Simplified and optimized multispectral imaging for 5-ALA-based fluorescence diagnosis of malignant lesions

5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is now clinically applied for accurate and ultrarapid diagnosis of malignant lesions such as lymph node metastasis during surgery. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA, protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue chromophores, such as collagen and flavins. In this study, we demonstrated PPIX fluorescence estimation with autofluorescence elimination for 5-ALA-based fluorescence diagnosis of malignant lesions by simplified and optimized multispectral imaging. We computationally optimized observation wavelength regions for the estimation of PPIX fluorescence in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen and flavins. By using the fluorescence intensities of the optimized wavelength regions, we verified quantitative detection of PPIX fluorescence by using chemical mixtures of PPIX, flavins, and collagen. Furthermore, we demonstrated detection capability by using metastatic and non-metastatic lymph nodes of colorectal cancer patients. These results suggest the potential and usefulness of the background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions, and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis

Osteoprotegerin Regulates Pancreatic β-Cell Homeostasis upon Microbial Invasion.

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-κB ligand (RANKL), antagonizes RANKL's osteoclastogenic function in bone. We previously demonstrated that systemic administration of lipopolysaccharide (LPS) to mice elevates OPG levels and reduces RANKL levels in peripheral blood. Here, we show that mice infected with Salmonella, Staphylococcus, Mycobacteria or influenza virus also show elevated serum OPG levels. We then asked whether OPG upregulation following microbial invasion had an effect outside of bone. To do so, we treated mice with LPS and observed OPG production in pancreas, especially in β-cells of pancreatic islets. Insulin release following LPS administration was enhanced in mice lacking OPG, suggesting that OPG inhibits insulin secretion under acute inflammatory conditions. Consistently, treatment of MIN6 pancreatic β-cells with OPG decreased their insulin secretion following glucose stimulation in the presence of LPS. Finally, our findings suggest that LPS-induced OPG upregulation is mediated in part by activator protein (AP)-1 family transcription factors, particularly Fos proteins. Overall, we report that acute microbial infection elevates serum OPG, which maintains β-cell homeostasis by restricting glucose-stimulated insulin secretion, possibly preventing microbe-induced exhaustion of β-cell secretory capacity

OPG inhibits insulin secretion from β-cells under inflammatory conditions.

<p>When β-cells are exposed to inflammatory stimuli, they secrete OPG, which blocks RANKL-RANK signaling. Both osteoblast- and β-cell-derived OPG negatively regulates insulin secretion. Lower panel was adopted from Wei and Karsenty (2015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146544#pone.0146544.ref045" target="_blank">45</a>]. Glu, undercarboxylated. Gla, carboxylated.</p

OPG expression in mouse β-cells and effect of OPG on glucose-stimulated insulin secretion.

<p>(A, B) Immunofluorescence analysis showing localization of OPG protein (red) with glucagon (green) (A) or insulin (green) (B) in pancreatic islets. OPG is predominantly expressed in β-cells based on co-localization with insulin. Scale bars, 20 μm. (C) Expression of <i>Opg</i>, <i>Rank</i>, and <i>Rankl</i> transcripts in isolated islets treated without or with LPS as measured by qPCR (n = 3–6). (D) Effect of LPS treatment on <i>Opg</i>, <i>Rank</i> and <i>Rankl</i> expression in MIN6 cells (n = 3). (E) Insulin secretion by MIN6 cells. Cells were untreated (n = 6) or treated with 100 ng/ml soluble RANKL (sRANKL, n = 6), 100 ng/ml recombinant OPG (rOPG, n = 6), 10 μg/ml LPS (n = 6), both LPS and sRANKL (n = 6), or both LPS and rOPG (n = 6), and then stimulated with 3, 9.8, or 20 mM glucose. Levels of secreted insulin were normalized to total cell protein. Shown are means ± SD. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Mice lacking OPG show altered glucose metabolism.

<p>(A) Fasting blood glucose levels (f.BGL) in control (<i>Opg</i>^+/±, n = 11, 8) and OPG knockout (<i>Opg</i>^-/-, n = 14, 9) mice 16 hr after injection of PBS or LPS. Shown are means ± SEM. ***<i>P</i> < 0.005. (B) Insulin signal intensity, calculated by insulin immunosignal intensity of cross-sections of pancreata of <i>Opg</i>^+/- and <i>Opg</i>^-/- mice 16 hr after injection of PBS or LPS (31Opg^+/± (n = 5, 4) and <i>Opg</i>^-/- (n = 8, 8) mice 16 hr after injection of PBS or LPS. Open circles indicate outliers. *<i>P</i> < 0.05. (D) Intraperitoneal glucose tolerance tests (IPGTT) of PBS- or LPS-injected <i>Opg</i>^+/- (n = 5, 4) or <i>Opg</i>^-/- (n = 6, 5) mice. Shown are means ± SEM. *<i>P</i> < 0.05. Blood glucose levels were measured at different time points, as indicated.</p

Bone homeostasis in mice after <i>Salmonella</i> infection.

<p>(A) TRAP staining of tibial sections of BALB/c mice infected for 5 days with <i>Salmonella enterica</i>. Cont, uninfected mice; PsB, periosteal bone; TB, trabecular bone. Scale bars in upper panels represent 500 μm, and in middle or lower panels, 200 μm. (B, C) The number of osteoclasts (N.OC) at the trabecular bone surface (TBS) and the periosteal bone surface (PsBS) in tibial sections. Shown are means ± SD. Sal, <i>Salmonella</i>-infected mice. (D, E) Serum OPG levels (D) or RANKL levels (E) in mice infected one week with the avirulent <i>Salmonella enterica</i> strains UF20, UF71 and UF110 (n = 6 for each group). Open circles indicate outliers. ***<i>P</i> < 0.005. (F, G) Femoral bone tissue mineral density (TMD) in mice infected one week with avirulent <i>Salmonella</i>. Tb, trabecular; Ct, cortical. Shown are means ± SD. *<i>P</i> < 0.05 versus each control.</p

Increased serum OPG levels in mice after microbial infection occurs via Fos family transcription factors.

<p>(A, B) Time-dependent elevation of OPG and IFN-β in serum, and colony forming units (CFU) in blood and spleen of 6-week-old C57BL/6J mice infected with <i>Salmonella enterica</i> χ3306 (A, n = 4 each point) or <i>Staphylococcus aureus</i> 92–1191 (B, n = 4 each point). “d” indicates death of one mouse. “ddd” indicates death of three mice. (C, D) Time-dependent elevation of OPG and IFN-β levels in serum after <i>Mycobacterium</i> (C, n = 4 each point) and influenza virus (D, n = 3 each point) infection of 6-week-old C57BL/6J mice. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005. (E, F) OPG serum levels in LPS-injected mice in c-Fos knockout mice (<i>Fos</i>^-/-) (E) or Fosl1 transgenic mice (<i>Fosl1</i>) (F) plus respective littermate controls (n = 3–4). Shown are means ± SD. *<i>P</i> < 0.05, **<i>P</i> < 0.01. LPS (4 μg/g body weight) was administered i.p. to 6-week-old C57BL/6J mice, and blood was collected 12 hr later.</p