an agonist for CCR-3-carrying cells, such as eosinophils and Th2 lymphocytes, mostly active in allergic asthma.

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

Meibomian gland dysfunction (MGD) is a highly prevalent condition and the most common cause of evaporative dry eye disease. Studying the proteome of MGD can result in important advances in the management of the condition. Here, we collected tear film samples from treatment naïve patients with MGD (n = 10) and age-matched controls (n = 11) with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography-tandem mass spectrometry. The proteins were considered differentially expressed if p < 0.05. A total of 88 proteins were significantly regulated. The largest change was observed in cystatin-SN, which was downregulated in MGD and correlated negatively with tear meniscus height. The downregulation of cystatin-SN was confirmed with targeted mass spectrometry by single reaction monitoring (SRM). Eighteen immunoglobulin components involved in B cell activation, phagocytosis, and complement activation were downregulated in MGD including Ig alpha-1 chain C region, immunoglobulin J chain, immunoglobulin heavy variable 3-15, and Ig mu chain C region. The changes in cystatin-SN and immunoglobulin chains are likely to result from the inflammatory changes related to tear film evaporation, and future studies may assess their association with the meibum quality.

Mastocytosis, a rare blood disorder characterized by the proliferation of clonal abnormal mast cells, has a variegated clinical spectrum and diagnosis is often difficult and delayed. Recently we proposed the cathepsin inhibitor cystatin D-R26 as a salivary candidate biomarker of systemic mastocytosis (SM). Its C26 variant is able to form multiprotein complexes (mPCs) and since protein-protein interactions (PPIs) are crucial for studying disease pathogenesis, potential markers, and therapeutic targets, we aimed to define the protein composition of the salivary cystatin D-C26 interactome associated with SM. An exploratory affinity purification-mass spectrometry method was applied on pooled salivary samples from SM patients, SM patient subgroups with and without cutaneous symptoms (SM+C and SM-C), and healthy controls (Ctrls). Interactors specifically detected in Ctrls were found to be implicated in networks associated with cell and tissue homeostasis, innate system, endopeptidase regulation, and antimicrobial protection. Interactors distinctive of SM-C patients participate to PPI networks related to glucose metabolism, protein S-nitrosylation, antibacterial humoral response, and neutrophil degranulation, while interactors specific to SM+C were mainly associated with epithelial and keratinocyte differentiation, cytoskeleton rearrangement, and immune response pathways. Proteins sensitive to redox changes, as well as proteins with immunomodulatory properties and activating mast cells, were identified in patients; many of them were involved directly in cytoskeleton rearrangement, a process crucial for mast cell activation. Although preliminary, these results demonstrate that PPI alterations of the cystatin D-C26 interactome are associated with SM and provide a basis for future investigations based on quantitative proteomic analysis and immune validation.

This study aims to evaluate the diagnostic value of human serum cysteine protease inhibitors (cystatin 4 [CST4]) in colorectal cancer (CRC) patients.
A total of 291 patients who were admitted to Zhuzhou Central Hospital for colonoscopy from January 2020 to December 2021 and met the inclusion criteria were selected. Serum samples of the patients were collected, and CST4 was detected by double-antibody sandwich enzyme-linked immunosorbent assay. Simultaneously, CEA and CA19-9 were detected, and the patients were divided into the CRC group, benign lesion group, and healthy control group. An attempt was made to construct a CRC prediction model including CST4 and draw a subject working characteristic curve as a diagnostic threshold for CRC prediction, and evaluate the diagnostic efficacy of the above indicators. At the same time, the expression analysis of CST4, CEA, and CA19-9 was verified by combining the data of CRC in the Tumor Genome Atlas (TCGA).
In this study, the levels of serum CST4, CEA, and CA19-9 in the CRC group were higher than those in the colorectal benign lesion group and healthy control group, with statistical significance (P < .001). The analysis results of the receiver operating characteristic curve showed that the area under the receiver operator characteristic curve (AUC) of CST4 was 0.7739, which was obviously larger than the AUC of CA19-9 and CEA. CRC data from the TCGA expression database showed that CST4 expression and CEA expression were higher in CRC patients than in normal samples. The combined model based on CST4 was successfully constructed, and the AUC for predicting the occurrence of CRC was 0.7851.
CST4 is a novel and improved diagnostic marker for CRC. The combined model based on CST4 has a certain potential value in terms of predicting the occurrence of intestinal cancer.

Acute liver failure (ALF) is a life-threatening disease that occurs secondary to drug toxicity, infection or a devastating immune response. Orthotopic liver transplantation is an effective treatment but limited by the shortage of donor organs, the requirement for life-long immune suppression and surgical challenges. Stem cell transplantation is a promising alternative therapy for fulminant liver failure owing to the immunomodulatory abilities of stem cells. Here, we report that when transplanted into the liver, human endoderm stem cells (hEnSCs) that are germ layer-specific and nontumorigenic cells derived from pluripotent stem cells are able to effectively ameliorate hepatic injury in multiple rodent and swine drug-induced ALF models. We demonstrate that hEnSCs tune the local immune microenvironment by skewing macrophages/Kupffer cells towards an anti-inflammatory state and by reducing the infiltrating monocytes/macrophages and inflammatory T helper cells. Single-cell transcriptomic analyses of infiltrating and resident monocytes/macrophages isolated from animal livers revealed dramatic changes, including changes in gene expression that correlated with the change of activation states, and dynamic population heterogeneity among these cells after hEnSC transplantation. We further demonstrate that hEnSCs modulate the activation state of macrophages/Kupffer cells via cystatin SN (CST1)-mediated inhibition of interferon signaling and therefore highlight CST1 as a candidate therapeutic agent for diseases that involve over-activation of interferons. We propose that hEnSC transplantation represents a novel and powerful cell therapeutic treatment for ALF.

Cystatin SN (CST1) and cystatin SA (CST2) are cysteine protease inhibitors that protect against allergen, viral, and bacterial proteases. Cystatins are overexpressed in the setting of allergic rhinitis and chronic rhinosinusitis with nasal polyps (CRSwNP); however, their role in promoting type 2 inflammation remains poorly characterized.
The purpose of this study was to use integrated poly-omics and a murine exposure model to explore the link between cystatin overexpression in CRSwNP and type 2 inflammation.
In this institutional review board- and institutional animal care and use committee-approved study, we compared tissue, exosome, and mucus CST1 and CST2 between CRSwNP and controls (n = 10 per group) by using matched whole exome sequencing, transcriptomic, proteomic, posttranslational modification, histologic, functional, and bioinformatic analyses. C57/BL6 mice were dosed with 3.9 μg/mL of CST1 or PBS intranasally for 5 to 18 days in the presence or absence of epithelial ABCB1a knockdown. Inflammatory cytokines were quantified by using Quansys multiplex assays or ELISAs.
Of the 1305 proteins quantified, CST1 and CST2 were among the most overexpressed protease inhibitors in tissue, exosome, and mucus samples; they were localized to the epithelial layer. Multiple posttranslational modifications were identified in the polyp tissue. Exosomal CST1 and CST2 were strongly and significantly correlated with eosinophils and Lund-Mackay scores. Murine type 2 cytokine secretion and TH2 cell infiltration increased in a time-dependent manner following CST1 exposure and was abrogated by epithelial knockdown of ABCB1a, a regulator of epithelial cytokine secretion.
CST1 is a potent upstream initiator of epithelial-derived type 2 inflammation in CRSwNP. Therapeutic strategies targeting CST activity and its associated posttranslational modifications deserve further interrogation.

The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1+ myofibroblasts with prognostic values and potential biological significance. CST1+ myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology.