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1.  Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen 
Journal of autoimmunity  2013;44:21-33.
Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4+ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared “poised” and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4+ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4+ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4+ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.
PMCID: PMC3743101  PMID: 23810578
Autoimmune uveitis; Immune privilege; TCR transgenic mouse; Spontaneous disease
3.  Rodent Models of Experimental Autoimmune Uveitis 
Methods in molecular biology (Clifton, N.J.)  2012;900:10.1007/978-1-60761-720-4_22.
The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and reproducible model that is easily followed and quantitated. It is inducible with synthetic peptides derived from retinal autoantigens in commonly available strains of rats and mice. The ability to induce EAU in various gene-manipulated, including HLA-transgenic, mouse strains makes the EAU model suitable for the study of basic mechanisms as well as in clinically relevant interventions.
PMCID: PMC3810964  PMID: 22933083
Uveitis; Uveoretinitis; EAU; Autoimmunity; T cells; Tolerance; Th1; Th2; IRBP; S-Ag
4.  High inter-reviewer variability of spike detection on intracranial EEG addressed by an automated multi-channel algorithm 
Clinical Neurophysiology  2011;123(6):1088-1095.
The goal of this study was to determine the consistency of human reviewer spike detection and then develop a computer algorithm to make the intracranial spike detection process more objective and reliable.
Three human reviewers marked interictal spikes on samples of intracranial EEGs from 10 patients. The sensitivity, precision and agreement in channel ranking by activity were calculated between reviewers. A computer algorithm was developed to parallel the way human reviewers detect spikes by first identifying all potential spikes on each channel using frequency filtering and then block scaling all channels at the same time in order to exclude potential spikes that fall below an amplitude and slope threshold. Its performance was compared to the human reviewers on the same set of patients.
Human reviewers showed surprisingly poor inter-reviewer agreement, but did broadly agree on the ranking of channels for spike activity. The computer algorithm performed as well as the human reviewers and did especially well at ranking channels from highest to lowest spike frequency.
Our algorithm showed good agreement with the different human reviewers, even though they demonstrated different criteria for what constitutes a ‘spike’ and performed especially well at the clinically important task of ranking channels by spike activity.
An automated, objective method to detect interictal spikes on intracranial recordings will improve both research and the surgical management of epilepsy patients.
PMCID: PMC3277646  PMID: 22033028
electrocorticography; interictal spike; epilepsy surgery; quantitative methods
5.  A study of clinical presentation and delays in management of acute myocardial infarction in community 
Indian Heart Journal  2012;64(3):295-301.
To assess the medico social demographics of acute myocardial infarction (AMI) in our community we studied 609 patients presenting between January 2008 to December 2008 with a detailed questionnaire in four centres of UP. Medical attention was sought late (> 6 hours) in 316 (51.6%), thrombolysis was obtained in 45.2% (275) and presentation was atypical in 16.3% (99). 36.2% (221) had pre-monitory symptoms of which 68% (150) ignored the same while of 32% (71) who did seek medical attention 47.9% (37) were brushed away as non-cardiac in origin. 20.3% (46/226) of hypertension, 23.2% (43/185) of diabetes and 83.4% (91/109) of hyperlipidaemia was diagnosed post event. We conclude that at least half of patients with AMI do not get definitive therapy, at least one in 10 patients do not have the classical symptoms, reasonable proportion are unaware of their risk factors, and a good majority have pre-monitory symptoms which get overlooked.
PMCID: PMC3860806  PMID: 22664814
Acute MI; Clinical presentation; Community study
6.  Ileal tuberculosis presenting as a case of massive rectal bleeding 
Massive rectal bleeding is an uncommon presentation of ileal tuberculosis. Fewer than 12 cases are reported in the literature. We report a case of ileal tuberculosis presenting at the emergency department with subacute intestinal obstruction and severe rectal bleeding. The case of the massive bleeding could be identified only after an exploratory laparotomy.
PMCID: PMC3108638  PMID: 21694836
massive rectal bleeding; ileum; intestinal tuberculosis
7.  Hydrodynamic Vaccination with DNA Encoding an Immunologically Privileged Retinal Antigen Protects from Autoimmunity through Induction of Regulatory T Cells1 
The eye is an immunologically privileged organ whose Ags serve as targets for experimental autoimmune uveitis (EAU), a model for human uveitis. We used a hydrodynamic i.v. injection of naked DNA to express the uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP) in the periphery, thus revoking its immune-privileged status. IRBP was expressed in the liver within hours of administration of as little as 10 μg of IRBP-DNA. Vaccinated mice were highly protected from EAU induced by immunization with IRBP for at least 10 wk after vaccination. Protection was partial in a reversal protocol. Mechanistic studies revealed specific hyporesponsiveness to IRBP without immune deviation, no evidence for apoptosis either by the Fas- or Bcl-2- regulated (mitochondrial) pathway and apparent lack of dependence on CD8+ cells, IL-10, or TGF-β. In contrast, depletion of CD25+ cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4+CD25high T cells could be cultured from vaccinated mice and transferred protection to unvaccinated, EAU-challenged recipients. In vitro characterization of these cells revealed that they are Ag specific, anergic, express FoxP3, CTLA-4, and glucocorticoid-induced TNFR, and suppress by contact. Thus, expression of IRBP in the periphery by DNA vaccination results in tolerance that acts at least in part through induction of IRBP-specific, FoxP3+CD4+CD25+ regulatory T cells. DNA vaccination may offer a new approach to Ag-specific therapy of uveitis.
PMCID: PMC2761821  PMID: 17911600
8.  Altered Chemokine Profile Associated with Exacerbated Autoimmune Pathology under Conditions of Genetic Interferon-γ Deficiency 
A prior study showed that mice deficient in IFN-γ (GKO) are more susceptible to experimental autoimmune uveitis (EAU) than are wild-type (WT) mice. Histopathology of uveitic eyes revealed that the ocular infiltrate in GKO mice was dominated by neutrophils and eosinophils rather than by mononuclear cells, as in WT mice. The present study was conducted to explore the differential expression of chemokine(s) likely to account for the distinct inflammatory cell composition in uveitic eyes of WT and GKO mice.
Mice were immunized to induce EAU. Lymph nodes draining the site of the immunization and the eyes were collected at different time points for chemokine analysis. Microarray, real-time PCR and protein analyses were performed to examine the expression of chemokines in WT and GKO mice.
Many chemokines were differentially upregulated in GKO versus WT mice. Expression of the Th1-associated chemokines CXCL10, CXCL9, CCL5, and CXCL11 was elevated in WT mice, whereas the Th2-associated chemokines CCL11, CCL17, and CCL1 and the Th17-associated chemokines CCL22 and CXCL2 were elevated in the GKO mice. Depletion of granulocytes abrogated EAU in both WT and GKO mice.
These results suggest that Th1-associated chemokines play a critical role in the attraction of mononuclear cells to the eyes in the presence of IFN-γ, while in the absence of this cytokine, Th2- and Th17-related chemokines may be the key elements for influx of granulocytes.
PMCID: PMC2756241  PMID: 17898285
9.  C57BL/6 Mice Genetically Deficient in IL-12/IL-23 and IFN-γ Are Susceptible to Experimental Autoimmune Myasthenia Gravis, Suggesting a Pathogenic Role of Non-Th1 Cells1 
Immunization with Torpedo acetylcholine receptor (TAChR) induces experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 (B6) mice. EAMG development needs IL-12, which drives differentiation of Th1 cells. The role of IFN-γ, an important Th1 effector, is not clear and that of IL-17, a proinflammatory cytokine produced by Th17 cells, is unknown. In this study, we examined the effect of simultaneous absence of IL-12 and IFN-γ on EAMG susceptibility, using null mutant B6 mice for the genes of both the IL-12/IL-23 p40 subunit and IFN-γ (dKO mice). Wild-type (WT) B6 mice served as control for EAMG induction. All mice were immunized with TAChR in Freund’s adjuvant. dKO mice developed weaker anti-TAChR CD4+T cells and Ab responses than WT mice. Yet, they developed EAMG symptoms, anti-mouse acetylcholine receptor (AChR) Ab, and CD4+ T cell responses against mouse AChR sequences similar to those of WT mice. dKO and WT mice had similarly reduced AChR content in their muscles, and IgG and complement at the neuromuscular junction. Naive dKO mice had significantly fewer NK, NKT, and CD4+CD25+Foxp3+ T regulatory (Treg) cells than naive WT mice. Treg cells from TAChR-immunized dKO mice had significantly less suppressive activity in vitro than Treg cells from TAChR-immunized WT mice. In contrast, TAChR-specific CD4+ T cells from TAChR-immunized dKO and WT mice secreted comparable amounts of IL-17 after stimulation in vitro with TAChR. The susceptibility of dKO mice to EAMG may be due to reduced Treg function, in the presence of a normal function of pathogenic Th17 cells.
PMCID: PMC2756237  PMID: 17513756
Invariant Natural Killer T cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). Here we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal antigen (Ag), IRBP. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with α-C-GalCer, which induces a strong IFN-γ but only a weak IL-4 production by iNKT cells, in contrast to the ligands α-GalCer (both IFN-γ and IL-4) and OCH (primarily IL-4). The protective effect of α-C-Gal-Cer was associated with a reduction of adaptive Ag specific IFN-γ and IL-17 production and was negated by systemic neutralization of IFN-γ. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-γ and a consequent dampening of the Th1 as well as the Th17 effector responses.
PMCID: PMC2597425  PMID: 18802082
NKT cells; autoimmune disease; cytokines
11.  Abrogation of Anti-Retinal Autoimmunity in IL-10 Transgenic Mice Due to Reduced T Cell Priming and Inhibition of Disease Effector Mechanisms1 
Experimental autoimmune uveitis (EAU) induced by immunization of animals with retinal Ags is a model for human uveitis. The immunosuppressive cytokine IL-10 regulates EAU susceptibility and may be a factor in genetic resistance to EAU. To further elucidate the regulatory role of endogenous IL-10 in the mouse model of EAU, we examined transgenic (Tg) mice expressing IL-10 either in activated T cells (inducible) or in macrophages (constitutive). These IL-10-Tg mice and non-Tg wild-type controls were immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein. Constitutive expression of IL-10 in macrophages abrogated disease and reduced Ag-specific immunological responses. These mice had detectable levels of IL-10 in sera and in ocular extracts. In contrast, expression of IL-10 in activated T cells only partially protected from EAU and marginally reduced Ag-specific responses. All IL-10-Tg lines showed suppression of Ag-specific effector cytokines. APC from Tg mice constitutively expressing IL-10 in macrophages exhibited decreased ability to prime naive T cells, however, Ag presentation to already primed T cells was not compromised. Importantly, IL-10-Tg mice that received interphotoreceptor retinoid-binding protein-specific uveitogenic T cells from wild-type donors were protected from EAU. We suggest that constitutively produced endogenous IL-10 ameliorates the development of EAU by suppressing de novo priming of Ag-specific T cells and inhibiting the recruitment and/or function of inflammatory leukocytes, rather than by inhibiting local Ag presentation within the eye.
PMCID: PMC2442578  PMID: 18390724
12.  Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity 
Susceptibility to experimental autoimmune uveitis (EAU), a model for human uveitis induced in mice with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP), is controlled by “natural” CD4+CD25+ regulatory T (T reg) cells. To examine whether endogenous expression of IRBP is necessary to generate these T reg cells, we studied responses of IRBP knockout (KO) versus wild-type (WT) mice. Unexpectedly, not only WT but also IRBP KO mice immunized with a uveitogenic regimen of IRBP in complete Freund's adjuvant (CFA) exhibited CD25+ regulatory cells that could be depleted by PC61 treatment, which suppressed development of uveitogenic effector T cells and decreased immunological responses to IRBP. These EAU-relevant T reg cells were not IRBP specific, as their activity was not present in IRBP KO mice immunized with IRBP in incomplete Freund's adjuvant (IFA), lacking mycobacteria (whereas the same mice exhibited normal T reg cell activity to retinal arrestin in IFA). We propose that mycobacterial components in CFA activate T reg cells of other specificities to inhibit generation of IRBP-specific effector T cells in a bystander fashion, indicating that effective T reg cells can be antigen nonspecific. Our data also provide the first evidence that generation of specific T reg cells to a native autoantigen in a mouse with a diverse T cell repertoire requires a cognate interaction.
PMCID: PMC2118294  PMID: 16585264
13.  Sexual dimorphism in immune response genes as a function of puberty 
BMC Immunology  2006;7:2.
Autoimmune diseases are more prevalent in females than in males, whereas males have higher mortality associated with infectious diseases. To increase our understanding of this sexual dimorphism in the immune system, we sought to identify and characterize inherent differences in immune response programs in the spleens of male and female mice before, during and after puberty.
After the onset of puberty, female mice showed a higher expression of adaptive immune response genes, while males had a higher expression of innate immune genes. This result suggested a requirement for sex hormones. Using in vivo and in vitro assays in normal and mutant mouse strains, we found that reverse signaling through FasL was directly influenced by estrogen, with downstream consequences of increased CD8+ T cell-derived B cell help (via cytokines) and enhanced immunoglobulin production.
These results demonstrate that sexual dimorphism in innate and adaptive immune genes is dependent on puberty. This study also revealed that estrogen influences immunoglobulin levels in post-pubertal female mice via the Fas-FasL pathway.
PMCID: PMC1402325  PMID: 16504066
14.  Retroviral gene therapy with an immunoglobulin-antigen fusion construct protects from experimental autoimmune uveitis 
Journal of Clinical Investigation  2000;106(2):245-252.
Immunoglobulins can serve as tolerogenic carriers for antigens, and B cells can function as tolerogenic antigen-presenting cells. We used this principle to design a strategy for gene therapy of experimental autoimmune uveitis, a cell-mediated autoimmune disease model for human uveitis induced with the uveitogenic interphotoreceptor retinoid-binding protein (IRBP). A retroviral vector was constructed containing a major uveitogenic IRBP epitope in frame with mouse IgG1 heavy chain. This construct was used to transduce peripheral B cells, which were infused into syngeneic recipients. A single infusion of transduced cells, 10 days before uveitogenic challenge, protected mice from clinical disease induced with the epitope or with the native IRBP protein. Protected mice had reduced antigen-specific responses, but showed no evidence for a classic Th1/Th2 response shift or for generalized anergy. Protection was not transferable, arguing against a mechanism dependent on regulatory cells. Importantly, the treatment was protective when initiated 7 days after uveitogenic immunization or concurrently with adoptive transfer of primed uveitogenic T cells. We suggest that this form of gene therapy can induce epitope-specific protection not only in naive, but also in already primed recipients, thus providing a protocol for treatment of established autoimmunity.
PMCID: PMC517488  PMID: 10903340

Results 1-14 (14)