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1.  Vaccination with the Recombinant Brucella Outer Membrane Protein 31 or a Derived 27-Amino-Acid Synthetic Peptide Elicits a CD4+ T Helper 1 Response That Protects against Brucella melitensis Infection  
Infection and Immunity  2005;73(12):8079-8088.
The immunogenicity and protective efficacy of the recombinant 31-kDa outer membrane protein from Brucella melitensis (rOmp31), administered with incomplete Freund's adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp31 conferred protection against B. ovis and B. melitensis infection. rOmp31 induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp31, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp31-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis of Brucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp31 immunization elicited specific CD4+ T cells that secrete IL-2 and gamma interferon, while CD8+ T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp31-elicited protection against B. melitensis infection is mediated by CD4+ T cells while the contribution of CD8+ T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp31 on the Brucella membrane, a peptide that contains amino acids 48 to 74 of Omp31. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+ T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp31 against B. melitensis but less protection than was induced by rOmp31 against B. ovis. Our results indicate that rOmp31 could be a useful candidate for the development of subunit vaccines against B. melitensis and B. ovis.
PMCID: PMC1307072  PMID: 16299302
2.  Preventive Effect of Dipeptidyl Peptidase-4 Inhibitor on Atherosclerosis Is Mainly Attributable to Incretin's Actions in Nondiabetic and Diabetic Apolipoprotein E-Null Mice 
PLoS ONE  2013;8(8):e70933.
Several recent reports have revealed that dipeptidyl peptidase (DPP)-4 inhibitors have suppressive effects on atherosclerosis in apolipoprotein E-null (Apoe−/−) mice. It remains to be seen, however, whether this effect stems from increased levels of the two active incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).
Nontreated Apoe−/− mice, streptozotocin-induced diabetic Apoe−/− mice, and db/db diabetic mice were administered the DPP-4 inhibitor vildagliptin in drinking water and co-infused with either saline, the GLP-1 receptor blocker, exendin(9–39), the GIP receptor blocker, (Pro3)GIP, or both via osmotic minipumps for 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages were determined.
Vildagliptin increased plasma GLP-1 and GIP levels without affecting food intake, body weight, blood pressure, or plasma lipid profile in any of the animals tested, though it reduced HbA1c in the diabetic mice. Diabetic Apoe−/− mice exhibited further-progressed atherosclerotic lesions and foam cell formation compared with nondiabetic counterparts. Nondiabetic and diabetic Apoe−/− mice showed a comparable response to vildagliptin, namely, remarkable suppression of atherosclerotic lesions with macrophage accumulation and foam cell formation in peritoneal macrophages. Exendin(9–39) or (Pro3)GIP partially attenuated the vildagliptin-induced suppression of atherosclerosis. The two blockers in combination abolished the anti-atherosclerotic effect of vildagliptin in nondiabetic mice but only partly attenuated it in diabetic mice. Vildagliptin suppressed macrophage foam cell formation in nondiabetic and diabetic mice, and this suppressive effect was abolished by infusions with exendin(9–39)+(Pro3)GIP. Incubation of DPP-4 or vildagliptin in vitro had no effect on macrophage foam cell formation.
Vildagliptin confers a substantial anti-atherosclerotic effect in both nondiabetic and diabetic mice, mainly via the action of the two incretins. However, the partial attenuation of atherosclerotic lesions by the dual incretin receptor antagonists in diabetic mice implies that vildagliptin confers a partial anti-atherogenic effect beyond that from the incretins.
PMCID: PMC3742603  PMID: 23967137
3.  Characterization of Conserved Combined T and B Cell Epitopes in Leptospira interrogans Major Outer Membrane Proteins OmpL1 and LipL41 
BMC Microbiology  2011;11:21.
Leptospira interrogans are bacterial pathogens of animal that cause zoonotic infections in human. Outer membrane proteins of leptospire are among the most effective antigens which can stimulate remarkable immune responses during the infection processes, and thus are currently considered leading candidate vaccine antigens. The objective of the present study is to predict and confirm major combined B and T cell epitopes of leptospiral outer membrane proteins OmpL1 and LipL41, as well as to evaluate their capacity in the induction of immune responses in BALB/c mice.
In this study, four epitopes from OmpL1 and four from LipL41 conserved regions were evaluated for their potential utilization in leptospire vaccines. Firstly, combined B and T cell epitopes were predicted by softwares and expressed using a phage display system. OmpL1 residues 87-98 and 173-191 (OmpL187-98 and OmpL1173-191) and LipL4130-48, LipL41233-256 of LipL41 were identified as immunodominant B cell epitopes by Western blot. Epitopes OmpL1173-191, OmpL1297-320 of OmpL1 and LipL41233-256, LipL41263-282 of LipL41 were identified as immunodominant CD4+ T cell epitopes through proliferation analysis of splenocytes from recombinant OmpL1 (rOmpL1) or recombinant LipL41 (rLipL41)-immunized BALB/c (H-2d) mice. These epitopes induced responses of CD4+ T cells and Th1 (T helper cells) type cytokine responses during the infection.
This work identified combined T and B cell immunodominant epitopes in outer membrane proteins OmpL1 and LipL41 of Leptospira interrogans. OmpL1173-191 of OmpL1 and LipL41233-256 of LipL41 could be useful in a vaccine against Leptospira. The findings could also contribute to the development of effective cross-protective vaccine strategies for leptospirosis.
PMCID: PMC3038132  PMID: 21269437
4.  Glucose-Dependent Insulinotropic Polypeptide Prevents the Progression of Macrophage-Driven Atherosclerosis in Diabetic Apolipoprotein E-Null Mice 
PLoS ONE  2012;7(4):e35683.
We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.
Nondiabetic Apoe−/− mice, streptozotocin-induced diabetic Apoe−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.
Diabetic Apoe−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe−/− mice of the same age. GIP infusion in diabetic Apoe−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro3]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.
Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.
PMCID: PMC3335006  PMID: 22536426
5.  Effector Mechanisms in Low-Dose Streptozotocin-induced Diabetes 
Developmental Immunology  1998;6(1-2):119-128.
The cellular and molecular requirements for β-cell damages in an immune-mediated toxininduced insulin-dependent diabetes mellitus have been studied in the model of multiple low-dose streptozotocin-induced diabetes in rats and mice. It was found that strain-related susceptibility to diabetes induction correlated with a higher level of IL-2, IFN-γ, and TNF-α production, whereas such differences were not observed when IL-1 and NO production by macrophages were analyzed; elimination of immunoregulatory RT6+T cells that increases IFN-γ production, enhances susceptibility to MLD-STZ-induced diabetes; mercury-induced Th-2 cells downregulated the disease; IFN-γ-mediated macrophage activation to produce proinflammatory cytokines rather than NO is an important event in early diabetogenic effects of invading macrophages; inhibition of IL-1 activity downregulates diabetes induction; and generation of NO in β cells appears to be important for diabetogenic effects. Taken together, data indicate that MLD-STZ diabetes is induced by Th-1 lymphocytes that secrete soluble effector molecules that activate macrophages and promote destruction of β cells possibly by both nitric oxide and nonnitric oxide-mediated mechanisms.
PMCID: PMC2276003  PMID: 9716913
Insulin-dependent diabetes mellitus; nitric oxide; RT^6+ T cells; interferon-γ; tumor necrosis factor; interleukin- 1; interleukin-1 inhibitor
6.  The Oral Histone Deacetylase Inhibitor ITF2357 Reduces Cytokines and Protects Islet β Cells In Vivo and In Vitro 
Molecular Medicine  2011;17(5-6):369-377.
In type 1 diabetes, inflammatory and immunocompetent cells enter the islet and produce proinflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); each contribute to β-cell destruction, mediated in part by nitric oxide. Inhibitors of histone deacetylases (HDAC) are used commonly in humans but also possess antiinflammatory and cytokine-suppressing properties. Here we show that oral administration of the HDAC inhibitor ITF2357 to mice normalized streptozotocin (STZ)-induced hyperglycemia at the clinically relevant doses of 1.25–2.5 mg/kg. Serum nitrite levels returned to nondiabetic values, islet function improved and glucose clearance increased from 14% (STZ) to 50% (STZ + ITF2357). In vitro, at 25 and 250 nmol/L, ITF2357 increased islet cell viability, enhanced insulin secretion, inhibited MIP-1α and MIP-2 release, reduced nitric oxide production and decreased apoptosis rates from 14.3% (vehicle) to 2.6% (ITF2357). Inducible nitric oxide synthase (iNOS) levels decreased in association with reduced islet-derived nitrite levels. In peritoneal macrophages and splenocytes, ITF2357 inhibited the production of nitrite, as well as that of TNFα and IFNγ at an IC50 of 25–50 nmol/L. In the insulin-producing INS cells challenged with the combination of IL-1β plus IFNγ, apoptosis was reduced by 50% (P < 0.01). Thus at clinically relevant doses, the orally active HDAC inhibitor ITF2357 favors β-cell survival during inflammatory conditions.
PMCID: PMC3105153  PMID: 21193899
7.  Immunotherapeutic effects of pentoxifylline in type 1 diabetic mice and its role in the response of T-helper lymphocytes 
Pentoxifylline is an immunomodulatory and anti-inflammatory agent and is used in vascular disorders. It has been shown that pentoxifylline inhibits proinflammatory cytokines production. The purpose of this study was to investigate the therapeutic effects of pentoxifylline on the treatment of autoimmune diabetes in mice.
Materials and Methods:
Diabetes was induced by multiple low dose of streptozotocin (MLDS) injection (40 mg/kg/day for 5 consecutive days) in male C57BL/6 mice. After induction of diabetes, mice were treated with pentoxifylline (100 mg/kg/day IP) for 21 days. Blood glucose levels and plasma levels of insulin were measured. Splenocytes were tested for proliferation by MTT test and cytokine production by ELISA.
Pentoxifylline treatment prevented hyperglycemia and increased plasma insulin levels in the diabetic mice. Aside from reducing lymphocyte proliferation, pentoxifylline significantly inhibited the production of proinflammatory interleukin 17 (IL-17) as well as interferon gamma (IFN-γ), while increased anti-inflammatory cytokine IL-10 as compared with those in MLDS group (diabetic control group).
These findings indicate that pentoxifylline may have therapeutic effect against the autoimmune destruction of the pancreatic beta-cells during the development of MLDS-induced type 1 diabetes in mice.
PMCID: PMC4414990  PMID: 25945237
Cytokine; Pentoxifylline; Type 1 diabetes mellitus
8.  Knockout of Toll-like Receptor-2 attenuates both the Pro-Inflammatory State of Diabetes and Incipient Diabetic Nephropathy 
Type 1 Diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy (DN) remains unknown.
Methods and Results
We induced T1DM in TLR2 knockout mice (TLR2−/−) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, kidneys were obtained for flow cytometry, Western blot, microscopy, cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88 dependent signaling were increased in diabetic mice (WT+STZ) compared to non-diabetic WT mice. These biomarkers were attenuated in diabetic TLR2−/− macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased TGF-β and laminin compared to WT mice. Nephrin, podocin and podocyte number and effacement were restored and TGF-β, laminin levels were decreased in TLR2−/−+ STZ mice kidneys versus WT+STZ. Peritoneal and Kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2−/−+STZ mice.
These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.
PMCID: PMC3148786  PMID: 21617141
TLR2; nephropathy; inflammation; diabetes; complications
9.  Blocking Type I Interferon Signaling Rescues Lymphocytes from Oxidative Stress, Exhaustion, and Apoptosis in a Streptozotocin-Induced Mouse Model of Type I Diabetes 
Elevated levels of type I interferon (IFN) during type 1 diabetes mellitus (T1D) are associated with a defective immune response. In the present study, we investigated whether blocking type I IFN signaling during streptozotocin- (STZ-) induced T1D in mice improves lymphocyte proliferation and escape from continuous apoptosis. Three groups of mice were examined: diabetic mice, type I IFN signaling-incompetent diabetic mice, and control nondiabetic mice. We first found that diabetes induction was accompanied by an elevation in the plasma levels of reactive oxygen species (ROS), hydroperoxide, malondialdehyde (MDN), and the proinflammatory cytokines IL-1α, IL-1β, IL-6, and CXCL10. Blocking type 1 IFN signaling in diabetic mice significantly decreased the levels of oxidative stress and proinflammatory cytokines. In addition, lymphocytes from diabetic mice exhibited a marked reduction in their proliferative capacity, increased apoptosis, upregulation of the exhaustion marker PD-1, and aberrant phosphorylation of STAT1, STAT2, AKT and IκB-α. Interestingly, following the blocking of type I IFN signaling in diabetic mice, the lymphocytes exhibited restored proliferative capacity, decreased apoptosis, normal expression of PD-1, and normal phosphorylation of STAT1, STAT2, AKT and IκB-α. Our data suggest that elevated levels of type I IFN during T1D trigger lymphocyte exhaustion and a defective lymphocyte-medicated immune response.
PMCID: PMC3606800  PMID: 23533683
10.  BMP4 is increased in the aortas of diabetic ApoE knockout mice and enhances uptake of oxidized low density lipoprotein into peritoneal macrophages 
BMP4, a member of the transforming growth factor-beta superfamily, is upregulated in the aortas of diabetic db/db mice. However, little is known about its role in diabetic atherosclerosis. Therefore, we examined the roles of BMP4 in the formation of diabetic atherosclerosis in apolipoprotein E knockout (ApoE KO) mice and in the uptake of oxidized low density lipoprotein (oxLDL) in peritoneal macrophages of wild-type mice.
To induce diabetes, ApoE KO mice were intraperitoneally injected with streptozotocin. Diabetic and non-diabetic ApoE KO mice were then fed a high-fat diet for 4 weeks. Next, to investigate a role of BMP4 in the peritoneal macrophages, we examined the uptake of oxLDL in BMP4-treated macrophages.
Diabetic ApoE KO mice showed accelerated progression of aortic plaques accompanied by increased luminal plaque area. Western blot analysis showed that BMP4 expression in the whole aorta was greatly increased in diabetic ApoE KO mice, than non-diabetic mice. Western blot analysis showed that the BMP4/SMAD1/5/8 signaling pathway was strongly activated in the aorta from diabetic ApoE KO mice, compared with control ApoE KO mice. Double immunofluorescence staining showed that BMP4 was expressed in MOMA2-labeled macrophage in the aortic lesions of ApoE KO mice. BMP4 significantly increased the uptake of oxLDL into peritoneal macrophages in vitro.
We show that in the aorta of diabetic ApoE KO mice, BMP4 is increased and activates SMAD1/5/8. Our in vitro findings indicate that BMP4 enhances oxLDL uptake in mouse peritoneal macrophages, suggesting BMP4 may be involved in aortic plaque formation in diabetic ApoE KO mice. Targeting BMP4 may offer a new strategy for inhibition of plaque progression and stabilization of atherosclerotic lesions.
PMCID: PMC3852969  PMID: 24107300
BMP4; Atherosclerosis; Diabetes; Macrophage; Oxidized low density lipoproteins
11.  Acinetobacter baumannii rOmpA Vaccine Dose Alters Immune Polarization and Immunodominant Epitopes 
Vaccine  2012;31(2):313-318.
The rOmpA vaccine has been shown to protect mice from lethal infection caused by extreme-drug-resistant (XDR) Acinetobacter baumannii. The role of dose in immunology of the rOmpA vaccine was explored.
Mice were vaccinated with various doses of rOmpA plus aluminum hydroxide (Al(OH)3) adjuvant. The impact of dose on antibody titers, cytokine production, and immunodominant epitopes were defined.
Anti-rOmpA IgG and IgG subtype titers were higher at larger vaccine doses (30 and 100 µg vs. 3 µg). The 3 µg dose induced a balanced IFN-γ-IL-4 immune response while the 100 µg dose induced a polarized IL-4/Type 2 response. Epitope mapping revealed distinct T cell epitopes that activated IFN-γ-, IL-4-, and IL-17-producing splenocytes. Vaccination with the 100 µg dose caused epitope spreading among IL-4-producing splenocytes, while it induced fewer reactive epitopes among IFN-γ-producing splenocytes.
Vaccine dose escalation resulted in an enhanced Type 2 immune response, accompanied by substantial IL-4-inducing T cell epitope spreading and restricted IFN-γ-inducing epitopes. These results inform continued development of the rOmpA vaccine against A. baumannii, and also are of general importance in that they indicate that immune polarization and epitope selectivity can be modulated by altering vaccine dose.
PMCID: PMC3557524  PMID: 23153442
Acinetobacter baumannii; OmpA; vaccine; type 1/type 2 immunity; epitope spreading
12.  Angiotensin II Type II Receptor Deficiency Accelerates the Development of Nephropathy in Type I Diabetes via Oxidative Stress and ACE2 
Experimental Diabetes Research  2011;2011:521076.
Since the functional role(s) of angiotensin II (Ang II) type II receptor (AT2R) in type I diabetes is unknown, we hypothesized that AT2R is involved in decreasing the effects of type I diabetes on the kidneys. We induced diabetes with low-dose streptozotocin (STZ) in both AT2R knockout (AT2RKO) and wild-type (WT) male mice aged 12 weeks and followed them for 4 weeks. Three subgroups nondiabetic, diabetic, and insulin-treated diabetic (Rx insulin implant) were studied. Systolic blood pressure (SBP), physiological parameters, glomerular filtration rate (GFR), renal morphology, gene expression, and apoptosis were assessed. After 4 weeks of diabetes, compared to WT controls, AT2RKO mice clearly developed features of early diabetic nephropathy (DN), such as renal hypertrophy, tubular apoptosis, and progressive extracellular matrix (ECM) protein accumulation as well as increased GFR. AT2RKO mice presented hypertension unaffected by diabetes. Renal oxidative stress (measured as heme oxygenase 1 (HO-1) gene expression and reactive oxygen species (ROS) generation) and intrarenal renin angiotensin system components, such as angiotensinogen (Agt), AT1R, and angiotensin-converting enzyme (ACE) gene expression, were augmented whereas angiotensin-converting enzyme2 (ACE2) gene expression was decreased in renal proximal tubules (RPTs) of AT2RKO mice. The renal changes noted above were significantly enhanced in diabetic AT2RKO mice but partially attenuated in insulin-treated diabetic WT and AT2RKO mice. In conclusion, AT2R deficiency accelerates the development of DN, which appears to be mediated, at least in part, via heightened oxidative stress and ACE/ACE2 ratio in RPTs.
PMCID: PMC3205615  PMID: 22110472
13.  Diabetic Nephropathy Is Accelerated by Farnesoid X Receptor Deficiency and Inhibited by Farnesoid X Receptor Activation in a Type 1 Diabetes Model 
Diabetes  2010;59(11):2916-2927.
The pathogenesis of diabetic nephropathy is complex and involves activation of multiple pathways leading to kidney damage. An important role for altered lipid metabolism via sterol regulatory element binding proteins (SREBPs) has been recently recognized in diabetic kidney disease. Our previous studies have shown that the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, modulates renal SREBP-1 expression. The purpose of the present study was then to determine if FXR deficiency accelerates type 1 diabetic nephropathy in part by further stimulation of SREBPs and related pathways, and conversely, if a selective FXR agonist can prevent the development of type 1 diabetic nephropathy.
Insulin deficiency and hyperglycemia were induced with streptozotocin (STZ) in C57BL/6 FXR KO mice. Progress of renal injury was compared with nephropathy-resistant wild-type C57BL/6 mice given STZ. DBA/2J mice with STZ-induced hyperglycemia were treated with the selective FXR agonist INT-747 for 12 weeks. To accelerate disease progression, all mice were placed on the Western diet after hyperglycemia development.
The present study demonstrates accelerated renal injury in diabetic FXR KO mice. In contrast, treatment with the FXR agonist INT-747 improves renal injury by decreasing proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis, and modulating renal lipid metabolism, macrophage infiltration, and renal expression of SREBPs, profibrotic growth factors, and oxidative stress enzymes in the diabetic DBA/2J strain.
Our findings indicate a critical role for FXR in the development of diabetic nephropathy and show that FXR activation prevents nephropathy in type 1 diabetes.
PMCID: PMC2963551  PMID: 20699418
14.  The altered expression of glucose-regulated proteins 78 in different phase of streptozotocin-affected pancreatic beta-cells 
Cell Stress & Chaperones  2008;14(1):43-48.
Endoplasmic reticulum (ER) stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cells and contributes to the development of type 1 diabetes. The chaperone molecule, glucose-regulated proteins 78 (Grp78), is required to maintain ER function during toxic insults. In this study, we investigated the changes of Grp78 expression in different phases of streptozotocin (STZ)-affected beta-cells to explore the relationship between Grp78 and the response of beta-cells to ER stress. An insulinoma cell line (NIT-1) treated with STZ for different time periods and STZ-induced diabetic Balb/C mice at different time points were used as the model system. The level of Grp78 and C/EBP homologous protein (CHOP) mRNA were detected by real-time polymerase chain reaction and their protein by immunoblot. Apoptosis and necrosis was measured by flow cytometry. In addition, the changes of Grp78 protein in STZ-treated nondiabetic mice were also detected by immunoblot. Grp78 expression significantly increased in the early phase but decreased in the later phase of affected beta-cells, while CHOP was induced and apoptosis occurred along with the decrease of Grp78. Interestingly, the Grp78 protein of STZ-treated nondiabetic mice increased stably compared with that of the control. From the results, we can conclude that Grp78 may contribute to the response of beta-cells to ER stress, and more attention should be paid to Grp78 in the improvement of diabetes.
PMCID: PMC2673903  PMID: 18597185
ER stress; Glucose-regulated proteins 78; Streptozotocin; Diabetic mice; Beta-cells
15.  Novel role of curcumin in the prevention of cytokine-induced islet death in vitro and diabetogenesis in vivo 
British Journal of Pharmacology  2008;155(5):702-713.
Background and purpose:
Oxidative stress caused by cytokine exposure is a major cause of pancreatic islet death in vitro and of diabetogenesis. Antioxidant compounds may prevent cytokine-induced damage to islet cells. Hence, we studied the potential of curcumin, an antioxidant and anti-inflammatory compound, in vitro to protect islets against pro-inflammatory cytokines and in vivo to prevent the progression of diabetes induced by multiple low doses of streptozotocin (MLD-STZ).
Experimental approach:
Pancreatic islets from C57/BL6J mice were pretreated with curcumin (10 μM) and then exposed to a combination of cytokines. Islet viability, reactive oxygen species (ROS), NO, inducible NO synthase and NF-κB translocation were studied. Curcumin pretreated (7.5 mg kg−1 day−1) C57/BL6J mice were given MLD-STZ (40 mg kg−1), and various parameters of diabetes induction and progression were monitored.
Key results:
Curcumin protected islets from cytokine-induced islet death in vitro by scavenging ROS and normalized cytokine-induced NF-κB translocation by inhibiting phosphorylation of inhibitor of kappa B alpha (IκBα). In vivo, curcumin also prevented MLD-STZ, as revealed by sustained normoglycaemia, normal glucose clearance and maintained pancreatic GLUT2 levels. Pro-inflammatory cytokine concentrations in the serum and pancreas were raised in STZ-treated animals, but not in animals pretreated with curcumin before STZ.
Conclusions and implications:
Here, we have demonstrated for the first time that curcumin in vitro protects pancreatic islets against cytokine-induced death and dysfunction and in vivo prevents STZ-induced diabetes.
PMCID: PMC2584917  PMID: 18695642
curcumin; cytokines; islet death; MLD-STZ
16.  Oral delivery of Brucella spp. recombinant protein U-Omp16 abrogates the IgE-mediated milk allergy 
Human Vaccines & Immunotherapeutics  2014;10(7):2015-2023.
Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.
Balb/c mice were sensitized with cholera toxin and cow’s milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response.
We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet.
These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine.
PMCID: PMC4186055  PMID: 25424811
milk allergy; U-Omp16; oral adjuvant; IgE; immunomodulation; intestinal mucosa
17.  Proanthocyanidin Attenuation of Oxidative Stress and NF-κB Protects Apolipoprotein E-Deficient Mice against Diabetic Nephropathy 
Hyperlipidemia and hyperglycemia result in oxidative stress and play a major role in the development of diabetic nephropathy (DN). We explored the effects of proanthocyanidin (PA) on the induction and progression of DN in apolipoprotein E-deficient mice. Diabetes Mellitus was induced in ten-week-old male apoE−/−mice using streptozotocin (STZ). Mice were fed with a high-fat diet in presence or absence of PA. PA treatment significantly reduced the high cholesterol levels, restored renal functions, and reduced albuminuria in the PA-treated diabetic mice compared with the diabetic untreated mice. In addition, the glomerular mesangial expansion in the diabetic mice was attenuated as a result of PA supplementation. Moreover, PA treatment restored the elevated levels of MDA and CML and the reduced activity of SOD and GSH in the diabetic mice. Furthermore, PA feeding reduced the activation and translocation of NF-κB to the nucleus compared with the diabetic untreated animals. Reduction of NF-κB activation resulted in the attenuation of the expression of IL-6, TGFβ, and RAGE which protected PA-treated mice against DN. The renoprotective effects of PA were found to be time independent regardless of whether the dietary feeding with PA was started pre-, co-, or post-STZ injection. In conclusion, part of the beneficial effects of PA includes the disruption of the detrimental AGE-RAGE-NFκB pathways.
PMCID: PMC3759251  PMID: 24023581
18.  Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice 
Journal of Clinical Investigation  1999;103(10):1431-1436.
Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic β cells is upregulated by cytotoxic cytokines like IL-1β. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent β-cell cytotoxicity. To evaluate the role of 12-LO gene expression in immune-mediated islet destruction, we used 12-LO knockout (12-LO KO) mice. Male homozygous 12-LO KO mice and control C57BL/6 mice received 5 consecutive daily injections of low-dose streptozotocin to induce immune-mediated diabetes. Fasting serum glucose and insulin levels were measured at 7-day intervals, and the mice were followed up for 28 days. 12-LO KO mice were highly resistant to diabetes development compared with control mice and had higher serum insulin levels on day 28. Isolated pancreatic islets were treated with IL-1β, TNF-α, and IFN-γ for 18 hours. Glucose-stimulated insulin secretion in cytokine-treated islets from C57/BL6 mice decreased 54% from that of untreated islets. In marked contrast, the same cytokine mix led to only a 26% decrease in islets from 12-LO KO mice. Furthermore, cytokine-induced 12-hydroxyeicosatetraenoic acid (12-HETE) production was absent in 12-LO KO islets but present in C57/BL6 islets. Isolated peritoneal macrophages were stimulated for 48 hours with IFN-γ + LPS and compared for nitrate/nitrite generation. 12-LO KO macrophages generated 50% less nitrate/nitrite when compared with C57BL/6 macrophages. In summary, elimination of leukocyte 12-LO in mice ameliorates low dose streptozotocin–induced diabetes by increasing islet resistance to cytokines and decreasing macrophage production of nitric oxide.
PMCID: PMC408453  PMID: 10330425
19.  A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection against B. melitensis and B. ovis Infection by Eliciting a Specific Cytotoxic Response  
Infection and Immunity  2005;73(10):6537-6546.
The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8+ T cells, which mediate cytotoxicity via perforins, but also CD4+ T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8+ T cells, although CD4+T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8+ T cells that eliminate Brucella-infected cells via the perforin pathway.
PMCID: PMC1230944  PMID: 16177328
20.  Liver X Receptor Modulates Diabetic Retinopathy Outcome in a Mouse Model of Streptozotocin-Induced Diabetes 
Diabetes  2012;61(12):3270-3279.
Endothelial progenitor cells (EPCs), critical for mediating vascular repair, are dysfunctional in a hyperglycemic and/or hypercholesterolemic environment. Their dysfunction contributes to the progression of diabetic macro- and microvascular complications. Activation of “cholesterol-sensing” nuclear receptors, the liver X receptors (LXRα/LXRβ), protects against atherosclerosis by transcriptional regulation of genes important in promoting cholesterol efflux and inhibiting inflammation. We hypothesized that LXR activation with a synthetic ligand would correct diabetes-induced EPC dysfunction and improve diabetic retinopathy. Studies were performed in streptozotocin (STZ)-injected DBA/2J mice fed a high-fat Western diet (DBA/STZ/WD) and treated with the LXR agonist GW3965 and in LXRα−/−, LXRβ−/−, and LXRα/β−/− mice. Retinas were evaluated for number of acellular capillaries and glial fibrillary acidic protein (GFAP) immunoreactivity. Bone marrow EPCs were analyzed for migratory function and gene expression. Compared with vehicle-treated DBA/STZ/WD mice, GW3965 treated mice showed fewer acellular capillaries and reduced GFAP expression. These mice also exhibited enhanced EPC migration and restoration of inflammatory and oxidative stress genes toward nondiabetic levels. LXRα−/−, LXRβ−/−, and LXRα/β−/− mice developed acellular capillaries and EPC dysfunction similar to the DBA/STZ/WD mice. These studies support a key role for LXR in retinal and bone marrow progenitor dysfunction associated with type 1 diabetes. LXR agonists may represent promising pharmacologic targets for correcting retinopathy and EPC dysfunction.
PMCID: PMC3501845  PMID: 22891211
21.  Arginase-2 Mediates Diabetic Renal Injury 
Diabetes  2011;60(11):3015-3022.
To determine 1) whether renal arginase activity or expression is increased in diabetes and 2) whether arginase plays a role in development of diabetic nephropathy (DN).
The impact of arginase activity and expression on renal damage was evaluated in spontaneously diabetic Ins2Akita mice and in streptozotocin (STZ)-induced diabetic Dilute Brown Agouti (DBA) and arginase-2–deficient mice (Arg2−/−).
Pharmacological blockade or genetic deficiency of arginase-2 conferred kidney protection in Ins2Akita mice or STZ-induced diabetic renal injury. Blocking arginases using S-(2-boronoethyl)-l-cysteine for 9 weeks in Ins2Akita mice or 6 weeks in STZ-induced diabetic DBA mice significantly attenuated albuminuria, the increase in blood urea nitrogen, histopathological changes, and kidney macrophage recruitment compared with vehicle-treated Ins2Akita mice. Furthermore, kidney arginase-2 expression increased in Ins2Akita mice compared with control. In contrast, arginase-1 expression was undetectable in kidneys under normal or diabetes conditions. Arg2−/− mice mimicked arginase blockade by reducing albuminuria after 6 and 18 weeks of STZ-induced diabetes. In wild-type mice, kidney arginase activity increased significantly after 6 and 18 weeks of STZ-induced diabetes but remained very low in STZ-diabetic Arg2−/− mice. The increase in kidney arginase activity was associated with a reduction in renal medullary blood flow in wild-type mice after 6 weeks of STZ-induced diabetes, an effect significantly attenuated in diabetic Arg2−/− mice.
These findings indicate that arginase-2 plays a major role in induction of diabetic renal injury and that blocking arginase-2 activity or expression could be a novel therapeutic approach for treatment of DN.
PMCID: PMC3198072  PMID: 21926276
22.  Diabetes-Induced Chemogenic Hypoalgesia is Paralleled by Attenuated Stimulus-Induced Fos Expression in the Spinal Cord of Diabetic Mice 
Chronic hyperglycemia in diabetes induces abnormal nerve pathologies, resulting in diabetic neuropathy (DN). Sensory symptoms of DN can manifest as positive (painful), negative (insensate), or both. Streptozotocin (STZ)-induced diabetic C57Bl/6 mice have reduced cutaneous innervation and display reduced behavioral responses to noxious stimuli, reflecting the insensate aspect of the human syndrome. Current studies were undertaken to determine whether the diabetes-induced deficits in pain responses are reflected by changes in spinal activation in this model of DN. Nocifensive responses of nondiabetic and diabetic mice to formalin injection were measured 1, 3, 5 and 7 weeks post-STZ, and at each time point formalin-induced spinal Fos expression was quantified. Responses of diabetic mice were significantly reduced during the second phase of the formalin test beginning 3 weeks post-STZ, and during Phase 1 beginning 5 weeks post-STZ. Consistent with the behavioral responses, the number of Fos-positive cells in the dorsal horn of diabetic animals was significantly reduced beginning 3 weeks post-STZ and continuing 5 and 7 weeks post-STZ. The deficits at 5 weeks post-STZ were restored by 2-week treatments with insulin or neurotrophins. These results demonstrate that the reduced sensation occurring from progressive peripheral axon loss results in functional deficits in spinal cord activation.
PMCID: PMC1994928  PMID: 17532267
Neuropathy; pain behavior; Fos; NGF; GDNF; Insulin
23.  High Pancreatic n-3 Fatty Acids Prevent STZ-Induced Diabetes in Fat-1 Mice: Inflammatory Pathway Inhibition 
Diabetes  2011;60(4):1090-1099.
Because of confounding factors, the effects of dietary n-3 polyunsaturated fatty acids (PUFA) on type 1 diabetes remain to be clarified. We therefore evaluated whether fat-1 transgenic mice, a well-controlled experimental model endogenously synthesizing n-3 PUFA, were protected against streptozotocin (STZ)-induced diabetes. We then aimed to elucidate the in vivo response at the pancreatic level.
β-Cell destruction was produced by multiple low-doses STZ (MLD-STZ). Blood glucose level, plasma insulin level, and plasma lipid analysis were then performed. Pancreatic mRNA expression of cytokines, the monocyte chemoattractant protein, and GLUT2 were evaluated as well as pancreas nuclear factor (NF)-κB p65 and inhibitor of κB (IκB) protein expression. Insulin and cleaved caspase-3 immunostaining and lipidomic analysis were performed in the pancreas.
STZ-induced fat-1 mice did not develop hyperglycemia compared with wild-type mice, and β-cell destruction was prevented as evidenced by lack of histological pancreatic damage or reduced insulin level. The prevention of β-cell destruction was associated with no proinflammatory cytokine induction (tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase) in the pancreas, a decreased NF-κB, and increased IκB pancreatic protein expression. In the fat-1–treated mice, proinflammatory arachidonic-derived mediators as prostaglandin E2 and 12-hydroxyeicosatetraenoic acid were decreased and the anti-inflammatory lipoxin A4 was detected. Moreover, the 18-hydroxyeicosapentaenoic acid, precursor of the anti-inflammatory resolvin E1, was highly increased.
Collectively, these findings indicate that fat-1 mice were protected against MLD-STZ–induced diabetes and pointed out for the first time in vivo the beneficial effects of n-3 PUFA at the pancreatic level, on each step of the development of the pathology—inflammation, β-cell damage—through cytokine response and lipid mediator production.
PMCID: PMC3064083  PMID: 21330635
24.  Protective Effect of Topiramate on Hyperglycemia-Induced Cerebral Oxidative Stress, Pericyte Loss and Learning Behavior in Diabetic Mice 
Diabetes mellitus-associated damage to the microvasculature of the brain is caused by hyperglycemia-induced oxidative stress, which results in pericyte loss, blood-brain barrier disruption, and impaired cognitive function. Oxidative stress, in diabetes, is caused by reactive oxygen species produced during accelerated respiration (mitochondrial oxidative metabolism of glucose). The rate of respiration is regulated by mitochondrial carbonic anhydrases (CAs). Inhibition of these enzymes protects the brain from diabetic damage. Previously, we reported that topiramate, a mitochondrial CA inhibitor, at a dose of 50 mg/kg/day protects the brain in diabetes by reducing oxidative stress and restoring pericyte numbers. Topiramate has high affinity for both mitochondrial CAs; therefore, it is conceivable that a much lower dose may inhibit these enzymes and thus protect the brain from hyperglycemia-induced oxidative damage. Therefore, in an effort to reduce the toxicity associated with higher doses of topiramate, the current study was designed to investigate the effect of 1.0 mg/kg topiramate on reducing oxidative stress, restoring pericyte numbers in the brain, and improving the impaired learning behavior in diabetic mouse. Diabetes was induced by a one-time injection of streptozotocin and topiramate was administered daily for 12 weeks. Levels of oxidative stress, reduced glutathione (GSH) and 4-hydroxy-2-trans-nonenal (HNE) were measured in the brain and pericyte/endothelial cell ratios in isolated brain microvessels. Learning behavior was assessed by T-maze foot shock avoidance test. A significant decrease in GSH (control, 12.2 ± 0.4 vs. diabetic, 10.8 ± 0.4 vs. diabetic + topiramate, 12.6 ± 0.6, p<0.05) and an increase in HNE (control, 100 ± 4.2, vs. diabetic, 127.3 ± 8.8 vs. diabetic + topiramate, 93.9 ± 8.4 p<0.05) in diabetic mice were corrected by topiramate treatment. Topiramate treatment also resulted in restoration of pericyte numbers in diabetic mice (control, 25.89 ± 0.85 vs. diabetic, 18.14 ± 0.66 vs. diabetic + topiramate, 24.35 ± 0.53, p<0.001) and improvement in learning behavior. In conclusion, these data clearly demonstrate that topiramate at 1.0 mg/kg protects the mouse brain from diabetic damage. A 1.0 mg/kg topiramate in the mouse translates to a 5.0 mg daily dose in a 60 kg human, which may help slow the onset and progression of diabetic complications in the human brain.
PMCID: PMC4479302  PMID: 26120599
Diabetes; Brain microvasculature; Cognition; Topiramate; Oxidative stress; Mitochondrial CAs
25.  Glycine therapy inhibits the progression of cataract in streptozotocin-induced diabetic rats 
Molecular Vision  2012;18:439-448.
The purpose of this paper was to investigate the effect of the oral administration of L-glycine (Gly) on the development of diabetic cataract induced by streptozotocin (STZ) in rats.
Two groups of male Wistar rats were intraperitoneally injected with a single dose of STZ (65 mg/kg bodyweight). Then, one group of diabetic rats and a control group were administered with 1% of Gly in drinking water for three months, ad libitum. Cataract development was monitored biweekly through ophthalmoscope inspection and was classified into four stages. At the end of 12 weeks, the animals were sacrificed and some biochemical parameters were determined in their lenses. The parameters include advanced glycation end products (AGEs), glycated proteins, total and soluble protein, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), aldose reductase (AR), and sorbitol dehydrogenase (SDH). Some parameters were also determined in the serum and blood of the rats.
Diabetic cataract gradually progressed in the STZ-administered group with no other treatment. Consequently, up to the end of the experiment, 2/3 of the animals in this group reached to the last stage of the cataract (mature cataract). The progress of this process was much slower in the diabetic group that was treated with Gly. At the end of the study, the visual cataract score was significantly lower in the diabetic group treated with Gly compared to those administered with STZ. Some lens parameters, including glycated proteins, AGEs, SOD, and AR activities, were increased while some others, including soluble and total protein, GSH level, and CAT activity, were decreased due to diabetes induction. After Gly treatment, all the above-named parameters had reverse changes except for the CAT activity. The SDH activity in the lenses had no changes due to diabetes or treatment. In addition, this treatment significantly decreased the amount of serum glucose (Glc), serum AGEs, and glycated hemoglobin (HbA1c) in the diabetic rats. Gly also increased the ferric reducing antioxidant power (FRAP) in the serum of diabetic rats. However, the decreased bodyweight of animals due to diabetes induction was not compensated by Gly administration. It is important to note that Gly had no effect on normal rat parameters.
The results indicated that the oral administration of Gly significantly delayed the onset and the progression of diabetic cataract in rats. These effects were due to its antiglycating action and to a lesser extent, due to the inhibition of oxidative stress and polyol pathway.
PMCID: PMC3283203  PMID: 22355255

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