Cutoff Values of Serum IgG4 and Histopathological IgG4+ Plasma Cells for Diagnosis of Patients with IgG4-Related Disease

IgG4-related disease is a new disease classification established in Japan in the 21st century. Patients with IgG4-related disease display hyper-IgG4-gammaglobulinemia, massive infiltration of IgG4+ plasma cells into tissue, and good response to glucocorticoids. Since IgG4 overexpression is also observed in other disorders, it is necessary to diagnose IgG4-related disease carefully and correctly. We therefore sought to determine cutoff values for serum IgG4 and IgG4/IgG and for IgG4+/IgG+ plasma cells in tissue diagnostic of IgG4-related disease. Patients and Methods. We retrospectively analyzed serum IgG4 concentrations and IgG4/IgG ratio and IgG4+/IgG+ plasma cell ratio in tissues of 132 patients with IgG4-related disease and 48 patients with other disorders. Result. Serum IgG4 >135  mg/dl demonstrated a sensitivity of 97.0% and a specificity of 79.6% in diagnosing IgG4-related disease, and serum IgG4/IgG ratios >8% had a sensitivity and specificity of 95.5% and 87.5%, respectively. IgG4+cell/IgG+ cell ratio in tissues >40% had a sensitivity and specificity of 94.4% and 85.7%, respectively. However, the number of IgG4+ cells was reduced in severely fibrotic parts of tissues. Conclusion. Although a recent unanimous consensus of all relevant researchers in Japan recently established the diagnostic criteria for IgG4-related disease, findings such as ours indicate that further discussion is needed

Augmented Growth Hormone Secretion and Stat3 Phosphorylation in an Aryl Hydrocarbon Receptor Interacting Protein (AIP)-Disrupted Somatotroph Cell Line

<div><p>Aryl hydrocarbon receptor interacting protein (<i>AIP</i>) is thought to be a tumor suppressor gene, as indicated by a mutational analysis of pituitary somatotroph adenomas. However, the physiological significance of <i>AIP</i> inactivation in somatotroph cells remains unclear. Using CRISPR/Cas9, we identified a GH3 cell clone (termed GH3-FTY) in which <i>Aip</i> was genetically disrupted, and subsequently investigated its character with respect to growth hormone (Gh) synthesis and proliferation. Compared with GH3, GH3-FTY cells showed remarkably increased Gh production and a slight increase in cell proliferation. Gh-induced Stat3 phosphorylation is known to be a mechanism of Gh oversecretion in GH3. Interestingly, phosphorylated-Stat3 expression in GH3-FTY cells was increased more compared with GH3 cells, suggesting a stronger drive for this mechanism in GH3-FTY. The phenotypes of GH3-FTY concerning Gh overproduction, cell proliferation, and increased Stat3 phosphorylation were significantly reversed by the exogenous expression of <i>Aip</i>. GH3-FTY cells were less sensitive to somatostatin than GH3 cells in the suppression of cell proliferation, which might be associated with the reduced expression of somatostatin receptor type 2. GH3-FTY xenografts in BALB/c nude mice (GH3-FTY mice) formed more mitotic somatotroph tumors than GH3 xenografts (GH3 mice), as also evidenced by increased Ki67 scores. GH3-FTY mice were also much larger and had significantly higher plasma Gh levels than GH3 mice. Furthermore, GH3-FTY mice showed relative insulin resistance compared with GH3 mice. In conclusion, we established a somatotroph cell line, GH3-FTY, which possessed prominent Gh secretion and mitotic features associated with the disruption of <i>Aip</i>.</p></div

Gh secretion in the cultured medium, <i>Gh</i> mRNA levels before and after exogenous <i>Aip</i> expression, and intracellular cAMP content in cultured GH3 and GH3-FTY cells.

<p>(<b>A</b>) Gh secretion in the media from cultured GH3 and GH3-FTY cells was measured and expressed as a fold increase. (<b>B</b>) <i>Gh</i> mRNA levels in cultured GH3 and GH3-GTY cells were calculated relative to <i>Actb</i> (internal control) using the ∆Ct method. (<b>C</b>) The effect of lentivirus (LV)-mediated forced expression of exogenous <i>gfp</i> as a control or <i>Aip</i> on <i>Gh</i> mRNA levels. GFP and Aip indicate LV-GFP and LV-Aip, respectively. (<b>D</b>) The intracellular cAMP content of both cultured cells was measured as described in the Methods. Data were compared using the unpaired two-tailed <i>t</i>-test. **<i>P</i><0.01. ns, not significant.</p

Combined Treatment with Exendin-4 and Metformin Attenuates Prostate Cancer Growth

Recently, the pleiotropic benefits of incretin-based therapy have been reported. We have previously reported that Exendin–4, a glucagon-like peptide–1 (GLP–1) receptor agonist, attenuates prostate cancer growth. Metformin is known for its anti-cancer effect. Here, we examined the anti-cancer effect of Exendin–4 and metformin using a prostate cancer model. experiments, LNCaP cells were transplanted subcutaneously into the flank region of athymic mice, which were then treated with Exendin–4 and/or metformin. TUNEL assay and immunohistochemistry were performed on tumors., Exendin–4 and metformin significantly decreased tumor size, and further significant tumor size reduction was observed after combined treatment. Immunohistochemistry on tumors revealed that the P504S and Ki67 expression decreased by Exendin–4 and/or metformin, and that metformin increased phospho-AMPK expression and the apoptotic cell number.These data suggest that Exendin–4 and metformin attenuated prostate cancer growth by inhibiting proliferation, and that metformin inhibited proliferation by inducing apoptosis. Combined treatment with Exendin–4 and metformin attenuated prostate cancer growth more than separate treatments

Plasma Gh and Igf-1 levels in GH3 and GH3-FTY mice.

<p>BALB/c-nu mice were inoculated with GH3 or GH3-FTY cells. Control mice were inoculated by the medium only. Plasma levels of Gh (<b>A</b>) and Igf-1 (<b>B</b>) at 8 weeks after inoculation. Data were compared using the one-way ANOVA with Tukey’s post-hoc test. *<i>P</i><0.05, ***<i>P</i><0.001. ns, not significant.</p

Comparison of <i>Sstr2</i>, and <i>Zac1</i> mRNA expression and Sstr2 expression levels by western blot in cultured GH3 and GH3-FTY cells.

<p>(<b>A</b>) <i>Sstr2</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR. (<b>B</b>) Western blot analysis of Sstr2 and beta-actin in cultured GH3 and GH3-FTY cells. Twenty μg protein from cell lysates was separated by SDS-PAGE and immunoblotted with antibodies against Sstr2 and beta-actin (upper panel). Statistical evaluation of Sstr2/ beta-actin expression between cultured GH3 cells and GH3-FTY cells (lower panel). (<b>C</b>) <i>Zac1</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR. (<b>D</b>) <i>Sstr2</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR after lentivirus (LV)-mediated exogenous expression of <i>Aip</i> (LV-Aip) or <i>gfp</i> (LV-GFP) as a control. GFP and Aip indicate LV-GFP and LV-Aip, respectively. (<b>E</b>) <i>Zac1</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR after lentivirus (LV)-mediated exogenous expression of <i>Aip</i> (LV-Aip) or <i>gfp</i> (LV-GFP) as a control. GFP and Aip indicate LV-GFP and LV-Aip, respectively. Data were compared using the unpaired two-tailed <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01. ns, not significant.</p

Western blot analysis of p-Stat3 (Tyr705) and Stat3 in cultured GH3 and GH3-FTY cells.

<p>(<b>A</b>) Western blot analysis of p-Stat3 (Tyr705) and Stat3. Twenty μg protein from cell lysates was separated by SDS-PAGE and immunoblotted with antibodies against p-Stat3, Stat3, and beta-actin. (<b>B</b>) Statistical evaluation of Stat3/ beta-actin expression between cultured GH3 and GH3-FTY cells. Data were compared using the unpaired two-tailed <i>t</i>-test. ns, not significant. The intensity of each detected band was analyzed using the image analysis software Quantity One (BIO-RAD), and the Stat3/ beta-actin ratio was calculated. (<b>C</b>) Statistical evaluation of p-Stat3/ beta-actin expression between cultured GH3 and GH3-FTY cells. Data were compared using the unpaired two-tailed <i>t</i>-test. **<i>P</i><0.01. (<b>D)</b> Western blot analysis of p-Stat3 and Stat3. The effect of lentivirus (LV)-mediated forced expression of exogenous <i>Aip</i> (LV-AIP) or <i>gfp</i> (LV-GFP) as a control on p-Stat3 expression was examined. Twenty μg protein from cell lysates was subjected to SDS-PAGE and immunoblotted with antibodies against p-Stat3 and beta-actin. (<b>E)</b> Statistical evaluation of p-Stat3. GFP and Aip indicate LV-GFP and LV-Aip, respectively. Data were compared using the unpaired two-tailed <i>t</i>-test. **<i>P</i><0.01. ns, not significant. (<b>F</b>) <i>Il6r</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR. (<b>G</b>) <i>Il6r</i> mRNA levels relative to <i>Actb</i> mRNA levels as determined by qPCR before and after lentivirus (LV)-mediated exogenous expression of <i>Aip</i> (LV-Aip) or <i>gfp</i> (LV-GFP) as a control. GFP and Aip indicate LV-GFP and LV-Aip, respectively. Data were compared using the unpaired two-tailed <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01. ns, not significant.</p