Partial retinal photoreceptor loss in a transgenic mouse model associated with reduced levels of interphotoreceptor retinol binding protein (IRBP, RBP3).

Organ-specific transgenic membrane expression of hen egg lysozyme (HEL) as a "neo-self antigen" has been used in several models to study immunological tolerance. In this study we report the changes which occur in the B10.BR mouse retina when membrane-bound HEL is expressed in photoreceptors under the control of the promoter for interphotoreceptor retinoid binding protein (IRBP, RBP3). On direct clinical examination of the single transgenic (sTg-IRBP:HEL) mouse fundus, a low-level increase in retinal degeneration compared to non-transgenic controls was observed, presenting as drusenoid deposits and occasional small patches of atrophy. On histological examination, there was an overall shortening of outer segments and loss of photoreceptor nuclei in sTg-IRBP:HEL mice, which was more pronounced in the retinal periphery, particularly inferiorly. The fundoscopically observed lesions did not correlate with the photoreceptor shortening/loss but appeared to be located at the level of the retinal pigment epithelium/choriocapillaris layer and were an exaggeration in size and number of similar age-related changes found in wild type (WT) mice. In addition, neither the atrophic lesions nor the photoreceptor shortening were associated with common retinal degeneration genes, nor were they caused by exposure to light damage since mice housed at both high and low ambient light levels had similar degrees of retinal degeneration. Instead, sTg-IRBP:HEL mice expressed reduced levels of soluble retinal IRBP compared to WT mice which were present from postnatal day16 (P16) and preceded development of photoreceptor shortening (onset P21). We propose that insertion of the HEL transgene in the photoreceptor membrane disrupted normal photoreceptor function and led to reduced levels of soluble IRBP and retinal thinning. A similar phenotype has been observed in IRBP deficient mice. Despite the retinal thinning, the amount of HEL expressed in the retina was sufficient to act as an autoantigenic target when the mice were crossed to the HEL T cell receptor Tg mouse, since double transgenic (dTg-IRBP:HEL) mice spontaneously developed a severe uveoretinitis with onset at weaning. We suggest that, although membrane expression of foreign transgene products is likely to modify the structure and function of tissues and cells, the technology provides useful models to investigate mechanisms of antigen-specific immunological tolerance.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Transmission Electron Microscopy Data on drusen-like deposits in the retinal degeneration sTg-IRBP: HEL mouse model.

Histology (H&E) and transmission electron microscopy (TEM) data are provided showing age-related changes in the retinal structure of sTg-IRBP:HEL mice. These include substantial photoreceptor loss, atrophy of the retinal pigment epithelium, Bruch׳s membrane disruption and thickening, along with the presence of drusenoid deposits and changes in basal laminar infoldings. These features resemble some of those key characteristics found in the course of human dry (atrophic) age-related macular degeneration (AMD), particularly with regard to drusen. Hence, we believe the sTg-IRBP:HEL mouse model represents a useful and promising archetype for future study of the mechanism of drusen formation in AMD.

Dehydroepiandrosterone attenuates allergic airway inflammation in Dermatophagoides farinae-sensitized mice.

Dehydroepiandrosterone, an androgen abundant in circulation, has important immunomodulating effects. In this study the therapeutic effect of dehydroepiandrosterone on established allergic inflammation was examined in a dust mite (Dermatophagoides farinae)-induced asthma model. Airway inflammation was provoked in D. farinae-sensitized BALB/c mice by repetitive intratracheal challenge (3 times, once a week). Three days after the first challenge, mice were fed a diet incorporated with 1.5% (w/w) dehydroepiandrosterone and were examined at days 3 and 6 after the last challenge. Airway challenge resulted in pulmonary eosinophilic inflammation accompanied by elevated blood eosinophil counts and elevated serum and bronchoalveolar lavage immunoglobulin E antibody levels in control diet-fed mice. However, the D. farinae-induced airway inflammation and blood eosinophilia was significantly reduced in dehydroepiandrosterone-fed mice, which was associated with a decrease in serum interleukin-4, interleukin-5, and interferon-gamma levels. Total immunoglobulin E antibody concentrations in serum and bronchoalveolar lavage fluids were not affected by the dehydroepiandrosterone treatment. These results demonstrated that dehydroepiandrosterone could suppress preexisting allergic airway inflammation.

Prevention of Der p2-induced allergic airway inflammation by Mycobacterium-bacillus Calmette Guerin.

Epidemiologic studies suggest an inverse correlation between infection and development of allergy. The purpose of this study was to test the hypothesis whether a preexisting T helper 1 (Th1)-type immune response elicited by Mycobacterium bovis-bacillus Calmette-Guerin (BCG) immunization could suppress allergic airway inflammation induced by the mite allergen Dermatophagoides pteronyssinus group 2 (Der p2) in an animal model. C57BL/6 mice were immunized with subcutaneous injection of BCG, then intraperitoneal Der p2 emulsified in alum. Der p2-specific immunoglobulin G1 and cytokine production from splenocytes were measured after Der p2 sensitization, and pulmonary function and airway inflammation were determined after inhalation challenge with Der p2. The intraperitoneal Der p2 with alum injection was able to induce Der p2-specific immunoglobulin G1 production, which could be downregulated by the pretreatment with BCG + Der p2. The inoculation of BCG + Der p2 caused splenocytes to produce more interferon-gamma, and this level was higher than that elicited by Der p2 or buffer alone. The positive interferon-gamma-staining CD4 cells were also increased after activation by phorbol myristate acetate and ionomycin. Lung pathology examination found decreased airway inflammation (associated with the best pulmonary function and least airway desquamation) in the mice inoculated with BCG + Der p2. In this Der p2-induced allergy model, BCG inoculation with Der p2 can cause a Th1-type immune response that hinders Der p2-induced allergic sensitization and the development of airway inflammation.

Effectiveness of Dp2 nasal therapy for Dp2- induced airway inflammation in mice: using oral Ganoderma lucidum as an immunomodulator.

Nasal immunotherapy with allergen has been reported to be effective for airway allergic disease. A group of 50 male Balb/c mice were immunized intraperitoneally with recombinant Dermatophagoides pteronyssinus group 2 (rDp2), then oral feeding with Ganoderma lucidum (known as "Ling Zhi," LZ OT) and intranasal therapy with native Dp2 (Dp2 NT) were given, the mice then received intratracheal challenge with rDp2 at 28 days and 35 days after immunization. Airway hypersensitivity to methacholine was measured 30 min (early phase) and 24 h (late phase) after the second challenge. The cytokine producing CD4 cells in PBL and interferon-gamma (IFN-gamma) concentrations in bronchoalveolar lavage fluid and sera were measured on 37 days after immunization. Both Dp2 NT and LZ OT downregulated total inflammatory cell infiltration in the airway. Dp2 NT reduced IL-5+/CD4+ cells and increased IFN-gamma+/CD4+ cells. When LZ OT was added to Dp2 NT, the reduction of IL-5+/CD4+ cells was diminished and the increment of IFN-gamma+/CD4+ cells was increased. LZ OT alone increased both IL-5+/CD4+ cells and IFN-gamma+/CD4+ cells. When LZ OT was added to Dp2 NT, IgG2a was further increased to a significant level. LZ OT alone significantly suppressed IgG1 and increased IgG2a production. When lung function was measured after therapy, early phase airway hypersensitivity to methacholine significantly suppressed by Dp2 NT, while late phase hypersensitivity was suppressed but not to a significant level. When LZ OT was added to Dp2 NT, the suppression of late phase airway hypersensitivity to methacholine reached a significant level. In this mouse model of Dp2-induced airway hypersensitivity, Dp2 NT downregulated airway inflammatory cell infiltration and decreased immediate airway hypersensitivity to methacholine. When LZ OT was coadministered, the airway lymphocytes and circulatory IFN-gamma+/CD4+ were both increased and late phase airway hypersensitivity was decreased. These results suggest that Dp2 NT might have a therapeutic effect on Dp2-induced airway hypersensitivity and LZ OT might also have an effect on Dp2 NT immunotherapy.

Postconditioning with far-infrared irradiation increases heme oxygenase-1 expression and protects against ischemia/reperfusion injury in rat testis.

AIMS: Studies have shown that heme oxygenase-1 (HO-1) has a protective role in the mechanism underlying hypoxic preconditioning. We used a far-infrared radiation (FIR) heater to investigate the postconditioning protective role of HO-1 against ischemia/reperfusion (I/R) injury in rat testis. MAIN METHODS: Forty rats were used. Testis ischemia was mimicked by total obstructive clamping of testis vessels for 1, 2, or 4 h, and concomitant postconditioning with 30 min FIR or heat light during initially 30 min reperfusion. HO-1 expression and apoptosis of testis tissues were examined by immunohistochemistry and in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, respectively. HO-1 protein level and caspase-3 activity were analyzed by Western blotting. KEY FINDINGS: There was less apoptotic activity in rat testis after FIR, as determined by TUNEL assay. Higher HO-1 protein expression was observed by immunohistochemistry and Western blotting (p<0.01) in testis cells after FIR postconditioning. In contrast, caspase-3 activity was significantly higher in heat light groups, as compared with FIR groups (p<0.01). SIGNIFICANCE: FIR postconditioning attenuated I/R injury in rat testis by inducing HO-1 expression, which might have a protective role in testis apoptosis after I/R injury.

Macrophage migration is controlled by Tribbles 1 through the interaction between C/EBPß and TNF-α.

In mammals, three Tribbles gene family members have been identified, Tribbles 1, 2 and 3 (Trib1, Trib2 and Trib3). All family members are considered to be pseudokinases in that they contain domains homologous to serine/threonine kinase catalytic cores, but they lack several conserved residues in the ATP-binding pocket. Trib1 is implicated in the inflammatory response pathway through its ability to regulate mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-κB) and CCAAT Enhancer Binding Protein (C/EBP). However, its role in macrophages function is unknown. Here, we investigated the functional role of Trib1 in Toll-like receptor-mediated inflammatory responses to IFN-γ in RAW264.7 cells. In gene knock-down experiments in macrophages using small interfering RNAs targeted to Trib1, it was observed that TNF-α production was increased following treatment with IFN-γ and/or TLR2 ligands. Finally, Trib1-silenced macrophages failed to show MCP-1 induced chemokinesis and indicating involvement of Trib1 in controlling of macrophage migration. This work demonstrates that Trib1 contributes to the pro-inflammatory response caused by TLR2 ligands and controls macrophage migration as well as being a biomarker in macrophage-related diseases in both human and veterinary medicine.

Simvastatin induces the expression of hemeoxygenase-1 against ischemia-reperfusion injury on the testes in rats.

We evaluate the protective role of simvastatin-induced HO-1 in remote preconditioning against testis ischemia-reperfusion (IR) injury in vivo. Simvastatin was intraperitoneally (i.p.) injected 24 h before IR injury. Testis was occluded in the right testis for 40 min and followed by 30 min of reperfusion to induce IR injury. Tin protoporphyrin (Snpp), a competitive inhibitor of hemeoxygenase, was i.p. injected 1 h before the IR injury in separate groups of rats. The rat testes were harvested 24 h later. Induction of HO-1 expression by simvastatin was significantly increased at 24 and 48 h. Rats pre-treated with simvastatin showed higher expression of HO-1 protein by Western blotting and immunohistochemistry (IHC), and presented lower caspases-3 activity by caspase-3 activity assay. TUNEL staining analysis revealed simvastatin pretreatment significantly reduced IR induced cellular apoptosis. Contrarily, the simvastatin-induced cytoprotective effect was entirely abolished by administrations of Snpp. Further, lower caspase-3 activities were also noted in simvastatin plus Snpp (SS) group than the control plus Snpp (CS) group. After IR injury, eNOS immunoreactivity was markedly increased in the germ cell and Leydig cell of testicular tissues. Pretreatment of simvastatin significantly decreased eNOS immunoreactivity in the germ cell of the tubules in the rat testes. In conclusion, we suggest HO-1 plays a protective role in IR-induced injury in the testes of rats.

Production of salivary immunoglobulin A and suppression of Dermatophagoides pteronyssinus-induced airway inflammation by local nasal immunotherapy.

BACKGROUND: Local nasal immunotherapy (LNIT) is an effective immunotherapeutic modality, especially when targeting a single immunodominant peptide from an allergen. However, the working mechanisms of LNIT are poorly understood. We hypothesized that LNIT with a mixture of group 2 allergens of Dermatophagoides pteronyssinus (Der p 2) protein and fungal immunomodulatory peptide (FIP) would generate suppression of Der p-induced airway inflammation through immunoglobulin (Ig) A secretion in the airways. METHOD: Mice were sensitized with recombinant Der p 2 (rDer p 2) and Der p followed by LNIT with rDer p 2 in conjunction with FIP. After intratracheal challenge with rDer p 2 and Der p, the airway inflammation was determined by analyzing the cell subpopulation and cytokine concentration in the bronchoalveolar lavage fluid. The allergen-specific IgE, IgG2a and IgG in the sera and IgA in the saliva were measured by ELISA. RESULTS: LNIT with rDer p 2 in conjunction with FIP could downregulate the lymphocyte infiltration in both rDer p 2- and Der p-induced airway inflammation. Both total and specific IgA in the saliva were increased after LNIT. Serum levels of IL-4, IL-10 and specific IgE were reduced and the specific IgG2a and IgG increased after LNIT. After LNIT, there was a reduction of airway hypersensitivity at 30 min after allergen challenge in the rDer p 2-and Der p-sensitized mice, with a significant decrease only in rDer p 2-sensitized mice. CONCLUSION: LNIT with rDer p 2 in conjunction with FIP was not only able to suppress rDer p 2-induced airway inflammation but also generate suppression of Der p-induced airway inflammation. The simultaneous reduction of IL-4 and IL-10 indicated that IL-10-producing cells were not activated by LNIT. The increment of IgA in the airway might play a role in the prevention of airway inflammation.

Efficacy of local nasal immunotherapy for Dp2-induced airway inflammation in mice: Using Dp2 peptide and fungal immunomodulatory peptide.

BACKGROUND: Local nasal immunotherapy (LNIT) is an effective immunotherapy. Peptides derived from the group 2 allergen of Dermatophagoides pteronyssinus, Dp2 28-40 and Dp2 28-40A, and fungal immunomodulatory peptide (FIP) have been shown to act as T(H)1 potential and response-inducing adjuvant. LNIT by the use of Dp2 peptides in conjunction with FIP were investigated. OBJECTIVE: We sought to determine whether Dp2-induced airway inflammation in mice could be downregulated by Dp2 peptides or a mixture of Dp2 peptides with FIP. METHOD: Mice were sensitized with rDp2 followed by LNIT with Dp2 peptides, FIP, or FIP and a mixture of Dp2 peptides. After intratracheal challenge with rDp2, the airway inflammation and hyperresponsiveness were determined by bronchoalveolar lavage fluid (BALF) analysis and methacholine challenge. RESULTS: Both Dp2 peptides and FIP were able to inhibit rDp2-induced airway inflammation and airway hyperresponsiveness. An increase in IFN-gamma and a decrease in IL-5 in BALF and sera were found after LNIT with Dp2 peptides, FIP, and mixtures of both. Serum levels of TGF-beta were reduced after LNIT with FIP and Dp2 28-40. Penh values were significantly decreased after methacholine challenge in both the early and late phase. CONCLUSIONS: LNIT with allergen-derived peptides and FIP can produce an anti-inflammatory effect on allergen-induced airway inflammation. LNIT with selected peptides and FIP might be a good alternative therapy for allergic airway disease.

Identification of the major allergenic components in Blomia tropicalis and the relevance of the specific IgE in asthmatic patients.

BACKGROUND: Blomia tropicalis has been reported to be a clinically important allergen in house dust. High prevalence of sensitization to B. tropicalis has been noted in asthmatic patients in Taiwan; however, the allergenic components and its impact on asthmatic patients remain to be clarified. OBJECTIVE: To analyze the prevalence of IgE against B. tropicalis and each allergenic component in asthmatic patients. METHODS: A series of recombinant allergenic components were used for skin tests. The B. tropicalis specific IgE in the serum were measured using the Pharmacia CAP System and immunoblot analysis. RESULTS: A total of 131 patients were included in this study: 44% of these 131 patients were allergic to B. tropicalis, 43% of the 80 B. tropicalis-sensitive patients were allergic to Blo t 5, and 75% of the 65 Blo t 5-sensitive patients were allergic to Blo t 5 fragment 3 (Blo t 5 70-117). The sera IgE binding activity to B. tropicalis was repeatedly tested after Dermatophagoides pteronyssinus absorption, and results showed that most patients were concurrently sensitized to D. pteronyssinus and B. tropicalis. In addition, in 2 (18%) of 11 patients, the B. tropicalis sensitization was caused by the cross-reactivity of D. pteronyssinus. CONCLUSION: A high prevalence of B. tropicalis sensitization was detected in our asthmatic patients, and most of them were concurrently sensitized to D. pteronyssinus and B. tropicalis. The major allergenic component and its IgE binding fragments in Blo t 5 have been identified. These allergenic components can be used for the allergenic determination in B. tropicalis and for further immunotherapy.