Papers by Clive Wasserfall
Pediatric Research, 2009
As the incidence of cystic fibrosis (CF) bone disease is increasing, we analyzed CF transmembrane... more As the incidence of cystic fibrosis (CF) bone disease is increasing, we analyzed CF transmembrane conductance regulator (CFTR) deficient mice (CF mice) to gain pathogenic insights. In these studies comparing adult (14 wk) CF and C57BL/6J mice, both bone length and total area were decreased in CF mice. Metaphyseal trabecular and cortical density were also decreased, as well as diaphyseal cortical and total density. Trabecular bone volume was diminished in CF mice. Female CF mice revealed decreased trabecular width and number compared with C57BL/6J, whereas males demonstrated no difference in trabecular number. Female CF mice had reduced mineralizing surface and bone formation rates. Conversely, male CF mice had increased mineralizing surface, mineral apposition, and bone formation rates compared with C57BL/6J males. Bone formation rate was greater in males compared with female CF mice. Smaller bones with decreased density in CF, despite absent differences in osteoblast and osteoclast surfaces, suggest CF transmembrane conductance regulator influences bone cell activity rather than number. Differences in bone formation rate in CF mice are suggestive of inadequate bone formation in females but increased bone formation in males. This proanabolic observation in male CF mice is consistent with other clinical sex differences in CF. (Pediatr Res 65: 311-316, 2009)
Blood, Jan 22, 2015
Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematological mal... more Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematological malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model we serendipitously discovered that MYXV prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 and soluble IL-2Rα, but unaffected IL-4 and IL-10. MYXV suppressed T cell proliferation in two patterns (full vs. partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymp...
Clinical immunology (Orlando, Fla.), 2015
We developed a novel poly(lactic-co-glycolic acid)-based, microparticle (MP) system providing con... more We developed a novel poly(lactic-co-glycolic acid)-based, microparticle (MP) system providing concurrent delivery of multiple encapsulated immuno-suppressive factors and antigen, for in vivo conditioning of dendritic cells (DCs) toward a tolerance promoting pathway. Subcutaneous administration prevents onset of type 1 diabetes (T1D) in NOD mice. Two MP sizes were made: phagocytosable MPs were fabricated encapsulating vitamin D3 or insulin B(9-23) peptide, while unphagocytosable MPs were fabricated encapsulating TGF-β1 or GM-CSF. The combination of Vit D3/TGF-β1 MPs confers an immature and LPS activation-resistant phenotype to DCs, and MP-delivered antigen is efficiently and functionally presented. Notably, two subcutaneous injections into 4week old NOD mice using the combination of MPs encapsulating Vit D3, Ins B, TGF-β1 and GM-CSF protected 40% of mice from T1D development, significant in comparison to the control. This work represents one of the first applications of a biomaterial...
PROTEOMICS, 2015
The need for biomarkers that illuminate the pathophysiology of type 1 diabetes (T1D), enhance ear... more The need for biomarkers that illuminate the pathophysiology of type 1 diabetes (T1D), enhance early diagnosis and provide additional avenues for therapeutic intervention is well recognized in the scientific community. We conducted a proteome-scale, two-stage serological AAb screening followed by an independent validation study. In the first stage, the immunoreactivity was compared between T1D cases and healthy controls against 6000 human proteins using the nucleic acid programmable protein array (NAPPA). Genes identified with higher signal intensities in patients were challenged with a larger sample set during the second stage. Statistical analysis revealed 26 novel autoantigens and a known T1D-associated autoantigen. During validation, we verified the presence of AAbs to dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) using the Luciferase ImmunoPrecipitation System (LIPS) assay (36% sensitivity, 98% specificity). The AUC for a combination of DYRK2A and the classical T1D AAb IA-2A was 0.90 compared to 0.72 for DYRK2A and 0.64 for IA-2A alone. This is the first systematic screening for seroreactivity against a large number of human proteins in T1D patients. We demonstrated the application of protein microarrays to identify novel autoantigens in T1D, expanded the current T1D "autoantigenome" and help fulfill the goal of searching for novel biomarker candidates for T1D.
Journal of materials chemistry. B, Materials for biology and medicine, Jan 7, 2014
Microparticulate systems are beginning to show promise for delivery of modulatory agents for immu... more Microparticulate systems are beginning to show promise for delivery of modulatory agents for immunotherapeutic applications which modulate dendritic cell (DC) functions. Co-administration of multiple factors is an emerging theme in immune modulation which may prove beneficial in this setting. Herein, we demonstrate that localized, controlled delivery of multiple factors can be accomplished through poly (lactic-co-glycolic acid) (PLGA) microparticle systems fabricated in two size classes of phagocytosable and unphagocytosable microparticles (MPs). The immunosuppressive ability of combinatorial multi-factor dual MP systems was evaluated by investigating effects on DC maturation, DC resistance to LPS-mediated maturation and proliferation of allogeneic T cells in a mixed lymphocyte reaction. Phagocytosable MPs (~2 μm) were fabricated encapsulating either rapamycin (RAPA) or all-trans retinoic acid (RA), and unphagocytosable MPs (~30 μm) were fabricated encapsulating either transforming ...
Diabetes, 2014
T-cell responses directed against insulin-secreting pancreatic β-cells are the key events highlig... more T-cell responses directed against insulin-secreting pancreatic β-cells are the key events highlighting type 1 diabetes (T1D). Therefore, a defective control of T-cell activation is thought to underlie T1D development. Recent studies implicated a B7-like negative costimulatory protein, V-set domain-containing T-cell activation inhibitor-1 (VTCN1), as a molecule capable of inhibiting T-cell activation and, potentially, an important constituent in experimental models of T1D. Here, we unravel a general deficiency within the VTCN1 pathway that is shared between diabetes-prone mice and a subset of T1D patients. Gradual loss of membrane-tethered VTCN1 from antigen-presenting cells combined with an increased release of soluble VTCN1 (sVTCN1) occurs in parallel to natural T1D development, potentiating hyperproliferation of diabetogenic T cells. Mechanistically, we demonstrate that the loss of membrane-tethered VTCN1 is linked to proteolytic cleavage mediated by the metalloproteinase nardilysin. The cleaved sVTCN1 fragment was detected at high levels in the peripheral blood of 53% T1D patients compared with only 9% of the healthy subjects. Elevated blood sVTCN1 levels appeared early in the disease progression and correlated with the aggressive pace of disease, highlighting the potential use of sVTCN1 as a new T1D biomarker, and identifying nardilysin as a potential therapeutic target.
Diabetes, 2015
Impaired regulatory B cell (Breg) responses are associated with several autoimmune diseases in hu... more Impaired regulatory B cell (Breg) responses are associated with several autoimmune diseases in humans; however, the role of Bregs in type 1 diabetes (T1D) remains unclear. We hypothesized that naturally occurring, interleukin-10 (IL-10)-producing Bregs maintain tolerance to islet autoantigens, and that hyperglycemic nonobese diabetic (NOD) mice and T1D patients lack these potent negative regulators. IgVH transcriptome analysis revealed that islet-infiltrating B cells in long-term normoglycemic (Lnglc) NOD, which are naturally protected from diabetes, are more antigen-experienced and possess more diverse B-cell receptor repertoires compared to those of hyperglycemic (Hglc) mice. Importantly, increased levels of Breg-promoting CD40(+) B cells and IL-10-producing B cells were found within islets of Lnglc compared to Hglc NOD. Likewise, healthy individuals showed increased frequencies of both CD40(+) and IL-10(+) B cells compared to T1D patients. Rituximab-mediated B-cell depletion foll...
Transplantation, 2002
Previous studies suggest that therapeutic expression of interleukin (IL)-4 by islet cells improve... more Previous studies suggest that therapeutic expression of interleukin (IL)-4 by islet cells improves their efficacy in transplantation models directed at reversing type 1 diabetes. We investigated the effects of introducing IL-4 into islets with recombinant adeno-associated virus (rAAV) on the reversal of hyperglycemia in a syngeneic marginal islet mass transplantation model. C57BL/6 islets were mock-transduced or transduced with rAAV expressing murine IL-4 (rAAV-IL-4) or rAAV expressing green fluorescent protein (rAAV-GFP) before transplantation of a marginal mass into diabetic mice. Normoglycemia was achieved in only 1/7 mice receiving rAAV-IL-4 transduced islets in comparison to 6/6 mock-transduced and 4/6 rAAV-GFP transduced animals. The failure of IL-4 expressing islets was not associated with cellular toxicity of rAAV or impairment of glucose-stimulated insulin release in vitro. Islet expression of IL-4 led to impaired metabolic function in mice receiving a marginal mass of syngeneic islets.
Pediatric Nephrology, 2009
The purpose of this study was to test the hypothesis that, in idiopathic minimal lesion nephrotic... more The purpose of this study was to test the hypothesis that, in idiopathic minimal lesion nephrotic syndrome (IMLNS), the T regulatory (T reg) cell suppressor mechanism is deficient, thereby enhancing cytokine release by T effector cells. Twenty-one patients with IMLNS, eight healthy controls and two patients with nephrotic syndrome and membranoproliferative glomerulonephritis were studied. The percentage of T reg cells was similar in the healthy controls and in patients with IMLNS in relapse or in remission. Thymidine incorporation in autologous T effector cells, as well as expression of the regulatory cytokine interleukin (IL)-10, was significantly reduced in patients in relapse when compared with patients in remission and healthy subjects. IL-2 expression was also reduced in patients in relapse but did not achieve statistical significance. In a different set of experiments, T cells, from subjects with IMLNS in remission, when stimulated with antiCD3-antiCD28 antibodies, secreted increased levels of cytokines. No such increase in cytokines was observed when cells from healthy controls were stimulated with same mitogen. The impaired T reg cell function observed in these patients may have pathogenic and therapeutic implications, because it could explain the persistence of the proposed pathogenic cytokines observed in the patients with IMLNS.
Journal of Translational Medicine, 2011
Background: Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory an... more Background: Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT) gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD) mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Methods: DBA/1 mice were immunized with bovine type II collagen (bCII) to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT). Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF), antibodies against both bovine (bCII) and mouse collagen II (mCII) were tested by ELISA. Results: Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8)-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially.
Human Gene Therapy, 2006
An imbalance of the immune-regulatory pathways plays an important role in the development of type... more An imbalance of the immune-regulatory pathways plays an important role in the development of type 1 diabetes. Therefore, immunoregulatory and antiinflammatory strategies hold great potential for the prevention of this autoimmune disease. Studies have demonstrated that two serine proteinase inhibitors, alpha1-antitrypsin (AAT) and elafin, act as potent antiinflammatory agents. In the present study, we sought to develop an efficient gene therapy approach to prevent type 1 diabetes. Cohorts of 4-week-old female nonobese diabetic (NOD) mice were injected intramuscularly with rAAV1-CB-hAAT, rAAV1-CB-hElafin, or saline. AAV1 vector mediated sustained high levels of transgene expression, sufficient to overcome a humoral immune response against hAAT. AAT gene therapy, contrary to elafin and saline, was remarkably effective in preventing type 1 diabetes. T cell receptor spectratyping indicated that AAT gene therapy altered T cell repertoire diversity in splenocytes from NOD mice. Adoptive transfer experiments demonstrated that AAT gene therapy attenuated cellular immunity associated with beta cell destruction. This study demonstrates that AAT gene therapy attenuates cell-mediated autoimmunity, alters the T cell receptor repertoire, and efficiently prevents type 1 diabetes in the NOD mouse model. These results strongly suggest that rAAV1-mediated AAT gene therapy may be useful as a novel approach to prevent type 1 diabetes.
Human Gene Therapy, 2005
Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in... more Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in acid alpha-glucosidase (GAA), and leads to cardiorespiratory failure by the age of 2 years. In this study, we investigate the impact of anti-GAA antibody formation on cross-correction of the heart, diaphragm, and hind-limb muscles from liver-directed delivery of recombinant adeno-associated virus (rAAV)5- and rAAV8-GAA vectors. GAA(-/-) mice receiving 1 x 10(12) vector genomes of rAAV5- or rAAV8-DHBV-hGAA were analyzed for anti-GAA antibody response, GAA levels, glycogen reduction, and contractile function. We demonstrate that restoration of GAA to the affected muscles is dependent on the presence or absence of the antibody response. Immune-tolerant mice had significantly increased enzyme levels in the heart and skeletal muscles, whereas immune-responsive mice had background levels of GAA in all tissues except the diaphragm. The increased levels of activity in immune-tolerant mice correlated with reduced glycogen in the heart and diaphragm and, overall, contractile function of the soleus muscle was significantly improved. These findings highlight the importance of the immune response to rAAV-encoded GAA in correcting GSDII and provide additional understanding of the approach to treatment of GSDII.
Hepatology, 1999
While patients with liver disease are known to have a higher prevalence of glucose intolerance, p... more While patients with liver disease are known to have a higher prevalence of glucose intolerance, preliminary studies suggest that hepatitis C virus (HCV) infection may be an additional risk factor for the development of diabetes mellitus. To further study the correlation of HCV infection and diabetes, we performed a retrospective analysis of 1,117 patients with chronic viral hepatitis and analyzed whether age, sex, race, hepatitis B virus (HBV) infection, HCV infection, and cirrhosis were independently associated with diabetes. In addition, a case-control study was conducted to determine the seroprevalence of HCV infection in a cohort of 594 diabetics and 377 clinic patients assessed for thyroid disease. In the former study after the exclusion of patients with conditions predisposing to hyperglycemia, diabetes was observed in 21% of HCV-infected patients compared with 12% of HBV-infected subjects (P ؍ .0004). Multivariate analysis revealed that HCV infection (P ؍ .02) and age (P ؍ .01) were independent predictors of diabetes. In the diabetes cohort, 4.2% of patients were found to be infected with HCV compared with 1.6% of control patients (P ؍ .02). HCV genotype 2a was observed in 29% of HCV-RNA-positive diabetic patients versus 3% of local HCV-infected controls (P F .005). In conclusion, the data suggest a relatively strong association between HCV infection and diabetes, because diabetics have an increased frequency of HCV infection, particularly with genotype 2a. Furthermore, it is possible that HCV infection may serve as an additional risk factor for the development of diabetes, beyond that attributable to chronic liver disease alone. (HEPATOLOGY 1999;29:328-333.)
Diabetes Care, 2009
OBJECTIVE -Interest continues to grow regarding the therapeutic potential for umbilical cord bloo... more OBJECTIVE -Interest continues to grow regarding the therapeutic potential for umbilical cord blood therapies to modulate autoimmune disease. We conducted an open-label phase I study using autologous umbilical cord blood infusion to ameliorate type 1 diabetes.
Diabetes, 2014
Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinica... more Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65370-575 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional β-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4(+)Foxp3(+)CD25(+) regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D.
Diabetes, 2011
OBJECTIVE-Previous studies have noted a specific association between type 1 diabetes and insuffic... more OBJECTIVE-Previous studies have noted a specific association between type 1 diabetes and insufficient levels of vitamin D, as well as polymorphisms within genes related to vitamin D pathways. Here, we examined whether serum levels or genotypes of the vitamin D-binding protein (VDBP), a molecule key to the biologic actions of vitamin D, specifically associate with the disorder.
Autoimmunity Reviews, 2006
Type 1 diabetes results from the autoimmune destruction of the insulin producing pancreatic beta-... more Type 1 diabetes results from the autoimmune destruction of the insulin producing pancreatic beta-cells. For years, the notion that T-lymphocytes played a crucial role in the disorder's formation was considered such sound dogma, that interest in B-lymphocytes and autoantibodies as pathogenic variables was largely relegated to second-class status. However, much of our knowledge regarding the pathogenesis and natural history of this disease has been afforded by analysis of subjects having type 1 diabetes associated autoantibodies. While autoantibodies to more than two dozen autoantigens have been associated with this disease, a majority of interest has been directed at four autoantibodies; islet cell cytoplasmic (ICA), insulin (IAA), glutamic acid decarboxylase (GADA), and IA2/ICA512 autoantigen (IA2A). These autoantibodies, combined with other metabolic and genetic markers, are extremely effective for predicting eventual development of type 1 diabetes in otherwise healthy individuals. These autoantibodies have also aided in our understanding of disease heterogeneity and suggest that the autoimmune processes underlying type 1 diabetes initiate in the earliest stages of life (e.g., initial autoantibody formation at 9-18 months of age). Additional improvements are needed to more accurately define the time to disease onset, response to therapeutic intervention, the pathogenic features of the autoimmune response, and perhaps even the quantity of residual beta cell function.
Pediatric Research, 2008
Neonatal diet may influence the development of type 1 diabetes (T1D) in susceptible individuals t... more Neonatal diet may influence the development of type 1 diabetes (T1D) in susceptible individuals through an intestinal mucosal inflammatory response, resulting in loss of self-tolerance. We tested the hypothesis that formula feeding during the neonatal period accelerates the development of T1D in diabetes-prone BioBreeding (BBDP) rats through regulation of CD4+CD25+ regulatory T lymphocytes (T(reg)) and anti-inflammatory cytokines. BBDP rat pups fed rat milk substitute (RMS) via a "pup-in-the cup" system were compared with mother-fed (MF) rats. The spleen and thymus were analyzed for Foxp3-expressing CD4+/CD25+ T cells. Multiplex enzyme-linked immunosorbent assays (ELISAs) were performed to measure cytokine-induced neutrophil chemoattractant (CINC), tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-10, and IL-18. Diabetes-free survival, time of disease onset, and T(reg)/total T lymphocyte ratios were not different. MF pups had higher ileal CINC (p < 0.001) and IL-18 (p = 0.002), but no differences in the liver. There were no differences in ileal cytokine concentrations of 75-d-old rats, but the formula-fed rats had greater liver TNF-alpha (p < 0.001), IFN-gamma, and IL-4 (p < 0.01) and lower IL-10 (p = 0.002) compared with MF animals. Formula versus maternal milk altered the hepatic cytokine profile at 75 d toward an inflammatory pattern but did not result in altered T(reg) cell frequencies or the development of T1D.
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Papers by Clive Wasserfall