SUMMARYWe previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation.

Background: Mast cells (MC) are involved in a wide spectrum of disorders characterized by neovascularization and fibroproliferation. We and others recently reported that human MC are a source of basic fibroblast growth factor (b FGF-2), a potent angiogenic and mitogenic polypeptide, in several disease conditions, such as chronic inflammation, hemangioma, and benign cutaneous mastocytosis. These findings suggest that FGF-2 may be an important mediator of cell proliferation and angiogenesis associated with MC. Since MC are heterogeneous across species, it is unknown whether FGF-2 expression is a feature common to all MC, or whether FGF-2 expression by MC can be regulated. We therefore examined FGF-2 expression by MC in mouse tissue and MC lines. Methods: Immunostaining, RT-PCR, ELISA, immunoblot and Northern blot analyses were employed to study four murine MC lines for FGF-2 expression and its regulation by transforming growth factor-β (TGF-β), stem cell factor (SCF), and tumor necrosis factor-α (TNF-α). Results: Mouse tissue MC and three of four murine MC lines (CFTL-12, CFTL-15, ABFTL-3) express FGF-2 as judged by immunostaining, ELISA, Western blot and Northern blot analyses, and reverse transcription-polymerase chain reaction. While TNF-α appeared to downregulate FGF-2 mRNA levels, treatment with SCF or TGF-β resulted in an increase in the expression of FGF-2 at mRNA level which can be attenuated by TNF-α. However, the concurrent increase in FGF-2 protein was negligible, possibly due to immaturity of these cell lines. Conclusion: Expression of FGF-2 may be a ubiquitous feature of MC in other species in addition to humans, and can be selectively regulated by SCF, TGF-β and TNF-α.

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Background: Programmed death-ligand 1 (PD-L1) is a crucial biomarker predicting efficacious treatment of immune checkpoint inhibitors (ICIs). Understanding the genetic association of PD-L1 expression provides insights into the tumor-immune interaction and further exploration of immunotherapeutic biomarkers. However, the diverse and dissimilar quantifying assays might lead to inconsonant and confusing results, not conducive to the following research. Previous studies identified distinct staining utility among multiple PD-L1 antibodies, however, whether the genomic correlate of stained PD-L1 is also impacted by different testing assays is largely unknown.Methods: We evaluated 873 Chinese LUAD patients with lung adenocarcinoma in whom FDA-approved PD-L1 testing (22C3, Dako, Glostrup, Denmark; SP263, Ventana, Tucson, AZ) and targeted next-generation sequencing (3D Medicines Inc.) was performed on the same tissue. Data from Memorial Sloan Kettering Cancer Center (MSKCC), The Cancer Genome Atlas (TCGA) and the OAK trial were analyzed for further validation.Results: In the 3DMed database (n¼873), PD-L1 expression in tumor cells was positively correlated with tumor mutational burden. (p<0.001). PD-L1 positivity was more common in metastatic lymph nodes compared to primary lung tumors and other metastatic samples (p ¼0.009). Deleterious mutations in KRAS, TP53, ALK and MET significantly associated with PD-L1 high expression (each p<0.05) and EGFR and ERBB2 mutations associated with PD-L1 negativity (each p<0.05). Comparing the results in the 3DMed, MSKCC, TCGA, and OAK cohorts using different methods to quantifying PD-L1 expression, most of the positive results were validated in at least two cohorts, except ERBB2.

Glial cells are components of the central and peripheral nervous systems, which provide physical support for neuron and modulate synaptic transmission. We and others previously showed that in mice, pancreatic glial cells are located in close association with the extensive neuro-endocrine-vascular network that surrounds the islet perimeter. It is well known that differences exist in mouse and human islet architecture, in particular in vascular density and innervation. We therefore sought to determine whether density and/or organization of pancreatic glial cells also differs between mouse and human. Glial fibrillary acidic protein (GFAP) was used to identify pancreatic glial cells and was examined by confocal microscopy. In mouse pancreas, GFAP+ cells were frequently identified around the islet periphery and were less frequently seen in the core of the islet. In human pancreas, however, most of the GFAP+ cells were found in the exocrine tissue, resulting in fewer peri-islet glial cells relative to mice (table). High-fat diet feeding significantly increased %peri-islet GFAP+ area in mice while no difference was seen between obese and lean human donors (table). These findings indicate that pancreatic glial cell distribution patterns differ between mouse and human, raising the possibility that these cells may affect islet-cell function differently between the two species. Disclosure J. Niu: None. D. J. Hackney: None. P. Ahmadi: None. M. Hara: None. R. L. Hull-meichle: Employee; Veterans Administration, Research Support; Casma Therapeutics, Cystic Fibrosis Foundation, NIH - National Institutes of Health, Veterans Administration. J. Tong: None. Funding University of Washington

Immunohistological study using EM technique offers several advantages over light microscopy (LM) such as unequivocal identification of cell type by morphology and accurate subcellular localization of antigenic molecules of interest. These are mainly attributed to fixation with strong cross-linking reagents and embedding in resin-based media. However, they also are associated with a major drawback -- greatly reduced antigenicity or completely abolished accessibility to tissue antigen due to extensive cross-linking and the hydrophobicity of the resin. As a result, the success rate of immunolabeling of routinely processed tissue for EM is far lower than that for LM using paraffin sections. Yet the process is far more laborious. To circumvent this problem, various etching or de-plasticizing methods have been introduced. However, most of them invariably lead to deterioration of the tissue morphology. A trade-off between morphology and antigenicity is generally considered necessary. Here we demonstrate that a brief enzymatic digestion prior to immunolabeling the routinely processed tissue samples for EM greatly improves the success rate of the immunostaining without noticeable effect on morphology. Our results suggest that enzymatic digestion is an effective antigenicity-restoring method for EM.