[Show abstract][Hide abstract] ABSTRACT: We had previously reported that stromal cell-derived factor 1 (SDF-1) mediates chemorepulsion of diabetogenic T cell adhesion to islet microvascular endothelium through unknown mechanisms in NOD mice. Here we report that SDF-1-mediated chemorepulsion occurs through slit homologue (SLIT)2-roundabout, axon guidance receptor, homologue 1 (Drosophila) (ROBO1) interactions.
C-X-C receptor (CXCR)4 and ROBO1 protein expression was measured in mouse and human T cells. Parallel plate flow chamber adhesion and detachment studies were performed to examine the molecular importance of ROBO1 and SLIT2 for SDF-1-mediated T cell chemorepulsion. Diabetogenic splenocyte transfer was performed in NOD/LtSz Rag1 (-/-) mice to examine the effect of the SDF-1 mimetic CTCE-0214 on adoptive transfer of diabetes.
CXCR4 and ROBO1 protein expression was elevated in diabetic NOD/ShiLtJ T cells over time and coincided with the onset of hyperglycaemia. CXCR4 and ROBO1 expression was also increased in human type 1 diabetic T cells, with ROBO1 expression maximal at less than 1 year post diagnosis. Cell detachment studies revealed that immunoneutralisation of ROBO1 prevented SDF-1-mediated chemorepulsion of NOD T cell firm adhesion to TNFα-stimulated islet endothelial cells. SDF-1 increased NOD T cell adhesion to recombinant adhesion molecules, a phenomenon that was reversed by recombinant SLIT2. Finally, we found that an SDF-1 peptide mimetic prevented NOD T cell adhesion in vitro and significantly delayed adoptive transfer of autoimmune diabetes in vivo.
These data reveal a novel molecular pathway, which regulates diabetogenic T cell recruitment and may be useful in modulating autoimmune diabetes.
[Show abstract][Hide abstract] ABSTRACT: Insulitis is an important pathological feature of autoimmune diabetes; however, mechanisms governing the recruitment of diabetogenic T-cells into pancreatic islets are poorly understood. Here, we determined the importance of leukocyte integrins beta(2)(Itgb2) and alphaL (ItgaL) in developing insulitis and frank diabetes.
Gene-targeted mutations of either Itgb2 or ItgaL were established on the NOD/LtJ mouse strain. Experiments were performed to measure insulitis and diabetes development. Studies were also performed measuring mutant T-cell adhesion to islet microvascular endothelial cells under hydrodynamic flow conditions. T-cell adhesion molecule profiles and adoptive transfer studies were also performed.
Genetic deficiency of either Itgb2 or ItgaL completely prevented the development of hyperglycemia and frank diabetes in NOD mice. Loss of Itgb2 or ItgaL prevented insulitis with Itgb2 deficiency conferring complete protection. In vitro hydrodynamic flow adhesion studies also showed that loss of Itgb2 completely abrogated T-cell adhesion. However, ItgaL deficiency did not alter NOD T-cell adhesion to or transmigration across islet endothelial cells. Adoptive transfer of ItgaL-deficient splenocytes into NOD/Rag-1 mice did not result in development of diabetes, suggesting a role for ItgaL in NOD/LtJ T-cell activation.
Together, these data demonstrate that genetic deficiency of Itgb2 or ItgaL confers protection against autoimmune diabetes through distinctly different mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Diabetogenic T-cell recruitment into pancreatic islets facilitates beta-cell destruction during autoimmune diabetes, yet specific mechanisms governing this process are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1) controls T-cell recruitment, and genetic polymorphisms of SDF-1 are associated with early development of type 1 diabetes.
Here, we examined the role of SDF-1 regulation of diabetogenic T-cell adhesion to islet microvascular endothelium. Islet microvascular endothelial cell monolayers were activated with tumor necrosis factor-alpha (TNF-alpha), subsequently coated with varying concentrations of SDF-1 (1-100 ng/ml), and assayed for T-cell/endothelial cell interactions under physiological flow conditions.
TNF-alpha significantly increased NOD/LtJ T-cell adhesion, which was completely blocked by SDF-1 in a dose-dependent manner, revealing a novel chemorepulsive effect. Conversely, SDF-1 enhanced C57BL/6J T-cell adhesion to TNF-alpha-activated islet endothelium, demonstrating that SDF-1 augments normal T-cell adhesion. SDF-1 chemorepulsion of NOD/LtJ T-cell adhesion was completely reversed by blocking G(i)alpha-protein-coupled receptor activity with pertussis toxin. CXCR4 protein expression was significantly decreased in NOD/LtJ T-cells, and inhibition of CXCR4 activity significantly reversed SDF-1 chemorepulsive effects. Interestingly, SDF-1 treatment significantly abolished T-cell resistance to shear-mediated detachment without altering adhesion molecule expression, thus demonstrating decreased integrin affinity and avidity.
In this study, we have identified a previously unknown novel function of SDF-1 in negatively regulating NOD/LtJ diabetogenic T-cell adhesion, which may be important in regulating diabetogenic T-cell recruitment into islets.
[Show abstract][Hide abstract] ABSTRACT: Electroencephalograms (EEG) and visual evoked potentials (VEP) in mice were recorded to evaluate loss of cortical function during the first 30 s after euthanasia by various methods. Tracheal cannulae (for positive-pressure ventilation, PPV) and cortical surface electrodes were placed in mice anesthetized with inhaled halothane. Succinylcholine was used to block spontaneous breathing in the mice, which then underwent continuous EEG recording. Photic stimuli (1 Hz) were presented to produce VEPs superimposed on the EEG. Anesthesia was discontinued immediately before euthanasia. Compared with that obtained before euthanasia, EEG activity during the 30-s study period immediately after euthanasia was significantly decreased after cervical dislocation (at 5 to 10 s), 100% PPV-CO2 (at 10 to 15 s), decapitation (at 15 to 20 s), and cardiac arrest due to KCl injection (at 20 to 25 s) but not after administration of 70% PPV-CO2. Similarly, these euthanasia methods also reduced VEP amplitude, although 100% PPV-CO2 treatment affected VEP amplitude more than it did EEG activity. Thus, 100% PPV-CO2 treatment significantly decreased VEP beginning 5 to 10 s after administration, with near abolition of VEP by 30 s. VEP amplitude was significantly reduced at 5 to 10 s after cervical dislocation and at 10 to 15 s after decapitation but not after either KCl or 70% PPV-CO2 administration. The data demonstrate that 100% PPV-CO2, decapitation, and cervical dislocation lead to rapid disruption of cortical function as measured by 2 different methods. In comparison, 70% PPV-CO2 and cardiac arrest due to intracardiac KCl injection had less rapid effects on cortical function.
Comparative medicine 01/2008; 57(6):570-3. · 0.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peripheral artery disease (PAD) is a prevalent cardiovascular disorder that results in tissue ischemia which can progress to critical limb ischemia. Restoration of tissue perfusion in the setting of chronic ischemia through stimulation of arteriogenesis and angiogenesis remains a key therapeutic target for PAD. However, experimental therapeutics, including growth factor and gene therapy, have had little clinical success indicating the need for a better understanding of molecular pathways required for therapeutic angiogenesis.
Here we report that phosphodiesterase-5 inhibition by sildenafil significantly increases vascular perfusion, tissue blood flow, and vascular density during chronic ischemia of the mouse hind limb. Importantly, sildenafil therapy did not alter any of these parameters in nonischemic limbs. Sildenafil increased tissue cGMP levels independently of increases in nitric oxide production, and sildenafil therapy stimulated angiogenesis in ischemic limbs of eNOS-/- and iNOS-/- mice. Lastly, sildenafil-mediated angiogenic activity was blocked by inhibition of protein kinase G using the PKG antagonist DT-3.
These data demonstrate that sildenafil therapy results in increased angiogenic activity through a PKG-dependent pathway that is independent of nitric oxide production or NOS activity and identify the angiogenic therapeutic potential of sildenafil for critical limb ischemia.
[Show abstract][Hide abstract] ABSTRACT: The adaptive immune system plays an important role in host defense against invading micro-organisms. Yet, mice deficient in T- and B-cells are surprisingly healthy and develop few spontaneous infections when raised under specific pathogen-free conditions (SPF). The objective of this study was to ascertain what role phagocyte-associated NADPH oxidase or myeloperoxidase (MPO) plays in host defense in mice lacking both T- and B-cells. To do this, we generated lymphopenic mice deficient in either NADPH oxidase or MPO by crossing gp91(phox)-deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko). We found that neither gp91 ko, MPO ko mice nor lymphocyte-deficient RAG ko mice developed spontaneous infections when raised under SPF conditions and all mice had life spans similar to wild-type (WT) animals. In contrast, gp91xRAG double-deficient (DKO) but not MPOxRAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Infections in the gp91xRAG DKO mice were characterized by granulomatous inflammation of the skin, liver, heart, brain, kidney, and lung. Addition of antibiotics to the drinking water attenuated the spontaneous infections and increased survival of the mice. Oyster glycogen-elicited polymorphonuclear neutrophils (PMNs) and macrophages obtained from gp91 ko and gp91xRAG DKO mice had no detectable NADPH oxidase activity whereas WT, RAG ko, and MPOxRAG DKO PMNs and macrophages produced large and similar amounts of superoxide in response to phorbol myristate acetate. The enhanced mortality of the gp91xRAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase activity (iNOS) as total numbers of elicited PMNs and macrophages as well as PMN- and macrophage-derived production of nitric oxide-derived metabolites in these mice were similar and not reduced when compared to that of WT mice. Taken together, our data suggest that that NADPH oxidase but not MPO (nor iNOS) is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each other's deficiency in providing resistance to spontaneous bacterial infections.
Biochemical and Biophysical Research Communications 05/2007; 355(3):801-6. DOI:10.1016/j.bbrc.2007.02.029 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox-sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A-induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A-mediated angiogenesis in ICAM-1(-/-) mice. Moreover, VEGF-A-dependent chemotaxis, eNOS phosphorylation, and nitric oxide production were impaired in ICAM-1(-/-) mouse aortic endothelial cells (MAEC) compared to WT MAEC. Decreasing intracellular GSH in ICAM-1(-/-) MAEC to levels observed in WT MAEC with 150 microM buthionine sulfoximine restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester mimicked defects observed in ICAM-1(-/-) cells. Deficient angiogenic responses in ICAM-1(-/-) cells were associated with increased expression of the lipid phosphatase PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A-induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.
Free Radical Biology and Medicine 04/2007; 42(5):720-9. DOI:10.1016/j.freeradbiomed.2006.12.010 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.
The Journal of Gene Medicine 09/2006; 8(9):1105-20. DOI:10.1002/jgm.935 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammatory bowel diseases (IBD) are chronic inflammatory disorders whose etiology remains unknown. Reports have shown that infiltration of leukocytes into intestinal tissue is a pathognomonic hallmark for this disease. Leukocyte beta(2) integrins are heterodimeric adhesion membrane proteins that are exclusively expressed on leukocytes and participate in immune cell adhesion and activation. In this study, we examined the pathophysiological role of the beta(2) integrins CD18, CD11a, and CD11b in the pathogenesis of dextran sodium sulfte (DSS)-induced experimental colitis. Disease activity was measured by daily assessment of clinical parameters including stool consistency, weight loss, occult blood, and gross rectal bleeding. Histopathological changes including severity of inflammation, surface epithelial/crypt damage, and depth of injury were also determined. The CD18 null and CD11a null mice had significantly lower disease activity and cumulative histopathological scores compared to wild-type mice. Interestingly, CD11b null mice did not show protection against DSS colitis and displayed increased disease activity compared to wild-type mice. Examination of specific leukocyte populations in the distal colon from various mice revealed significant attenuation of neutrophil and macrophage infiltrates in CD18, CD11a, and CD11b null mice. Surprisingly, the CD11b null mice showed a significant increase in plasma cell infiltration in response to DSS suggesting that this molecule may influence plasma cell function during colitis. This study demonstrates that genetic loss of CD18 or CD11a is protective during experimental colitis and that CD11b may serve a regulatory role during development of disease.
[Show abstract][Hide abstract] ABSTRACT: Current cancer gene therapies are hindered by poor gene delivery and lack of tumor specificity. In order to enhance tumor specificity, the therapeutic suicide gene HSV-1 thymidine kinase (TK) sequence was modified with a 5’ upstream-untranslated region (5’-UTR) from the rat bFGF mRNA. This modification restricts protein translation of the suicide gene and cytotoxicity to cancer cells previously shown to express high levels of the translation initiation factor eIF4E. MicroPET scans utilizing a MicroPET rodent four-ring system (modelR4) from CTI Concord Microsystems, LLC (Knoxville, TN) imaging the radiolabled HSV1-TK substrate 18F-penciclovir were used to test suicide gene delivery and selective expression in tumor cells with in vitro cell cultures and in vivo animal models. A non- lytic adenovirus vector containing the HSV1-TK gene (Ad-TK) or the HSV1-TK gene modified with a 5’-UTR (Ad-UTK) was used to deliver the suicide gene in both model systems. In vitro model studies compared TK expression from the two vectors in human breast epithelial cells MCF-10A, and MCF-10A cells stably transfected to express high levels of eIF4E (MCF-10A-4E). Administration of Ad-TK and Ad-UTK to non-tumor bearing nu/nu mice and nu/nu mice bearing human breast cancer tumors was used to compare in vivo expression of TK in non-tumor and tumor tissues, respectively. Analysis of emission data from these studies showed that Ad-TK administration resulted in broad expression of TK activity in normal and tumor tissues, whereas expression of suicide gene activity from Ad-UTK vector administration was restricted to the human breast epithelial cells expressing high levels of eIF4E (MCF-10A-4E) and the human breast cancer tumor tissues.
[Show abstract][Hide abstract] ABSTRACT: Leukocyte recruitment into pancreatic islets is believed to play an important pathophysiological role in autoimmune diabetes. Previous reports have suggested that several different adhesion molecules may be involved in leukocyte recruitment during autoimmune diabetes, including members of the leukocyte beta(2) integrins. Here we report that a gene-targeted deficiency of the beta(2) integrin, CD18, protects against multiple low-dose streptozotocin-induced autoimmune diabetes. CD18 null mice displayed lower blood glucose values throughout the study, with only 10% of these mice eventually developing diabetes compared to 95% in the control group. Importantly, the development of insulitis was markedly absent in the CD18 null mice, suggesting that members of this integrin subfamily predominately regulate leukocyte infiltration into pancreatic islets. This study demonstrates that the beta(2) integrins play a key pathophysiological role in the development of multiple low-dose streptozotocin-induced autoimmune diabetes.
American Journal Of Pathology 01/2005; 165(6):1849-52. DOI:10.1016/S0002-9440(10)63237-3 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rodent pinworms rarely cause clinical disease, but infestation can affect experimental results. Our facility maintained a colony of Wistar rats for behavioral pharmacology studies that had been infested with Syphacia muris for > 15 years. The laboratory in which the animals were housed encompassed several rooms and contained a variety of complex behavioral equipment, including > 60 operant chambers. Several prior attempts to eliminate the pinworms were unsuccessful because of inadequate duration of treatment and incomplete environmental decontamination. Many of the rats in this colony were food-restricted as part of behavioral studies. Pinworms were eliminated from these animals by treating them with 450 ppm fenbendazole-containing feed for 3 consecutive weeks followed by 6 weeks of alternating every other week with standard rodent diet. Rats not on food restriction protocols were treated on the same schedule with 150 ppm fenbendazole-containing feed. Environmental decontamination of eggs from the behavioral equipment was not attempted. One year after treatment, the colony has remained free of S. muris. We adapted previously published protocols to our situation, including the problem of food-restricted rats and unfeasible environmental decontamination, to eradicate S. muris from our colony.
Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science 01/2005; 44(1):23-5. · 0.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The contribution of pericellular proteolysis to tumor progression is well documented. To better understand protease biology and facilitate clinical translation, specific proteolytic systems need to be better defined. In particular, the precise role of endogenous protease inhibitors still needs to be deciphered. We reported previously that cystatin M, a potent endogenous inhibitor of lysosomal cysteine proteases, significantly suppressed in vitro cell proliferation, migration, and Matrigel invasion. Here, we show that scid mice orthotopically implanted with breast cancer cells expressing cystatin M show significantly delayed primary tumor growth and lower metastatic burden in the lungs and liver when compared with mice implanted with mock controls. The incidence of metastasis, however, appeared to be unaltered between the cystatin M group and the control group. Experimental metastasis assays suggest that cystatin M suppressed tumor cell proliferation at the secondary site. By using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we found consistent expression of cystatin M in normal human breast epithelial cells, whereas expression was decreased by 86% in invasive ductal carcinoma (IDC) cells of stage I to IV patients. Complete loss of expression of cystatin M was observed in two of three IDCs from stage IV patients. Immunohistochemical studies confirmed that expression of cystatin M in IDCs was partially or completely lost. We propose cystatin M as a novel candidate tumor suppressor gene for breast cancer.
Cancer Research 11/2004; 64(19):6957-64. DOI:10.1158/0008-5472.CAN-04-0819 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD18-deficient PL/J mice develop dermatitis characterized by hyperkeratosis, and a mixed dermal and epidermal inflammatory infiltrate. The development of this disease requires low-level CD18 expression and at least two PL/J loci. Currently, the mechanisms by which decreased beta(2) integrin expression on leukocytes promotes skin inflammation in PL/J mice are unknown. In these studies, we investigated the role of microbial infection and T lymphocytes in the pathogenesis of this disease. We found that germ-free CD18(-/-) PL/J mice developed dermatitis indistinguishable from that of mice raised in pathogen-free conditions. Adoptive transfer of CD18(-/-) PL/J splenocytes into skin disease-resistant CD18(+/-) PL/J mice failed to induce skin inflammation. However, transfer of CD18(+/-) splenocytes blocked the progression and ultimately led to resolution of skin disease in the majority of CD18(-/-) recipients. Depletion of both CD4(+) and CD8(+) T cells mice prior to onset of the disease significantly delayed the appearance of inflammatory skin disease. In contrast, single depletions of these T cells did not inhibit disease development. These studies show that dermatitis in CD18-deficient PL/J mice is not the consequence of infection, does not require bacterial superantigens, and is mediated by both CD4(+) and CD8(+) T lymphocytes. Furthermore, they suggest that one possible mechanism for skin disease development in these mice may involve the absence or dysfunctional activity of a regulatory T cell population. These mice may therefore be useful in identifying potential mechanisms of pathogenesis and genetic predisposition in human inflammatory skin diseases.
International Immunology 03/2004; 16(2):345-51. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Itgb2(tm1Bay) PL/J mice express low levels of the beta(2) integrins and, unlike Itgb2(tm1Bay) C57BL/6J mice, spontaneously develop psoriasiform dermatitis with several similarities to human psoriasis. To define the genetic requirements for skin disease susceptibility we analyzed more than 500 F2 progeny from an Itgb2(tm1Bay) (PL/J x C57BL/6J) intercross. We found that 23.5% developed chronic inflammatory skin disease, although significant differences in severity were observed. Another CD18 mutation, Itgb2(tm2Bay), has now been generated that completely eliminates CD18 expression. Surprisingly, of 10 Itgb2(tm2Bay) homozygote PL/J N4 mice generated, none showed clinical or histopathological evidence of disease. However, Itgb2(tm1Bay)/Itgb2(tm2Bay) PL/J mice developed dermatitis indistinguishable from Itgb2(tm1Bay) PL/J mice. In addition, approximately half of Itgb2(tm1Bay)/Itgb2(tm2Bay) (C57BL/6J x PL/J)F1 mice were found to develop mild psoriasiform dermatitis identical to the early stages of disease seen in Itgb2(tm1Bay) PL/J mice. Collectively, these results suggest a complex inheritance pattern of psoriasiform dermatitis in this model that involves lowered, but not absent, CD18 expression and at least two additional PL/J loci for the development of severe disease. The susceptibility allele can act in either a heterozygous or homozygous state, dependent on the level of CD18 expression.
American Journal Of Pathology 08/2003; 163(1):197-202. DOI:10.1016/S0002-9440(10)63643-7 · 4.59 Impact Factor