Reg2 treatment is protective but the induced Reg2 autoantibody is destructive to the islets in NOD mice.

Regenerating family protein 2 (Reg2) is a trophic factor which stimulates ß-cell replication and resists islet destruction. However, Reg2 also serves as an islet autoantigen, which makes it complicated to judge the effectiveness in treating diabetes. How Reg2 treatment behaves in non-obese diabetic (NOD) mice is to be investigated. NOD mice were treated with recombinant Reg2 protein, Complete Freund's adjuvant (CFA) + PBS and CFA+Reg2 vaccinations, CFA+PBS- and CFA+Reg2-immunized antisera, and single chain variable fragment (scFv)-Reg2 and mIgG2a-Reg2 antibodies. Glycemic level, bodyweight, serum Reg2 antibody titer, glucose tolerance, and insulin secretion were determined. Islet morphological characteristics, insulitis, cell apoptosis, islet cell components, and T cell infiltration were analyzed by histological examinations. The autoantigenicity of constructed Reg2C and Reg2X fragments was determined in healthy BALB/c mice, and the bioactivity in stimulating cell proliferation and survival was assessed in insulinoma MIN6 cells. Reg2 administration alleviated diabetes in NOD mice with improved glucose tolerance and insulin secretion but elevated serum Reg2 autoantibodies. Histomorphometry showed reduced inflammatory area, TUNEL signal and CD8 + T cell infiltration, and increased ß-cell proportion in support of the islet-protective effect of Reg2 treatment. CFA+PBS and CFA+Reg2 immunizations prevented diabetic onset and alleviated insulitis while injections of the antisera offered mild protections. Antibody treatments accelerated diabetic onset without increasing the overall incidence. Reg2C fragment depletes antigenicity, but reserves protective activity in streptozotocin (STZ)-treated MIN6 cells. In conclusion, Reg2 treatment alleviates type 1 diabetes (T1D) by preserving islet ß-cells, but induces Reg2 autoantibody production which poses a potential risk of accelerating diabetic progression.

Combined Immunotherapy and Tyrosine Kinase Inhibitor Treatment for Metastatic Renal Cell Carcinoma With Unknown Primary Origin: A Report of Two Cases and Literature Review.

BACKGROUND/AIM: Renal cell carcinoma (RCC) of unknown primary origin is rarely identified and accounts for only 5% of cancers of unknown primary origin (CUP). The disease prognosis is typically poor because of no standard and effective therapy. Our review indicated that 23 cases have been reported and treated with conventional chemotherapy or tyrosine-kinase inhibitors alone; accordingly, most patients showed partial response or progression diseases with short survival time. CASE REPORT: Herein, we present two cases of metastatic RCC of unknown primary origin. One case was papillary type and the other was clear cell type. According to the recent clinical trials in patients with metastatic RCC, a combination of immunotherapy and tyrosine-kinase inhibitors exhibited better response than conventional therapy or tyrosine-kinase inhibitors alone. Both present cases accepted a combination treatment with immunotherapy and tyrosine-kinase inhibitor and showed stable diseases. The radiological progression-free time for the case with metastatic papillary RCC was 5 months, and that with clear cell RCC was 6 months until now. CONCLUSION: The combination of immunotherapy and tyrosine-kinase inhibitors is at least as effective as a tyrosine-kinase inhibitor alone, and superior to conventional chemotherapy for treating metastatic RCC of unknown primary origin.

Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro.

BACKGROUND: Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein. METHODS: This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay. RESULTS: The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed. CONCLUSION: The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

Recombinant Reg3ß protein protects against streptozotocin-induced ß-cell damage and diabetes.

Regenerating genes (Reg) have been found during the search for factors involved in pancreatic islet regeneration. Our recent study discovered that pancreatic ß-cell-specific overexpression of Reg3ß protects against streptozotocin (Stz) -induced diabetes in mice. To investigate its potential roles in the treatment of diabetes, we produced a recombinant Reg3ß protein and provided evidence that it is active in promoting islet ß-cell survival against Stz- triggered cell death. Though ineffective in alleviating preexisting diabetes, pretreatment of recombinant Reg3ß was capable of minimizing the Stz-induced hyperglycemia and weight loss, by preserving serum and pancreatic insulin levels, and islet ß-cell mass. No obvious changes were observed in the rate of cell proliferation and hypertrophy in α- or acinar-cells after treatment with recombinant Reg3ß. The underlying mechanism of Reg3ß-mediated protection seems to involve Akt activation which upregulates Bcl-2 and Bcl-xL levels and consequently promotes cell survival.

Recombinant Reg3α protein protects against experimental acute pancreatitis in mice.

Regenerating gene 3α (Reg3α) protein is a trophic factor that stimulates cell and tissue proliferation, neogenesis and also acts against apoptosis and necrosis. In order to explore the potential roles of recombinant Reg3α (rReg3α), we produced a mature rReg3α polypeptide for direct administration in l-arginine (L-Arg) induced acute pancreatitis (AP) in mice. Our results showed that rReg3α stimulated cell proliferation through Erk1/2 and p38 phosphorylation and also cyclin D1 upregulation mediated by Akt/ATF-2 signaling. Moreover, rReg3α administration significantly reduced the pancreatic damage caused by L-Arg injection, as shown in histological examination and serum amylase, lipase and C-reactive protein (CRP) assays. Not only acinar cell necrosis but also apoptosis found in the pancreas of AP mice were alleviated by rReg3α. Finally, upregulated Bcl-2 and Bcl-xL and suppressed poly (ADP-ribose) synthetase/polymerase (PARP) levels were detected as being relevant to the mechanism of rReg3α protection. We therefore conclude that rReg3α acts as a protective polypeptide against AP in mice by enhancing Bcl-2 and Bcl-xL expressions and suppressing PARP level.