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Biomedical subjects

B Wiggert

Publications and source records attributed to B Wiggert.

At least 19 recordsLinked to original sources
PubMed1997

Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a T helper-1-like or a T helper-2-like response.

This study examines whether genetic susceptibility vs resistance to experimental autoimmune uveoretinitis (EAU) in mice is associated with dominant type 1 vs type 2 cytokine response profiles. Mice from six strains were immunized with the uveitogenic retinal Ag IRBP. EAU was evaluated by histopathology. As judged by disease scores, three of the strains were susceptible, one was minimally susceptible, and two were resistant. Ag-specific type 1 vs type 2 cytokine responses (protein and/or mRNA) in draining lymph node cells, and IgG2a vs IgG1 Ab isotypes to IRBP, were measured as indicators of Th1-like vs Th2-like responses, respectively. The three susceptible strains (B10.A, C57BL/10, and BALB/k) showed a dominant Th1-like response profile characterized by high IFN-gamma and IL-12p40 (but not IL-4) responses, and a predominance of IgG2a Abs. The minimally susceptible strain (A/J) had an IFN-gamma response detectable only at the mRNA level, but produced predominantly IgG2a Abs. One of the two resistant strains (BALB/c) showed a characteristic Th2-like response with dominant Ag-specific IL-4 and IL-10 responses but no IFN-gamma, and predominantly IgG1 Abs. However, the other resistant strain (AKR) did not show a Th2-dominated response pattern, in that it had low, or no, IL-4 and IL-10 responses, and made predominantly IgG2a Abs to IRBP. These results suggest that whereas a Th1 response is required for susceptibility, resistance is not dependent on a Th2 response pattern. We suggest that regulatory influences other than skewing the response toward the Th2 pathway may be equally effective at conferring genetic resistance to EAU.

Animals↗
PubMed1997

IFN-gamma-deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response.

Experimental autoimmune uveitis (EAU) is a T cell-mediated disease that targets the neural retina and serves as a model of human uveitis. Uveitogenic effector T cells have a Th1-like phenotype (high IFN-gamma, low IL-4), and genetic susceptibility to EAU is associated with an elevated Th1 response. Here we investigate whether the ability to produce IFN-gamma is necessary for the development of EAU by immunizing IFN-gamma-deficient (GKO) mice with the uveitogenic protein interphotoreceptor retinoid binding protein (IRBP) and characterize the associated immunologic responses. GKO mice developed EAU comparable in severity and incidence to that of their wild-type littermates. However, the cytokine profile in their uveitic eyes as well as the cytokines produced by primed lymph node cells in response to IRBP showed a distinct profile: undiminished TNF-alpha and elevated IL-5, IL-6, IL-10, and lymphotoxin (but not IL-4) responses. The inflammatory infiltrate in GKO eyes contained an excess of granulocytes and IL-5- and IL-6-producing cells, but uveitic GKO mice did not up-regulate inducible nitric oxide synthase. GKOs had enhanced lymphocyte proliferation and delayed-type hypersensitivity responses to IRBP. Histology of the delayed-type hypersensitivity lesion in GKO had superimposed elements of an allergic-like response. Anti-IRBP Ab isotypes of GKO mice showed a reduction of IgG2a, but no enhancement of IgG1. Comparison of responses in +/+ and +/- wild-type mice revealed some limited evidence of a gene-dose effect. We conclude that IFN-gamma is not required for priming of pathogenic T cells or for effecting the retinal damage and photoreceptor loss typical of EAU. However, what appears to be a grossly similar disease is caused in the GKO by a deviant type of effector response.

Animals↗
PubMed1997

Experimental autoimmune uveitis in HLA-B27 transgenic mice.

The major histocompatibility complex (MHC) gene, HLA-B27 is strongly associated with auto-immune uveitis and spondyloarthropathies in humans. Experimental mouse models of autoimmune uveitis involve systemic immunization with the retinal autoantigen interphotoreceptor retinoid binding protein (IRBP). To assess possible roles of HLA-B27 in autoimmune uveitis, as well as to investigate a possible new animal model of human uveitis, inbred strains of C57BL/6 and C57BL/6 possessing the human HLA-B27 or HLA-A2 transgene were immunized with IRBP emulsified in complete Freund's adjuvant (CFA). Dilated eye examinations were performed to assess the timing and clinical course of any ensuing uveitis. Mice were sacrificed 3 to 4 weeks postinjection and the eyes submitted for histopathologic analysis. CFA alone did not produce any clinical uveitis. Fifty percent of eyes from the background C57BL/6 strain developed uveitis as early as 10 days postinjection. Of the eyes demonstrating uveitis, an average clinical score of 2.5 was present. Pathologically, a moderate scleritis and anterior uveitis was present. Fifty percent of A2 transgenic eyes developed uveitis as early as 14 days postinjection with an average clinical score of 2.0. Pathologically, a mild vitritis was present. Uveitis developed in only 20% of B27 transgenic mice and reached a peak on day 28. The average EAU score in diseased animals was 4.5. A dense retinitis and panuveitis was associated with severe vitritis. We conclude that the presence of the B27 gene is associated with a decreased incidence and slower rate of onset of EAU following immunization with IRBP; however, EAU may be more severe in the HLA-B27 expressing animals who do develop disease.

Animals↗
PubMed1996

Molecular characterization and developmental expression of a retinoid- and fatty acid-binding glycoprotein from Drosophila. A putative lipophorin.

A detailed understanding of the mechanism of lipid transport in insects has been hampered by the inability to identify the proapolipophorin gene that encodes apolipophorins I and II, the principal protein components of lipophorin, the lipid transport vehicle. Here we provide the first molecular description of the Drosophila gene encoding a retinoid- and fatty acid-binding glycoprotein (RFABG) and present evidence that it is a member of the proapolipophorin gene family. The gene, localized to the chromosome 4 (102 F region), encodes a 3351-amino acid protein that could serve as the precursor for the approximately 70-kDa and >200-kDa polypeptides associated with RFABG. The N-terminal sequence of the approximately 70-kDa polypeptide and that predicted for the >200-kDa polypeptide showed high sequence similarity to blowfly apolipophorin II and apolipophorin I, respectively. The RFABG precursor contains a signal peptide and exhibits a multidomain mosaic protein structure, which is typical of extracellular proteins. It has structural domains similar to lipid-binding proteins, namely vitellogenins and apolipoprotein B. The protein also contains a domain similar to the D domain of von Willebrand factor and mucin. The gene is expressed in the Drosophila embryo during development in cells that make up the amnioserosa and fat bodies. Immunolocalizations using specific antibodies against RFABG reveal that the protein is initially dispersed through the embryonic amnioserosa sac and latter concentrated at skeletal muscle-epidermis apodemeal contact junctions during larval development. This novel gene may play an important role in the transport of lipids, including retinoids and fatty acids, in insects.

Amino Acid Sequence↗
PubMed1996

Establishment and characterization of a murine CD4+ T cell line and clone that induce experimental autoimmune uveoretinitis in B10.A mice.

B10.A mice develop experimental autoimmune uveoretinitis after active immunization with the interphotoreceptor retinoid-binding protein (IRBP). CD4+ T cells play an important role in the development of the disease. In this study we have isolated and characterized a CD4+ T cell line and a T cell clone that induce experimental autoimmune uveoretinitis when transferred into naive B10.A mice. The cell line was isolated from draining lymph nodes of IRBP-immunized animals by repeated cycles of IRBP stimulation. The line was shown to be pathogenic after 4 rounds of in vitro stimulation with IRBP at 5 x 10(6) cells/mouse. A T cell clone derived from this line by limiting dilution was shown to be pathogenic when the same number of cells was injected; incidence and severity of disease, however, were much lower. After 16 rounds of IRBP-specific stimulation the cell line was pathogenic at 10(5) cells/mouse. Analysis of the V beta repertoire revealed that at this point the line was mostly composed of V beta 8.2- and V beta 6-positive cells (> 80% of the population). The uveitogenic clone expressed V beta 8.2. Both the T cell line and the clone elaborated an unrestricted lymphokine profile in vitro. However, when these cells were adoptively transferred into naive recipients, mRNA isolated from the uveitic retina showed only Th1 type cytokines. These data help to characterize the nature of pathogenic cells involved in ocular autoimmunity.

Animals↗
PubMed1996

Cytokine-dependent modulation of oral tolerance in a murine model of autoimmune uveitis.

In summary, our data suggest that oral tolerance in the mouse EAU model may occur by anergy/deletion or by suppression, depending on the feeding regimen. Tolerance involving putative regulatory cells appears to require the ability to produce both IL-4 and IL-10, whereas induction of tolerance involving anergy may not require the presence of Il-4 and IL-10. We propose that regulatory cells induced by three feedings of IRBP can be selectively enhanced through the use of cytokines. From the point of view of clinical therapy, it would be worthwhile to explore postimmunization feeding regimens involving administration of IL-4 and IL-10.

Administration, Oral↗
PubMed1996

Anti-tumor necrosis factor alpha therapy suppresses the induction of experimental autoimmune uveoretinitis in mice by inhibiting antigen priming.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) serves as a model for several immune-mediated diseases that affect the eye in humans. Previous studies indicated that tumor necrosis factor alpha (TNF-alpha) has an important proinflammatory role in EAU and possibly in human uveitis. In this study, the authors investigated the effect of anti-TNF-alpha therapy on EAU in mice. METHODS: Experimental autoimmune uveoretinitis was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP). The mice were treated with 100 or 300 microliters rabbit antiserum or polyclonal antibodies to human TNF-alpha. The treatment spanned either the afferent or the efferent stage of EAU (days -1, 1, 3, 5, 7, or days 8, 10, 12, 14, 16, respectively). Control animals were injected with preimmune rabbit serum at the corresponding times or were not treated. Three weeks after immunization, EAU was assessed by clinical evaluation and by histopathology. Immunologic responses were assessed by delayed-type hypersensitivity (DTH), lymphocyte proliferation to IRBP, and relative abundance of IRBP-primed splenocytes. RESULTS: The treatment with rabbit anti-TNF-alpha serum significantly ameliorated disease when given during the afferent stage but had no effect when given during the efferent stage of EAU. The effect on DTH, lymphocyte proliferation, and abundance of antigen-reactive cells roughly paralleled the effect on disease. CONCLUSIONS: Neutralization of systemic TNF ameliorates EAU. The effectiveness of afferent treatment in comparison to the treatment during the efferent stage, together with the reduced proliferation and the reduced abundance of IRBP-responsive cells, suggest that interference with afferent-acting processes such as antigen priming is important to achieve protection from EAU by anti-TNF treatment.

Animals↗
PubMed1995

Transforming growth factor-beta inhibits the cytokine-mediated expression of the inducible nitric oxide synthase mRNA in human retinal pigment epithelial cells.

Human retinal pigment epithelial (RPE) cells in culture respond to a mixture of cytokines (IFN-gamma, IL-1 beta, TNF-alpha) by producing large amounts of nitric oxide. Transforming growth factor-beta, unlike other growth factors, was found to inhibit this response by more than 75%. The expression of mRNA for the inducible form of nitric oxide synthase in RPE cells treated with cytokines was demonstrated by reverse transcription-polymerase chain reaction, sequencing of the PCR product and northern blotting. Transforming growth factor-beta was highly effective in inhibiting (by 75%) the cytokine-induced nitric oxide synthase mRNA expression. This response by RPE may play an important role in the etiology of infectious and inflammatory diseases affecting retina.

Base Sequence↗
PubMed1995

Induction of heme oxygenase 1 in the retina by intense visible light: suppression by the antioxidant dimethylthiourea.

The effect of intense visible light (light damage) on the expression of heme oxygenase 1 (HO-1), a protein induced by oxidative stress, was investigated in the rat retina. A sensitive reverse transcription-PCR assay demonstrated the expression of mRNA for HO-1 as well as HO-2, the noninducible HO form, in the normal retina. As analyzed by Northern blotting, however, HO-1 mRNA was barely detectable under normal circumstances. After exposure to intense visible light, retinas had markedly higher HO-1 mRNA levels than unexposed controls, with increases up to 52- and 98-fold at 12 and 24 hr of exposure, respectively. Intense light exposure also resulted in an increase in HO-1 protein. In contrast, no appreciable change in HO-2 mRNA or protein was observed. The increase in HO-1 message was more pronounced in rats previously reared in the dark than in those reared in a weak cyclic-light environment. A marked decrease from the high level of HO-1 mRNA induced by light insult was observed when the animals were allowed to recover in the dark for 24 hr after light exposure. Most important, treatment of animals with 1,3-dimethylthiourea, a synthetic antioxidant, prior to light exposure effectively blocked the increase in HO-1 mRNA. Thus, HO-1 is a sensitive marker for assessing light-induced insult in the retina. Since increased expression of HO-1 is thought to be a cellular defense against oxidative damage, its expression may play an important role in protecting the retina against light damage.

Amino Acid Sequence↗
PubMed1995

Induction of heme oxygenase-1 mRNA and protein in neocortex and cerebral vessels in Alzheimer's disease.

Previous studies demonstrated the specific association of heme oxygenase (HO)-1 protein to the neurofibrillary pathology of Alzheimer's disease (AD). In this study, we used reverse transcription-polymerase chain reaction methods to show the increased expression of HO-1 but not HO-2 mRNA transcripts in cerebral cortex and cerebral vessels from subjects with AD compared with age-matched non-AD controls. Neither the HO-1 nor the HO-2 mRNA levels was altered in the cerebellum, a brain region usually spared from the pathological alterations of AD. There was no clear evidence that the expression of HO-1 in these tissues was related to postmortem interval, cause of death, or the age of the subjects studied. Using immunoblotting methods, we further showed that HO-1 protein content was increased in neocortical and vascular samples from AD subjects compared with controls. Our findings suggest the specific induction of HO-1 mRNA and protein in the cerebral cortex and cerebral vessels but not HO-2 mRNA or protein in association with the pathological lesions of the disease.

Aged↗
PubMed1995

Photoreceptor cells in the vitiligo mouse die by apoptosis. TRPM-2/clusterin expression is increased in the neural retina and in the retinal pigment epithelium.

PURPOSE: To determine the mechanism of photoreceptor cell death in the vitiligo mouse, a model of retinal degeneration in which the genetic defect is not retina specific but is instead caused by single point mutation in the microphthalmia (mi) gene that codes for a basic helix-loop-helix DNA transcription factor. METHODS: Detection of apoptotic cells was performed in fixed retinal tissue using the TUNEL assay in animals 1, 2, 4, 6, 8, 16, 32, 40, and 52 weeks. Electron microscopic analysis was used to confirm the morphologic hallmarks of apoptosis, and Southern blot analysis was used to detect internucleosomal DNA fragmentation. Additionally, the expression of a gene associated with apoptosis, TRPM-2/clusterin, was examined. RESULTS: At ages beyond the time of normal retinal programmed cell death, vitiligo retinas had significantly more TUNEL-positive photoreceptor cells and more photoreceptor cells with condensed chromatin than controls. DNA internucleosomal fragmentation ladders were present in vitiligo retinas even as late as 15 weeks, a time well beyond developmental apoptosis in controls. TRPM-2/clusterin mRNA levels in vitiligo neural retinas were similar to controls initially but were two times greater than controls by 12 weeks. Surprisingly, TRPM-2/clusterin mRNA levels were elevated in the retinal pigment epithelium in the mutant; the expression at one week was two times greater than normals and remained elevated for many months, even though retinal pigment epithelial cells showed no morphologic evidence of apoptosis. CONCLUSIONS: The morphologic and biochemical data suggest that photoreceptor cells die by apoptosis in vitiligo mice. The increased retinal TRPM-2/clusterin mRNA levels may be a direct response to these events. The increased expression of this gene in the retinal pigment epithelium, however, may reflect its role in tissue regression and membrane remodeling. Mechanisms by which the mi gene defect might result in the vitiligo retinopathy are proposed.

Aging↗
PubMed1995

Identification of a major pathogenic epitope in the human IRBP molecule recognized by mice of the H-2r haplotype.

PURPOSE: Mice of the H-2b, H-2k, and H-2r haplotypes develop experimental autoimmune uveoretinitis (EAU) after immunization with interphotoreceptor retinoid-binding protein (IRBP) of bovine or monkey origin. The purpose of this study was to identify putative pathogenic epitope(s) of IRBP and to establish their immunodominance within the IRBP molecule. METHODS: Overlapping 20-amino acid peptides, spanning the entire human IRBP molecule, were synthesized and used to immunize C57BL/10 (H-2b), B10.BR (H-2k), and B10.RIII (H-2r) mice. Bovine IRBP was used as a positive control. Experimental autoimmune uveoretinitis was examined by histopathology 21 days after immunization. Immunologic responses were assessed by delayed-type hypersensitivity (DH) and lymphocyte proliferation assays. RESULTS: Peptide 161-180, spanning the sequence SGIPYIISYLHPGNTILHVD, was found to be highly pathogenic for B10.RIII mice but not for the other strains. A dose-response curve showed that peptide 161-180 was maximally pathogenic at 50 micrograms, but incidence and scores were reduced at 10 micrograms. The truncated 13-mer 165-177 was also highly pathogenic (100 to 200 micrograms), suggesting that it contained the pathogenic epitope. Mice immunized with the peptide, or with whole IRBP, had positive DH and lymphocyte responses to the immunizing as well as to the reciprocal antigen. A cell line derived to peptide 161-180 was also pathogenic for B10.RIII mice after adoptive transfer and responded (proliferation) to native IRBP. CONCLUSIONS: High incidence and high severity scores, as well as immunologic cross-recognition of peptide 161-180 and native IRBP in vivo and in vitro, suggest that this peptide contains a major epitope recognized as pathogenic by B10.RIII mice.

Amino Acid Sequence↗
PubMed1994

Characterization of human B cell proteins binding specifically to uveitopathogenic peptide 1169-1191 of bovine IRBP.

Peptide 1169-1191 is a major uveitopathogenic determinant of bovine Interphotoreceptor Retinoid Binding Protein (IRBP) in Lewis rats. Previously, we identified two proteins with approximate molecular masses of 72 and 74 kDa and one with a molecular mass of 40 kDa from B cells of naive Lewis rats and EBV-transformed B cells from a human patient with ocular Behçet's disease that bind to bovine IRBP peptide 1169-1191. In this study, we have partially characterized these proteins. The two proteins with molecular masses 72 and 74 kDa belong to the HSP 70 family of proteins and the 40-kDa protein is actin.

Adenosine Triphosphate↗
PubMed1994

Retinoid processing in retinal pigment epithelium of toad (Bufo marinus).

The formation of 11-cis-[3H]retinal in the retinal pigment epithelium (RPE) and its release to extracellular medium containing interphotoreceptor retinoid-binding protein (IRBP) were studied in the RPE eyecup of the toad (Bufo marinus). The RPE was labeled with all-trans-[3H]retinol during an initial 1-h incubation. In phase 2 of the incubation (0-2 h), the extracellular medium contained initially ligand-free IRBP (0-26 microM). Retinoids subsequently extracted from the extracellular medium and RPE were analyzed by high performance liquid chromatography and scintillation counting. IRBP increased both the molar amount and specific radioactivity of 11-cis-retinal released by the RPE during phase 2. The molar amount of 11-cis-retinal in the RPE was small relative to that of retinal released with high IRBP. With 21 microM IRBP and phase 2 incubations of > or = 10 min, the specific radioactivity of released 11-cis-retinal exceeded that of all-trans-retinyl ester in the RPE. The specific radioactivity of 11-cis-retinyl ester was less than that of all-trans-ester, independent of IRBP concentration. The results indicate that IRBP promotes the formation (from all-trans-precursor) as well as the release of 11-cis-retinal and suggest the preferred utilization of recently incorporated and esterified all-trans-retinol in 11-cis-retinal synthesis in a "last in/first out" manner.

Animals↗
PubMed1994

Increase in retinyl palmitate concentration in eyes and livers and the concentration of interphotoreceptor retinoid-binding protein in eyes of vitiligo mutant mice.

Retinyl esters play an important role in the visual cycle because they are involved in regeneration of 11-cis-retinal for use in rhodopsin formation. In the present study, retinyl ester concentrations were significantly elevated in eyes and livers of mice homozygous for the vitiligo mutation (mivit/mivit). Vitiligo mice demonstrate a slowly progressing retinal degeneration characterized by gradual loss of photoreceptor cells and rhodopsin as well as uneven pigmentation of the retinal pigment epithelium (RPE). Analysis of retinoids by h.p.l.c. indicated that the retinyl palmitate level was increased fivefold in eyes of affected mice at 10 weeks postnatally and was threefold higher at 22 weeks of age. Accumulation of retinyl palmitate occurred in the RPE rather than the neural retina. Furthermore, the concentration of all-trans-retinol was elevated in the RPE of vitiligo mice. Levels of interphotoreceptor retinoid binding protein (IRBP) were increased in vitiligo mice between ages 4 and 14 weeks, but returned to normal by 16 weeks. Increased IRBP levels were not due to increased protein synthesis because IRBP mRNA levels did not differ significantly between control and affected animals. To examine possible systemic involvement in vitiligo mice, retinoids were evaluated in liver and plasma. Mean hepatic total vitamin A levels in affected mice were approximately 1.7 times higher than controls. Analysis of esterified and non-esterified retinoids in liver showed that the concentration of retinyl palmitate was elevated. Plasma retinol levels were normal. This study provides the first evidence of altered systemic retinoid metabolism in vitiligo mice, which occurs, significantly, under normal dietary conditions.

Animals↗