No abstract

Class II major histocompatability molecules are the primary susceptibility locus for many autoimmune disorders including type 1 diabetes. Human DQ8 and I-Ag7, in the non-obese diabetic (NOD) mouse model of spontaneous autoimmune diabetes, confers diabetes risk by modulating presentation of specific islet peptides in the thymus and periphery. We utilized an in silico molecular docking program to screen a large “drug-like” chemical library to define small molecules capable of occupying specific structural pockets along the I-Ag7 binding groove with the objective of influencing presentation of the autoantigen insulin B:9–23 to T cells. In this study we demonstrate using both murine and human cells that small molecules can enhance or inhibit specific T cell receptor (TCR) signaling in the presence of cognate target peptides based upon the structural pocket targeted. The influence of compounds on the TCR response was pocket dependent with pocket 1 and 6 compounds inhibiting responses and molecules directed at pocket 9 enhancing response to peptide. At nanomolar concentrations, the inhibitory molecules block insulin B:9–23 peptide, endogenous insulin, and islet stimulated T cell responses. Glyphosine, a pocket 9 compound, enhances insulin peptide presentation to T cells at concentrations as low as 10 nM, upregulates IL-10 secretion, and prevents diabetes in NOD mice. These studies present a novel method for identifying small molecules capable of both stimulating and inhibiting T cell responses with potentially therapeutic applications.

Evidence is mounting that not only microangiopathy, but also neurodegenerative events occur in the retinas of humans and rodents with early diabetes. Diverse pathologies are known to alter the amount and/or location of glial expression of the water-selective channels aquaporins (AQPs) 1 and 4. However, the temporal relationships among glial activation, the altered expression of the AQP proteins and neuronal death in the retinas of diabetic animals remains to be investigated. Male spontaneously diabetic Torii (SDT) rats reportedly develop diabetes by 40 weeks of age at the latest and manifest proliferative diabetic retinopathy at 50 weeks or later. This study compared temporal changes in neuroretinal apoptosis, glial fibrillary acidic protein (GFAP) expression and the expression of AQPs 1 and 4 between SDT rat retinas and age-matched Sprague-Dawley (SD) rat retinas. Cell death was detected by terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick end-labeling on retinal flatmounts and activated caspase 3 immunofluorescence of retinal cryosections. The expression of GFAP and AQPs 1 and 4 was assessed by immunohistochemistry of cryosections and retinal flatmounts. Diabetes started to develop around 15 weeks in SDT rats. Apoptotic cells in the ganglion cell layer and the inner nuclear layer were significantly more numerous in 40-week-old SDT rat retinas than in either age-matched SD rat retinas or 10-week-old SDT rats. GFAP immunoreactivity was confined to the nerve fiber layer both in SD and SDT rats at 10 weeks, whereas it spanned the whole retina in SDT rats, but not in SD rats, at 40 weeks. AQP1 was expressed in the outer retina, whereas AQP4 was expressed in the perivascular and end feet of Müller cells and astrocytes in the inner retina in the control SD rats and the SDT rats at 10 weeks. The perivascular AQPs shifted from AQP4 to AQP1 in 40-week-old SDT rats that exhibited marked hyperglycemia. Thus, the development of diabetes increases neuroretinal apoptosis, and this coincides with an altered expression pattern of GFAP and water-selective channels AQPs 1 and 4 in SDT rats.

Cancer stem‐like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P‐CSLCs). A P‐CSLC‐enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by 2‐D electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P‐CSLCs compared to parental cells. Flow cytometry analysis indicated that CRT was mostly localized to the surface of P‐CSLCs and did not correlate with the levels of CD44v9, another P‐CSLC biomarker. Furthermore, the side population in the CRThigh/CD44v9low population was much higher than that in the CRTlow/CD44v9high population. Calreticulin expression was also assessed by immunohistochemistry in pancreatic cancer tissues (n = 80) obtained after radical resection and was found to be associated with patients' clinicopathological features and disease outcomes in the Cox proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and postoperative therapy. Our results suggest that CRT can serve as a biomarker of P‐CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be considered as a therapeutic target for cancer immunotherapy.