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1.  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
2.  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
3.  Immunization with Recombinant Brucella Species Outer Membrane Protein Omp16 or Omp19 in Adjuvant Induces Specific CD4+ and CD8+ T Cells as Well as Systemic and Oral Protection against Brucella abortus Infection▿  
Infection and Immunity  2008;77(1):436-445.
Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in the outer membrane proteins (OMPs) of B. abortus: Omp16 and Omp19. In this study, we assessed the use of these proteins as vaccines against Brucella in BALB/c mice. Immunization with lipidated Omp16 (L-Omp16) or L-Omp19 in incomplete Freund's adjuvant (IFA) conferred significant protection against B. abortus infection. Vaccination with unlipidated Omp16 (U-Omp16) or U-Omp19 in IFA induced a higher degree of protection than the respective lipidated versions. Moreover, the level of protection induced after U-Omp16 or U-Omp19 immunization in IFA was similar to that elicited by live B. abortus S19 immunization. Flow cytometric analysis showed that immunization with U-Omp16 or U-Omp19 induced antigen-specific CD4+ as well as CD8+ T cells producing gamma interferon. In vivo depletion of CD4+ or CD8+ T cells in mice immunized with U-Omp16 or U-Omp19 plus IFA resulted in a loss of the elicited protection, indicating that both cell types are mediating immune protection. U-Omp16 or U-Omp19 vaccination induced a T helper 1 response, systemic protection in aluminum hydroxide formulation, and oral protection with cholera toxin adjuvant against B. abortus infection. Both immunization routes exhibited a similar degree of protection to attenuated Brucella vaccines (S19 and RB51, respectively). Overall these results indicate that U-Omp16 or U-Omp19 would be a useful candidate for a subunit vaccine against human and animal brucellosis.
PMCID: PMC2612246  PMID: 18981242
4.  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
5.  Improved Immunogenicity of a Vaccination Regimen Combining a DNA Vaccine Encoding Brucella melitensis Outer Membrane Protein 31 (Omp31) and Recombinant Omp31 Boosting▿  
In the present study, we report an attempt to improve the immunogenicity of the Omp31 antigen by a DNA prime-protein boost immunization regimen. We immunized BALB/c mice with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant and characterized the resulting immune responses and the protective efficacy against Brucella ovis and B. melitensis infection. Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization. In conclusion, pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis.
PMCID: PMC1951060  PMID: 17428946
6.  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
7.  Anti-oxidant effect of gold nanoparticles restrains hyperglycemic conditions in diabetic mice 
Oxidative stress is imperative for its morbidity towards diabetic complications, where abnormal metabolic milieu as a result of hyperglycemia, leads to the onset of several complications. A biological antioxidant capable of inhibiting oxidative stress mediated diabetic progressions; during hyperglycemia is still the need of the era. The current study was performed to study the effect of biologically synthesized gold nanoparticles (AuNPs) to control the hyperglycemic conditions in streptozotocin induced diabetic mice.
The profound control of AuNPs over the anti oxidant enzymes such as GSH, SOD, Catalase and GPx in diabetic mice to normal, by inhibition of lipid peroxidation and ROS generation during hyperglycemia evidence their anti-oxidant effect during hyperglycemia. The AuNPs exhibited an insistent control over the blood glucose level, lipids and serum biochemical profiles in diabetic mice near to the control mice provokes their effective role in controlling and increasing the organ functions for better utilization of blood glucose. Histopathological and hematological studies revealed the non-toxic and protective effect of the gold nanoparticles over the vital organs when administered at dosage of 2.5 mg/kilogram.body.weight/day. ICP-MS analysis revealed the biodistribution of gold nanoparticles in the vital organs showing accumulation of AuNPs in the spleen comparatively greater than other organs.
The results obtained disclose the effectual role of AuNPs as an anti-oxidative agent, by inhibiting the formation of ROS, scavenging free radicals; thus increasing the anti-oxidant defense enzymes and creating a sustained control over hyperglycemic conditions which consequently evoke the potential of AuNPs as an economic therapeutic remedy in diabetic treatments and its complications.
PMCID: PMC2914719  PMID: 20630072
8.  Depressed immunological defence mechanisms in mice with experimentally induced diabetes. 
Infection and Immunity  1980;28(1):127-131.
Persistent diabetes mellitus with marked hyperglycemia was induced in mice by the administration of streptozotocin. In these streptozotocin-induced diabetic mice, resistance to tubercle bacillus challenge and primary as well as secondardy humoral immune responses against foreign erythrocytes were markedly depressed. The T-cell function in delayed hypersensitivity to 2,4-dinitro-1-fluorobenzene and bacterial phagocytic activity or peritoneal macrophages were markedly depressed. In contrast, the B-cell function in antibody production against T-independent antigen and the intracellular killing of bacteria in peritoneal macrophages were intact. We concluded that depression of the T-cell function or the phagocytic activity of macrophages or both may be the main immunological defect in these mice.
PMCID: PMC550901  PMID: 6966615
9.  Synthetic peptide segments from the Escherichia coli porin OmpF constitute leukocyte activators. 
Infection and Immunity  1990;58(8):2719-2724.
After characterization of the porin OmpF and selection of molecular structures responsible for leukocyte activation by using computer-assisted epitope analysis, the analogs OmpF (153-174) (containing amino acids 153 to 174), OmpF (157-174), and OmpF (275-285) were synthesized and tested. Like the native protein, the segments were mitogenic for BALB/c splenocytes and induced B lymphocyte differentiation into antibody-producing plasma cells and tumor cytotoxicity of macrophages against the fibroblast cell line L929. We thus demonstrated that defined peptide segments are responsible for the leukocyte-activating properties of a major bacterial surface protein.
PMCID: PMC258883  PMID: 1695209
10.  Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis 
BMC Microbiology  2011;11:39.
The osmotic regulator OmpR in Escherichia coli regulates differentially the expression of major porin proteins OmpF and OmpC. In Yersinia enterocolitica and Y. pseudotuberculosis, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in Y. pestis are not yet fully understood.
Y. pestis OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The ompR mutation likely did not affect the virulence of Y. pestis strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the ompR mutation, indicating the global regulatory role of OmpR in Y. pestis. Real-time RT-PCR or lacZ fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. ompC, F, X, and R were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner.
OmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. Y. pestis OmpR regulates ompC, F, X, and R directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and × at high osmolarity in Y. pestis, in contrast to the reciprocal regulation of them in E. coli. The main difference is that ompF expression is not repressed at high osmolarity in Y. pestis, which is likely due to the absence of a promoter-distal OmpR-binding site for ompF.
PMCID: PMC3050692  PMID: 21345178
11.  Polymicrobial Sepsis Enhances Clearance of Apoptotic Immune Cells by Splenic Macrophages 
Surgery  2007;142(2):253-261.
Macrophage phagocytosis of apoptotic cells induces an anti-inflammatory macrophage phenotype. Immune cell apoptosis is widespread in sepsis; however, it is unknown whether sepsis alters the capacity of macrophages to clear this expanded population. We hypothesize that sepsis will enhance splenic macrophage phagocytosis of apoptotic immune cells, potentially contributing to immunosuppression.
Sepsis was induced in C57BL/6J mice by cecal ligation and puncture (CLP). Apoptosis was induced in mouse thymocytes by dexamethasone incubation. At multiple time points following CLP/sham, splenic and peritoneal macrophages were isolated, plated on glass coverslips, co-incubated with apoptotic thymocytes, fixed, and the coverslips were then Giemsa stained. Splenic macrophages were also isolated 48 hours after CLP/sham, stained with the red fluorescent dye PKH26, co-incubated with green fluorescent dye CFSE-stained apoptotic thymocytes and then coverslips were fixed and counterstained with DAPI. The macrophage phagocytic index (PI) was calculated for both staining methods.
The PI of CLP splenic macrophages was significantly higher than sham by 24 hours, and this difference was sustained through 48 hours.
Studies suggest that apoptotic cell clearance leads to an anti-inflammatory macrophage condition, which together with our findings in septic macrophages, may point at a process that contributes to septic immune suppression.
PMCID: PMC2023968  PMID: 17689693
Sepsis; mice; apoptosis; phagocytosis; macrophage
12.  The Protective Role of Nrf2 in Streptozotocin-Induced Diabetic Nephropathy 
Diabetes  2010;59(4):850-860.
Diabetic nephropathy is one of the major causes of renal failure, which is accompanied by the production of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls the antioxidant response essential for maintaining cellular redox homeostasis. Here, we report our findings demonstrating a protective role of Nrf2 against diabetic nephropathy.
We explore the protective role of Nrf2 against diabetic nephropathy using human kidney biopsy tissues from diabetic nephropathy patients, a streptozotocin-induced diabetic nephropathy model in Nrf2−/− mice, and cultured human mesangial cells.
The glomeruli of human diabetic nephropathy patients were under oxidative stress and had elevated Nrf2 levels. In the animal study, Nrf2 was demonstrated to be crucial in ameliorating streptozotocin-induced renal damage. This is evident by Nrf2−/− mice having higher ROS production and suffering from greater oxidative DNA damage and renal injury compared with Nrf2+/+ mice. Mechanistic studies in both in vivo and in vitro systems showed that the Nrf2-mediated protection against diabetic nephropathy is, at least, partially through inhibition of transforming growth factor-β1 (TGF-β1) and reduction of extracellular matrix production. In human renal mesangial cells, high glucose induced ROS production and activated expression of Nrf2 and its downstream genes. Furthermore, activation or overexpression of Nrf2 inhibited the promoter activity of TGF-β1 in a dose-dependent manner, whereas knockdown of Nrf2 by siRNA enhanced TGF-β1 transcription and fibronectin production.
This work clearly indicates a protective role of Nrf2 in diabetic nephropathy, suggesting that dietary or therapeutic activation of Nrf2 could be used as a strategy to prevent or slow down the progression of diabetic nephropathy.
PMCID: PMC2844833  PMID: 20103708
13.  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
14.  Expression of Recipient CD47 on Rat Insulinoma Cell Xenografts Prevents Macrophage-Mediated Rejection through SIRPα Inhibitory Signaling in Mice 
PLoS ONE  2013;8(3):e58359.
We have previously proven that the interspecies incompatibility of CD47 is responsible for in vitro phagocytosis of xenogeneic cells by host macrophages. Utilizing an in vivo model in the present study, we investigated whether genetically engineered expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophage-mediated xenograft rejection. INS-1E cells transfected with the pRc/CMV-mouse CD47 vector (mCD47-INS-1E) induced SIRPα-tyrosine phosphorylation in mouse macrophages in vitro, whereas cells transfected with the control vector (cont-INS-1E) did not. When these cells were injected into the peritoneal cavity of streptozotocin-induced diabetic Rag2−/−γ chain −/− mice, which lack T, B, and NK cells, the expression of mouse CD47 on the INS-1E cells markedly reduced the susceptibility of these cells to phagocytosis by macrophages. Moreover, these mice became normoglycemic after receiving mCD47-INS-1E, whereas the mice that received cont-INS-1E failed to achieve normoglycemia. Furthermore, injection of an anti-mouse SIRPα blocking monoclonal antibody into the mouse recipients of mCD47-INS-1E cells prevented achievement of normoglycemia. These results demonstrate that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of the expression of recipient CD47 on xenogeneic donor cells could provide inhibitory signals to recipient macrophages via SIPRα; this constitutes a novel approach for preventing macrophage-mediated xenograft rejection.
PMCID: PMC3589424  PMID: 23472187
15.  Diabetes increases mortality after myocardial infarction by oxidizing CaMKII 
The Journal of Clinical Investigation  2013;123(3):1262-1274.
Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.
PMCID: PMC3673230  PMID: 23426181
16.  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
17.  Oxidative Stress and Free-Radical Oxidation in BCG Granulomatosis Development 
Background. Little is known about the role of free-radical and oxidative stress signaling in granuloma maturation and resolution. We aimed to study the activity of free-radical oxidation processes in the dynamics of BCG-induced generalized granulomatosis in mice. Methods. Chronic granulomatous inflammation was induced in male BALB/c mice by intravenously injecting the BCG vaccine, and the production of oxidative stress (activity of free-radical oxidation processes) and histological changes in the lungs, liver, and peritoneal exudate were measured 3, 30, 60, and 90 days after infection. Results. The tuberculous granuloma numerical density and diameter continuously increased from day 30 to day 90, and the macrophage content within the granulomas progressively diminished with a concomitant elevation in the number of epithelioid cells. The activity of the free-radical oxidation processes in the liver (i.e., the intensity of the homogenate chemiluminescence) reached a maximum at postinfection day 60 and subsequently began to decrease. The peak generation of reactive oxygen species by phagocytes in the peritoneal exudate (measured using flow cytometry) was also shifted in time and fell on day 30. Conclusions. The rise in the steady-state concentration of H2O2 in the liver of mice with BCG-induced granulomatosis is not related to local H2O2 production by phagocytes, and a decrease in the severity of generalized inflammation precedes the resolution of local inflammation.
PMCID: PMC3655644  PMID: 23738038
18.  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
19.  Outer membrane characteristics of Salmonella enteritidis phage type 4 growing in chickens. 
Epidemiology and Infection  1993;111(3):449-454.
Strains of Salmonella enteritidis belonging to phage type 4 (SE4) were grown in the peritoneal cavities of chickens, and without subculture on laboratory media examined for inducible in vivo phenotypic characteristics. These bacteria expressed three major outer membrane proteins (OMPs) of 33, 35 and 36 kilodaltons (kDa), and iron regulated OMPs of 74, 78 and 81 kDa. Bacteria growing in vivo did not express flagella, or fimbriae with a subunit molecular mass of 14 kDa (14 kDa fimbriae). Two OMPs of 55 and 23 kDa, expressed during culture in nutrient broth, were repressed during growth in chickens. Possession of a 38 MDa 'mouse virulence' plasmid did not influence the expression of OMPs, flagella or fimbriae. It was concluded that strains of SE4 growing in chicken tissues, use an enterobactin mediated iron uptake system to obtain ferric ions, do not express flagella or 14 kDa fimbriae and appear not to express novel OMPs involved in survival in vivo.
PMCID: PMC2271258  PMID: 7903640
20.  Dual Inhibition of Classical Protein Kinase C-α and Protein Kinase C-β Isoforms Protects Against Experimental Murine Diabetic Nephropathy 
Diabetes  2013;62(4):1167-1174.
Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy with proteinuria and peritubular extracellular matrix production. We have previously shown that the PKC isoforms α and β mediate different cellular effects. PKC-β contributes to hyperglycemia-induced renal matrix production, whereby PKC-α is involved in the development of albuminuria. We further tested this hypothesis by deletion of both isoforms and used a PKC inhibitor. We analyzed the phenotype of nondiabetic and streptozotocin (STZ)-induced diabetic homozygous PKC-α/β double-knockout mice (PKC-α/β−/−). After 8 weeks of diabetes mellitus, the high-glucose–induced renal and glomerular hypertrophy as well as transforming growth factor-β1) and extracellular matrix production were diminished in the PKC-α/β−/− mice compared with wild-type controls. Urinary albumin/creatinine ratio also was significantly reduced, however, it was not completely abolished in diabetic PKC-α/β−/− mice. Treatment with CGP41252, which inhibits PKC-α and PKC-β, is able to prevent the development of albuminuria and to reduce existing albuminuria in type 1 (STZ model) or type 2 (db/db model) diabetic mice. These results support our hypothesis that PKC-α and PKC-β contribute to the pathogenesis of diabetic nephropathy, and that dual inhibition of the classical PKC isoforms is a suitable therapeutic strategy in the prevention and treatment of diabetic nephropathy.
PMCID: PMC3609593  PMID: 23434935
21.  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
22.  Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo. 
Infection and Immunity  1989;57(7):2136-2140.
The ompC, ompD, and ompF genes encode the three major porins of Salmonella typhimurium. ompR encodes a positive regulator required for the expression of ompC and ompF. Transposon-generated mutations in ompC, ompD, ompF, and ompR were introduced into the S. typhimurium mouse virulent strain SL1344 by P22-mediated transduction. Following preliminary characterization in vitro, the strains were used to challenge BALB/c mice by using the oral or intravenous route. Strains harboring ompC or ompF mutations were as virulent as SL1344 after oral challenge. Strains harboring ompD mutations had a slight reduction in virulence. In contrast, ompR mutants failed to kill BALB/c mice after oral challenge and the intravenous 50% lethal dose was reduced by approximately 10(5). The ompR mutants persisted in murine tissues for several weeks following oral or intravenous challenge. Furthermore, mice orally immunized with these ompR mutant strains were well protected against challenge with virulent SL1344.
PMCID: PMC313852  PMID: 2543631
23.  nNOS-Dependent Reactivity of Cerebral Arterioles in Type 1 Diabetes 
Brain research  2007;1184:365-371.
Our goals were to determine whether Type 1 diabetes (T1D) alters neuronal nitric oxide synthase (nNOS) dependent reactivity of cerebral arterioles and to identify a potential role for oxidative stress in T1D-induced impairment in nNOS-dependent responses of cerebral arterioles. Rats were injected with vehicle (sodium citrate buffer) or streptozotocin (50 mg/kg IP) to induce T1D. Two to three months later, we measured functional responses of cerebral arterioles to nNOS-dependent (NMDA and kainate) and -independent (nitroglycerin) agonists in nondiabetic and diabetic rats before and during inhibition of oxidative stress using tempol (100 μM). In addition, we measured superoxide anion production under basal conditions, during stimulation with NMDA and kainate, and during treatment with tempol. We found that nNOS-dependent, but not -independent, vasodilatation was impaired in diabetic compared to nondiabetic rats. In addition, treatment of the cerebral microcirculation with tempol restored impaired nNOS-dependent vasodilatation in diabetic rats towards that observed in nondiabetic rats. Further, the production of superoxide anion (lucigenin chemiluminescence) was increased in parietal cortical tissue of diabetic rats under basal conditions. Application of NMDA and kainate did not increase superoxide anion production in nondiabetic or diabetic rats. However, tempol decreased basal production of superoxide anion in diabetic rats. Our findings suggest that T1D impairs nNOS dependent dilatation of cerebral arterioles by a mechanism that appears to be related to the formation of superoxide anion.
PMCID: PMC2174607  PMID: 17991456
Diabetes; Brain; Nitric Oxide; Superoxide Anion; NMDA; Kainate
24.  Immunomodulatory properties of porins of some members of the family Enterobacteriaceae. 
Infection and Immunity  1997;65(6):2382-2388.
The outer membrane protein (OMP) preparation of Salmonella typhi was observed to have several immunomodulatory properties. Treatment of mice with an intraperitoneal injection of the OMP preparation enhanced both cellular and humoral responses of the mice to an unrelated antigen, a killed vaccine of Mycobacterium vaccae; both the delayed-type hypersensitivity (DTH) response and the antibody titers were enhanced. The predominant isotype of the antibody shifted from immunoglobulin G1 (IgG1) to IgG2a upon treatment with OMP. Treatment of mice with the OMP preparation improved the efficiency of in vitro antigen presentation by the peritoneal cells and also induced the cells to secrete interleukin-1. Treatment with the lipopolysaccharide (LPS) preparation of S. typhi had the opposite effect; i.e., the DTH response to M. vaccae was suppressed. Treatment with OMP neutralized the suppressive effects of LPS. The OMP preparation also had an enhancing effect on the innate immune mechanisms of the mice. Intraperitoneal injection of the OMP preparation enhanced the microbicidal activity of the peritoneal cells, and production of nitric oxide intermediates was stimulated. Injection of the OMP preparation into footpads of naive nonimmune mice induced a sustained hypersensitivity response that peaked at 24 h. Purified porins of the OMP preparation could induce both immunomodulation and hypersensitivity. Porins prepared from five different Salmonella strains and a strain of normal fecal Escherichia coli also exhibited immunomodulatory and hypersensitivity-inducing activities.
PMCID: PMC175330  PMID: 9169778
25.  Diabetes Adversely Affects Macrophages During Atherosclerotic Plaque Regression in Mice 
Diabetes  2011;60(6):1759-1769.
Patients with diabetes have increased cardiovascular risk. Atherosclerosis in these patients is often associated with increased plaque macrophages and dyslipidemia. We hypothesized that diabetic atherosclerosis involves processes that impair favorable effects of lipid reduction on plaque macrophages.
Reversa mice are LDL receptor–deficient mice that develop atherosclerosis. Their elevated plasma LDL levels are lowered after conditional knockout of the gene encoding microsomal triglyceride transfer protein. We examined the morphologic and molecular changes in atherosclerotic plaques in control and streptozotocin-induced diabetic Reversa mice after LDL lowering. Bone marrow–derived macrophages were also used to study changes mediated by hyperglycemia.
Reversa mice were fed a western diet for 16 weeks to develop plaques (baseline). Four weeks after lipid normalization, control (nondiabetic) mice had reduced plasma cholesterol (−77%), plaque cholesterol (−53%), and plaque cells positive for macrophage marker CD68+ (−73%), but increased plaque collagen (+116%) compared with baseline mice. Diabetic mice had similarly reduced plasma cholesterol, but collagen content increased by only 34% compared with baseline; compared with control mice, there were lower reductions in plaque cholesterol (−30%) and CD68+ cells (−41%). Diabetic (vs. control) plaque CD68+ cells also exhibited more oxidant stress and inflammatory gene expression and less polarization toward the anti-inflammatory M2 macrophage state. Many of the findings in vivo were recapitulated by hyperglycemia in mouse bone marrow–derived macrophages.
Diabetes hindered plaque regression in atherosclerotic mice (based on CD68+ plaque content) and favorable changes in plaque macrophage characteristics after the reduction of elevated plasma LDL.
PMCID: PMC3114401  PMID: 21562077

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